+ All Categories
Home > Documents > Silica Sol-Gel Entrapment of the Enzyme Chloroperoxidase

Silica Sol-Gel Entrapment of the Enzyme Chloroperoxidase

Date post: 13-Feb-2017
Category:
Upload: vohuong
View: 226 times
Download: 0 times
Share this document with a friend
11
Research Article Silica Sol-Gel Entrapment of the Enzyme Chloroperoxidase Tuan Le, Selina Chan, Bassem Ebaid, and Monika Sommerhalter Department of Chemistry and Biochemistry, California State University East Bay, 25800 Carlos Bee Boulevard, Hayward, CA 94542, USA Correspondence should be addressed to Monika Sommerhalter; [email protected] Received 3 June 2015; Revised 24 August 2015; Accepted 31 August 2015 Academic Editor: Jiazhi Yang Copyright © 2015 Tuan Le et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. e enzyme chloroperoxidase (CPO) was immobilized in silica sol-gel beads prepared from tetramethoxysilane. e average pore diameter of the silica host structure (3 nm) was smaller than the globular CPO diameter (6 nm) and the enzyme remained entrapped aſter sol-gel maturation. e catalytic performance of the entrapped enzyme was assessed via the pyrogallol peroxidation reaction. Sol-gel beads loaded with 4 g CPO per mL sol solution reached 9–12% relative activity compared to free CPO in solution. Enzyme kinetic analysis revealed a decrease in cat but no changes in or . Product release or enzyme damage might thus limit catalytic performance. Yet circular dichroism and visible absorption spectra of transparent CPO sol-gel sheets did not indicate enzyme damage. Activity decline due to methanol exposure was shown to be reversible in solution. To improve catalytic performance the sol-gel protocol was modified. e incorporation of 5, 20, or 40% methyltrimethoxysilane resulted in more brittle sol-gel beads but the catalytic performance increased to 14% relative to free CPO in solution. e use of more acidic casting buffers (pH 4.5 or 5.5 instead of 6.5) resulted in a more porous silica host reaching up to 18% relative activity. 1. Introduction Silica nanostructures can be fabricated using a room tem- perature sol-gel process that is compatible with biomolecules [1, 2]. e entrapment of enzymes inside these silica nanostructures has facilitated diverse applications in bio- catalysis and biosensing [3]. Here, we demonstrate the use of sol-gel technology to make a biocatalyst based on entrap- ment of the enzyme chloroperoxidase (CPO) inside a silica nanostructure. CPO (EC 1.11.1.10) is one of the most versatile heme enzymes known to date. It can be obtained as a secreted, glycosylated protein from the marine fungus Caldariomyces fumago [4, 5]. CPO catalyzes halogenation, sulfoxidation, hydroxylation, and epoxidation reactions with high substrate promiscuity under environmentally benign conditions [6]. CPO catalyzed oxidative transformations are based on hydro- gen peroxide or an alkyl peroxide as oxidant, whereas the equivalent traditional chemical reactions require stoichio- metric amounts of heavy metal salts [7]. Another advanta- geous feature of CPO is its ability to perform these reactions in a highly enantioselective manner [8]. Transforming CPO into a practical biocatalyst for oxidative transformations in biosynthetic chemistry is therefore highly desirable. Fur- ther possible biotechnological applications of CPO include wastewater treatment or upgrading petroleum products [9– 11]. e use of CPO as biocatalyst, however, is hampered by its loss of activity in the presence of organic solvents, deactivation at high concentrations of the oxidant H 2 O 2 , and instability at elevated temperatures [8, 12]. Various approaches have been attempted to improve the stability and productivity of CPO in solution [13–16] as well as in immobilized form [12, 17–21]. In our study, CPO was entrapped in a hydrogel prepared from the precursor tetramethoxysilane (TMOS) with or without the addition of methyltrimethoxysilane (MTMS). Enzyme immobilization offers key advantages, most notably ease of enzyme recovery and reusability [22]. Silica-based sol-gels furnish chemically inert and optically transparent immobilization matrices [2, 23]. However, sol-gel immobilization is oſten accompanied by a loss of enzymatic activity. is loss can be due to enzyme damage, leakage of enzyme from the immobilization host, or limited material transport of substrates and products within the sol-gel nanostructure. We examined each of these possible reasons using absorption and circular dichroism spec- troscopy, porosimetry, and an enzyme activity assay for CPO, Hindawi Publishing Corporation Journal of Nanotechnology Volume 2015, Article ID 632076, 10 pages http://dx.doi.org/10.1155/2015/632076
Transcript
Page 1: Silica Sol-Gel Entrapment of the Enzyme Chloroperoxidase

Research ArticleSilica Sol-Gel Entrapment of the Enzyme Chloroperoxidase

Tuan Le Selina Chan Bassem Ebaid and Monika Sommerhalter

Department of Chemistry and Biochemistry California State University East Bay 25800 Carlos Bee BoulevardHayward CA 94542 USA

Correspondence should be addressed to Monika Sommerhalter monikasommerhaltercsueastbayedu

Received 3 June 2015 Revised 24 August 2015 Accepted 31 August 2015

Academic Editor Jiazhi Yang

Copyright copy 2015 Tuan Le et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The enzyme chloroperoxidase (CPO) was immobilized in silica sol-gel beads prepared from tetramethoxysilane The average porediameter of the silica host structure (sim3 nm) was smaller than the globular CPO diameter (sim6 nm) and the enzyme remainedentrapped after sol-gel maturationThe catalytic performance of the entrapped enzymewas assessed via the pyrogallol peroxidationreaction Sol-gel beads loaded with 4 120583g CPO per mL sol solution reached 9ndash12 relative activity compared to free CPO insolution Enzyme kinetic analysis revealed a decrease in 119896cat but no changes in119870119872 or119870

119868 Product release or enzyme damage might

thus limit catalytic performance Yet circular dichroism and visible absorption spectra of transparent CPO sol-gel sheets did notindicate enzyme damage Activity decline due to methanol exposure was shown to be reversible in solution To improve catalyticperformance the sol-gel protocol was modifiedThe incorporation of 5 20 or 40methyltrimethoxysilane resulted in more brittlesol-gel beads but the catalytic performance increased to 14 relative to free CPO in solutionThe use of more acidic casting buffers(pH 45 or 55 instead of 65) resulted in a more porous silica host reaching up to 18 relative activity

1 Introduction

Silica nanostructures can be fabricated using a room tem-perature sol-gel process that is compatible with biomolecules[1 2] The entrapment of enzymes inside these silicananostructures has facilitated diverse applications in bio-catalysis and biosensing [3] Here we demonstrate the useof sol-gel technology to make a biocatalyst based on entrap-ment of the enzyme chloroperoxidase (CPO) inside a silicananostructure

CPO (EC 111110) is one of the most versatile hemeenzymes known to date It can be obtained as a secretedglycosylated protein from the marine fungus Caldariomycesfumago [4 5] CPO catalyzes halogenation sulfoxidationhydroxylation and epoxidation reactions with high substratepromiscuity under environmentally benign conditions [6]CPO catalyzed oxidative transformations are based on hydro-gen peroxide or an alkyl peroxide as oxidant whereas theequivalent traditional chemical reactions require stoichio-metric amounts of heavy metal salts [7] Another advanta-geous feature of CPO is its ability to perform these reactionsin a highly enantioselective manner [8] Transforming CPOinto a practical biocatalyst for oxidative transformations in

biosynthetic chemistry is therefore highly desirable Fur-ther possible biotechnological applications of CPO includewastewater treatment or upgrading petroleum products [9ndash11] The use of CPO as biocatalyst however is hamperedby its loss of activity in the presence of organic solventsdeactivation at high concentrations of the oxidant H

2O2 and

instability at elevated temperatures [8 12]Various approaches have been attempted to improve the

stability and productivity of CPO in solution [13ndash16] aswell as in immobilized form [12 17ndash21] In our study CPOwas entrapped in a hydrogel prepared from the precursortetramethoxysilane (TMOS) with or without the addition ofmethyltrimethoxysilane (MTMS) Enzyme immobilizationoffers key advantages most notably ease of enzyme recoveryand reusability [22] Silica-based sol-gels furnish chemicallyinert and optically transparent immobilization matrices [223] However sol-gel immobilization is often accompaniedby a loss of enzymatic activityThis loss can be due to enzymedamage leakage of enzyme from the immobilization host orlimited material transport of substrates and products withinthe sol-gel nanostructureWe examined each of these possiblereasons using absorption and circular dichroism spec-troscopy porosimetry and an enzyme activity assay for CPO

Hindawi Publishing CorporationJournal of NanotechnologyVolume 2015 Article ID 632076 10 pageshttpdxdoiorg1011552015632076

2 Journal of Nanotechnology

that is the peroxidation of pyrogallol The effect of methanolonCPOwas also investigated sincemethanol is released fromthe precursor TMOS during the hydrolysis and condensationreactions of the sol-gel process In our attempts to optimizethe catalytic performance of sol-gel immobilized CPO weexploited the stability of CPO in acidic solutions [24] anddeviated from the pH range of 60ndash70 which is typicallyemployed in protein sol-gel immobilization procedures Theuse of more acidic solutions in the sol-gel process might alsobe beneficial for various sol-gel applications based on otherproteins that either withstand or thrive in acidic conditions

2 Materials and Methods

21 Materials CPO from C fumago was purchased fromSigma-Aldrich The turbid suspensions were centrifuged at15000 rpm for 5 minutes in an Eppendorf microcentrifugeThe concentrations of the CPO supernatants were deter-mined via the intensity of the Soret band [25] and confirmedwith protein assays [26] The CPO samples used in this studyhad concentrations in the range of 9 to 15mgmL All otherreagentswere purchased fromSigma-Aldrich or Fisher Scien-tific Aqueous solutions were prepared with deionized waterobtained from a Millipore Milli-Q device

22 Sol-Gel Entrapment A sol solution was prepared bymixing 50120583L deionized water 47 120583L 0010M hydrochloricacid and 235 120583L TMOS in a microcentrifuge tube If MTMSwas included TMOS and MTMS were premixed The molarratio ofMTMSwas either 5 20 or 40of the totalMTMSandTMOS precursor mixtureThe sol solution was sonicated in aBranson 1510 sonicator bath filled with ice water for 30 min-utes and either used immediately thereafter or stored for upto a week at 4∘C In amicrocentrifuge tube 140 120583L sol 480 120583Lcasting buffer and 500120583L diluted CPO solution were mixedThe casting buffers were prepared by mixing 01M citric acidand 02M dibasic sodium phosphate to reach pH values of45 55 60 or 65 CPO was diluted in 20mM potassiumphosphate-potassium hydroxide buffer pH 60 Small beadsof 50120583L volume were prepared by pipetting the mixtureonto parafilm sheets As the pH value of the casting bufferdecreased the gelation time increased from approximately 15minutes (pH 65) to 240min (pH 45)The CPO sol-gel beadswere transferred into glass tubes in sets of three to eight beadsand covered with a storage buffer composed of 21mL deion-izedwater and 032mL 01M citric acid-02Mdibasic sodiumphosphate buffer pH 42 This storage buffer was typicallyexchangedwith a newportion of storage buffer after one hourand thereafter each day for seven or ten days The storagetemperature was 4∘C

23 Activity Assays Activity assays were typically conductedin 01M citric acid-02M dibasic sodium phosphate bufferpH 42 with 27 or 86mM H

2O2 35mM pyrogallol and

02 120583gmL CPO CPO was added last to start the reactionThe assay volume was 3mL Control reactions without theenzyme were performed in parallel The hydrogen peroxideand pyrogallol solutions were prepared fresh on the dayof the measurements and pyrogallol was kept in the dark

The absorbance increase at 420 nm was monitored every 3seconds for 2 minutes using a NanoDrop 2000c (ThermoFisher Scientific) with cuvette option A molar absorptivityvalue of 2640Mminus1 cmminus1 for the product purpurogallin wasused to calculate the enzyme activity [24] One InternationalUnit (IU) corresponds to the formation of one micromolepurpurogallin in oneminute at room temperature andpH42

For enzyme kinetic studies the concentrations of thesubstrates pyrogallol and H

2O2were varied The substrate

inhibition model described by (1) was used to fit the depen-dence of the initial reaction velocity V

0 on the substrate

concentration [119878] The fit parameters are the Michaelis-Menten constant 119870

119872 the substrate inhibition constant

119870

119868 and the maximum initial reaction velocity Vmax The

parameter Vmax depends on the turnover number 119896cat and theenzyme concentration [119864] The fit was carried out with theprogram EnzFitter from Biosoft

V0=

Vmax sdot [119878]

119870

119872+ [119878] + [119878]

2119870

119868

with Vmax = 119896cat sdot [119864] (1)

To detect how much CPO leaked from sol-gel beadsactivity assays were conducted with the storage buffers thatwere removed and replaced during the maturation phase ofthe sol-gel beads These assays were conducted in a similarmanner as described above with the following exception theassay was started with the addition of H

2O2as no extra CPO

was addedActivity assays with CPO sol-gel beads were performed

with longer time intervals of 20 or 30 seconds for a total dura-tion of 5 minutes A Milton Roy Spectronic 20D instrumentwas used to monitor the absorbance at 420 nm Agitation ofthe reaction solution and the CPO sol-gel beads via inversionor pipetting was crucial to ensure good mixing in betweenabsorbance recordings Control reactions with sol-gel beadsthat did not contain CPO confirmed that the sol-gel beadsthemselves do not catalyze the peroxidation of pyrogallol

24 Spectroscopic Studies (CD andUVVIS) CD spectra wererecorded with an AVIV Model 215 circular dichroism spec-trometer at 25∘CThe temperature was controlled with a ther-mostat We employed the sample preparation method devel-oped by Eggers andValentine [27] A buffer of low concentra-tion (2mM sodium phosphate pH 70) was chosen to mini-mize total sample absorbance in the UV region To achieveefficient gelation despite the low concentration of the buffera pH value of 70 was selected A sol solution was preparedby sonicating 737mL TMOS 092mL dH

2O and 088mL

001M hydrochloric acid for 20 minutes in an ice-water bathA portion of 175mL of this sol-gel solution was gently mixedwith 2625mL CPO solution containing 12120583MCPO in 2mMsodium phosphate pH 70 and cast into a plastic cassetteof 1mm thickness The cassettes were purchased from LifeTechnologies For the first four days the storage buffer ontop of the sol-gel sheet was exchanged daily For completematuration inside the more enclosed cassettes the CPO sol-gel sheets were stored for onemonth at 4∘C Prior to the actualCD measurements the cassette housing the CPO sol-gel wasopened A small piece was cut out with a razor blade and

Journal of Nanotechnology 3

transferred into a CD quartz cuvette of 2mm thickness Theremaining space was filled with 2mM sodium phosphatebuffer pH 70

UVVIS spectra were recorded using a UV mini 1240Shimadzu single beam spectrophotometer The CPO sol-gelmaterial was prepared as described in Section 22 Howeverinstead of casting beads the solution was poured into 1mmthick plastic cassettes and a higher CPO concentration yield-ing 16mgmL CPO in the final sol-gel sheet was used TheCPO sol-gel sheet was stored in the cassette for one month at4∘C A rectangular piece was cut out and placed onto one wallof a plastic cuvette of 10mm thicknessThe CPO sol-gel sheetremained attached to the plastic wall after the cuvette wasfilled with 50mMpotassiumphosphate buffer pH 60 To testthe effect of changing the pH value of buffers surrounding theCPO sol-gel sheets the molarity of the potassium phosphatebuffers at various pH values was increased to 05M

25 Influence of Methanol on CPOrsquos Catalytic Activity andSpectroscopic Properties CPO dissolved at concentrations ofup to 9 120583M in 01M citrate-02M phosphate buffer pH 42was incubated with 11 vv methanol After specific timeintervals sample aliquots were removed to record UVVISor CD spectra or to measure the enzymatic activity viathe pyrogallol peroxidation assay Reference samples withoutmethanol were prepared in parallel To test whether anyeffects caused by methanol exposure might be reversiblemethanol treated samples were dialyzed for three hours ina Pierce Slide-A-Lyzer (10000 MWCO) against 1 L of 001Mcitrate-002M phosphate buffer pH 42 at room temperature

26 Porosimetry Measurements All porosimetry measure-ments were conducted with an ASAP 2020 physisorptionanalyzer from Micromeritics using nitrogen gas To preparethe samples the solvent exchange procedure described byHarreld and coworkers was used [28]The sol-gel beads (typ-ically 8) were placed in sim4mL acetone overnightThe acetonewas exchanged each day for three days For the final two daysn-pentane was used instead of acetone N-pentane and ace-tone are miscible with each other but n-pentane evaporatesmore readily than acetone This technique yields dry beadswhile minimizing the risk of pore collapse The beads wereplaced in the sample degas station of the physisorption ana-lyzer for approximately 8 hours at 37∘C until constant pres-sure was reached Nitrogen adsorption and desorption curveswere recorded and analyzed with the Brunauer-Emmett-Teller (BET) algorithm Proper performance of the instru-ment was confirmed with a silica-alumina standard providedby Micromeritics

3 Results and Discussion

31 Peroxidation of Pyrogallol Catalyzed by Free and Sol-GelEntrapped CPO The catalytic activity of CPO was assessedby monitoring the peroxidation reaction of pyrogallol Todetermine optimal reaction conditions we varied the pHvalue of the assay buffer and the concentrations of pyrogalloland hydrogen peroxide Our observations for free CPO agreewell with previous studies [24 29] Acidic conditions with

pH values in the range of 35 to 45 and pyrogallol concentra-tions at approximately 35mM resulted in optimum catalyticperformance Very high pyrogallol concentrations gave riseto substrate inactivation but the enzyme was even moresensitive towards inactivation by the cosubstrate H

2O2 The

H2O2concentration should not exceed 10mM The enzyme

kinetic data obtained for sol-gel entrapped CPO displayedsimilar features albeit at much lower specific activity Notablysol-gel entrapment did not abolish the detrimental effect ofH2O2 All data displayed in Figure 1 was thus fitted with a

substrate inhibition model (see (1))The enzyme kinetic parameters for the peroxidation of

pyrogallol catalyzed by free and sol-gel entrapped CPO aresummarized in Table 1 Significantly smaller Vmax and thus119896cat values were obtained for sol-gel entrapped CPO com-pared to free CPOThe parameters119870

119898and119870

119868did not change

significantly in response to sol-gel entrapment for either sub-strate The poor quality of the fit shown in Figure 1(b) for thedata on sol-gel entrapped CPOmight be caused by large datapoint variations and the limitations of the substrate inhibitionmodel Manoj et al [29] demonstrated that the substrateinhibition model used here (see (1)) can yield acceptable fitsfor individual substrate variations but global fit parameterswere shown to be unattainable [29] For example the119870

119872and

119870

119868values of one substrate depended on the concentration of

the other cosubstrate All kinetic parameters determined hereare thus only apparent values and the parameters for sol-gelentrapped CPO are further influenced by additional factorsFor example 119896cat was calculated based on the assumption thatall entrapped CPO molecules are able to participate in thecatalytic process This will not be the case for CPOmoleculesthat are entrapped in closed silica pores or are otherwisepermanently shielded and for CPO molecules with compro-mised functionality due to enzyme damage The decrease in119896cat (and also Vmax)might thus signify an apparent decrease inaccessible andor active CPO molecules upon sol-gel entrap-ment Notably hindered product release can also cause adecrease in 119896catThe 119896cat value is either determined by the rateof catalytic turnover or the rate of product release dependingon which of the two steps is slower and thus rate-limitingThe silica nanostructure does not seem to impose anysignificant diffusional constraints on themolecules pyrogalloland possiblyH

2O2 If these substratemolecules were encoun-

tering substantial diffusional barriers an increase in theirapparent119870

119872and119870

119868values would be expected

32 Entrapment and Catalytic Performance of CPO in Sol-GelBeads To further assess the entrapment and catalytic perfor-mance of the immobilized enzyme different amounts of CPOwere immobilized inside sol-gel beads corresponding to finalconcentrations of 40120583gmL 8 120583gmL and 4 120583gmL per totalsol-gel solution The storage buffer was exchanged to removemethanol originating from the hydrolysis and condensationsteps of the sol-gel formation CPO activity was measured inthese exchanged buffer samples and compared to a referencesample with free CPO in solution to quantify enzyme leakage(see Figure 2) Higher enzyme loading resulted in more

4 Journal of Nanotechnology

0

500

1000

1500

0 20 40 60 80 100 120 140

Activ

ity o

f fre

e CPO

(IU

mg)

Pyrogallol (mM)

0

50

100

150

Activ

ity o

f sol

-gel

CPO

(IU

mg)

(a)

0 20 40 60 80 100H2O2 (mM)

2500

2000

Activ

ity o

f fre

e CPO

(IU

mg)

1500

1000

500

0 0

50

100

150

200

250

Activ

ity o

f sol

-gel

CPO

(IU

mg)

(b)

Figure 1 Peroxidation of pyrogallol catalyzed by free CPO (open squares 0575 120583g CPO in 3mL total assay volume) and sol-gel entrappedCPO (grey circles 0575120583g CPO entrapped in three sol-gel beads placed in 3mL total assay volume) The CPO sol-gel beads were preparedfrom TMOS with a pH 60 casting buffer and matured for one week with a buffer exchange on every other day The activity assays wereperformed in 01M citric acid-02M dibasic sodium phosphate buffer pH 42 with constant concentrations of either 27mM H

2O2(a) or

35mM pyrogallol (b) All data were measured in triplicate and are displayed as mean values plusmn one standard deviation The parameters of thecurve fits are summarized in Table 1

Table 1 Enzyme kinetic parameters for the CPO catalyzed peroxidation of pyrogallollowast

119870

119872(mM) 119870

119868(mM) Vmax (IUmg) 119896cat (1sec)

PyrogallolFree CPO (1198772 = 0983) 11 plusmn 10 160 plusmn 20 2000 plusmn 80 1400 plusmn 60Sol-gel entrapped CPO (1198772 = 0847) 93 plusmn 26 190 plusmn 74 100 plusmn 12 70 plusmn 84

H2O2

Free CPO (1198772 = 0921) 17 plusmn 62 64 plusmn 22 9100 plusmn 2500 6370 plusmn 1750Sol-gel entrapped CPO (1198772 = 0657) 27 plusmn 39 61 plusmn 86 680 plusmn 830 480 plusmn 580

lowastEnzyme kinetic parameters were obtained by fitting the data shown in Figure 1(a) (pyrogallol) and Figure 1(b) (H2O2) to a substrate inhibition model (see(1)) All parameters are presented as value of the fit plusmn one standard error of the fit

0

1

2

3

4

5

6

7

Leak

age (

)

Time1d1h 2d 3d 4d 6d

Figure 2Three sets of CPO sol-gel beads with CPO concentrationsof 40 120583gmL (dark grey bars) 8 120583gmL (grey bars) and 4 120583gmL(light grey bars) were aged for one week The storage buffer wasreplaced in the time intervals indicated on the graph and tested forCPO leakage Each set was replicated three times and containedthree beads per sample tube The specific activity of the referencesample with free CPO in solution was 1436plusmn39 IUmgThe columnheights represent the mean value and the error bars represent plusmn onestandard deviation

enzyme leakage As the sol-gel material matured and moreconnections formed within the silica mesh enzyme leakagedeclined

Table 2 contains the results of the activity measurementswith the CPO sol-gel beads and summarizes the cumulativeleakage over the first six days The activity measurementswere performed with CPO sol-gel beads that were seven daysold Each assay comprised 01M citric acid-02M dibasicsodium phosphate buffer pH 42 27mM H

2O2 and 35mM

pyrogallol The CPO sol-gel beads with the highest CPOloading of 40 120583g CPO per mL sol-gel yielded the highestabsolute activity values of 249 plusmn 33mIU A comparisonamong specific activity values reported per mg of initiallyloaded CPO however clearly showed that the CPO sol-gelbeads with a lower enzyme content of 8 or 4120583gmL perfor-med better At a higher loading of CPOmore CPOmoleculesmight be obstructed by either other enzyme molecules orthe silica nanostructure Also some of these CPO moleculesmight be entrapped in closed pores

Another set of three tubes each containing three beadsloaded with 4120583gmL CPO was aged for one week and testedfor reusability (see Figure 3) The CPO sol-gel beads can bereused up to three times in a convenient manner by replacingthe liquid phase composed of buffer and product moleculeswith new buffer and substrate However a further decline incatalytic performance was apparent We also noticed that allCPO sol-gel beads adopted the yellow-orange color of theproduct purpurogallin after the first use Gentle washing with

Journal of Nanotechnology 5

Table 2 Catalytic performance of sol-gel beads loaded with different CPO amounts

CPO loading of sol-gel beads (120583gmL) 40 8 4Activity (mIU)lowast 249 plusmn 33 138 plusmn 18 76 plusmn 4Specific activity (IUmg)lowast 41 plusmn 6 115 plusmn 15 127 plusmn 6Relative activity compared to free CPO () 29 80 880Cumulative leakage for six days ()dagger 15 12 7lowastAbsolute activity values in mIU (times10minus3 International Units) and specific activity values per mg initially loaded CPO are presented as mean values plusmn onestandard deviation All sample sets were prepared in triplicate The relative activity was based on a reference assay with free CPO in solution The referencevalue was 1436 plusmn 39 IUmg daggerThe cumulative leakage over six days of maturation corresponds to the summation of the leakage data shown in Figure 2

0

50

100

150

200

First use

Activ

ity (I

Um

g )

Time1d 3d 4d 5d 11d 12d

Figure 3 Reusability test for CPO sol-gel beads loaded with4 120583gmL CPOThree sets each with three beads per tube were usedin this test The column heights represent the mean specific activityvalue and the error bars representplusmn one standard deviationThe firstmeasurement was taken after the beads were aged for one week withdaily buffer exchanges The number of days that passed before thenext reuse is listed on the 119909-axis

the storage buffer diminished the coloration only slightlyThus clogging of the sol-gel nanostructure with productmolecules was one factor that hampered the reusability of theCPO sol-gel beads Furthermore the peroxidation reactionscatalyzed by CPO are prone to substrate inhibition (seeFigure 1) Trapped pyrogallol and H

2O2molecules could

therefore interfere with effective reuse Jung and Hartmanndemonstrated that in situ generation of hydrogen peroxidevia coimmobilization of glucose oxidase can improve thereusability of cross-linked CPO molecules entrapped inmesoporous molecular sieves [30]

The specific activity of three independently prepared setsof sol-gel beads all containing sim4 120583gmL CPO was different70 plusmn 15 IUmg (Figure 1) 127 plusmn 6 IUmg (Table 2) and171 plusmn 14 IUmg (Figure 3) The CPO sol-gel materials usedfor generating the data presented in Table 2 and Figure 3were prepared from the same CPO vial with a free CPOreference value of 1436 plusmn 39 IUmg The reference value forthe first CPO vial was only 1280 plusmn 80 IUmg However manyother experimental parameters such as the exact sol-gelcomposition the quality of all starting materials humiditytemperature and duration of sol-gel drying phase can alsoinfluence the properties of the final sol-gel material There-fore all CPO sol-gel beads that are compared within onetable or graph were prepared on the same day with the samereagent batches in parallel

33Modification of Sol-Gel Procedure with respect toMethanolRelease Next we tested whether minor modifications in thesol-gel procedure that influence the retention of methanolreleased from the sol-gel precursor TMOS would result inany significant changes On the same day three differentsets of CPO sol-gel beads all containing 4 120583gmL CPO wereprepared in triplicateThe first set was prepared with a proce-dure that facilitates methanol release by using an open vesselwhile sonicating the sol solution and performing daily bufferexchanges during the first week of sol-gel maturation Thesecond and third sets were prepared using a closed sonicationvessel and no buffer exchange was performed for the thirdset Despite these modifications all three sets prepared onthe same day yielded virtually identical specific activity valuesof 167 plusmn 18 IUmg 164 plusmn 10 IUmg and 178 plusmn 21 IUmgrespectively A more vigorous procedure presented by Ferreret al [31] involves rotavaporization of the sol solution prior toaddition of the buffered enzyme solution In our laboratorythis method was not successful as sol solidification started toset in too rapidly to achieve consistent gelation

Overall the catalytic performance of the best set of sol-gel beads was only 125plusmn15 in comparison to the referenceassay with free CPO in solution Possible reasons for thedecline in catalytic performance upon entrapment include (1)loss of enzyme due to leakage from the sol-gel matrix (2)damage to the enzyme caused by the entrapment procedureor (3) hindered substrate or product diffusion within the sol-gel nanostructure As mentioned above enzyme leakage didoccur but it can only account for a small loss of approximately10 In the experiments described below we tested for thetwo remaining possible reasons for the decline in catalyticperformance of the sol-gel entrappedCPOand also examinedthe effect of methanol on CPO

34 CD and UVVIS Spectroscopy with Sol-Gel EntrappedCPO To monitor possible enzyme damage we exploited thefact that sol-gels prepared from TMOS are transparent Cir-cular dichroism and visible absorbance spectra of free CPOin solution and CPO in sol-gel entrapped form are shown inFigures 4 and 5 respectively The spectra of free CPO andsol-gel entrapped CPO are virtually identical to each otherMinor changes in intensity of the spectroscopic signals aremost likely due to scattering effects from the sol-gel surfaceor the shrinkage of the sol-gel material during thematurationprocess Shrinkage slightly raises the concentration of thesample but also decreases the spectroscopic path length TheCD spectra are typical for a protein with high alpha helical

6 Journal of Nanotechnology

minus20

minus15

minus10

minus5

0

5

200 210 220 230 240 250 260Wavelength (nm)

CD si

gnal

(mde

g)

Figure 4 CD spectra of sol-gel entrapped CPO (black squares)and free CPO (white squares) The CPO concentration was 7120583Min the silica sol-gel sheet of sim1mm thickness The solution samplecontaining 7 120583M CPO in 2mM sodium phosphate buffer pH 70was measured in a cuvette of 1mm path length

0

01

02

03

04

05

300 400 500 600 700 800 900

Abso

rban

ce

Wavelength (nm)

Figure 5 Absorbance spectra of sol-gel entrapped CPO (solid line)and free CPO (dashed line) The silica sol-gel sheet was sim1mmthick and contained 16mgmL CPO The sheet was placed onthe wall of a 10mm wide plastic cuvette and immersed in 50mMpotassium phosphate buffer pH 60 The solution reference samplewas prepared from the same CPO stock via dilution to 016mgmL(4 120583M) with 50mM potassium phosphate buffer pH 60 in a 10mmthick plastic cuvette

content This finding agrees with the X-ray protein structureof CPO [32] and previously determined CD data [15 33]

The visible absorbance spectra shown in Figure 5 aretypical for the active form of CPO with a five-coordinateiron center in the heme chromophore [34] As the pH valueincreases above pH 70 CPO is inactivated the iron centerin the heme group becomes six-coordinate and the Soretband shifts to a longer wavelength [34] This spectroscopictransition can also be observed in CPO sol-gel sheets despitethe entrapment of the enzyme inside the silica matrixFigure 6 displays the absorption spectra of CPO sol-gelsheets immersed in 05M phosphate buffers at pH values of60 80 and 100 In comparison to solution spectra morealkaline conditions are necessary to achieve the Soret peakcharacteristic for sixfold coordinated heme iron centers It isconceivable that the silanol groups in the sol-gel impose anadditional buffering effect According toDunn and Zink [35]

0005

01015

02025

03035

04045

300 320 340 360 380 400 420 440 460 480 500

Abso

rban

ce

Wavelength (nm)

Figure 6 Absorbance spectra of sol-gel entrapped CPO immersedin 05M potassium phosphate buffer with pH values of 60 (solidline) 80 (dashed line) and 100 (dotted line)The silica sol-gel sheetsof 1mm thickness loaded with 16mgmL CPO were placed on thewall of plastic cuvette with a path length of 10mm

the pH can be up to one pH unit lower inside a sol-gel porethan in the surrounding aqueous buffer

The low apparent 119896cat values for sol-gel entrapped CPO(see Table 1) indicate that a significant number of entrappedCPO molecules were unable to catalyze the peroxidation ofpyrogallol in an effective manner CD and visible absorptionspectra however did not indicate any enzyme damage Itshould be noted that both spectroscopic methods can onlyaddress specific features of the enzymeThese features are theoverall secondary structure of the protein the electronic con-figuration of the active site chromophore and the ability torearrange the coordination sphere of the heme iron center inresponse to an external change in pH value

35 Influence of Methanol on the Spectroscopic Features andActivity of Free CPO Methanol is released from the sol-gelprecursor TMOS If we assume complete hydrolysis of TMOSand no evaporation of methanol the protein is exposed toa methanol concentration of 11 vv as the buffered enzymesolution and the sol are mixed and pipetted onto the parafilmfor gelation and drying The transfer into storage bufferand the subsequent exchange of buffer drastically lower themethanol content during the maturation phase of the sol-gelAfter the first buffer exchange the methanol concentration isonly 003 vv

To study the influence of methanol on the spectroscopicfeatures of CPO the enzyme was incubated with 11 vvmethanol andUVVIS andCD spectrawere recordedWedidnot detect a change in the spectroscopic features of the CDspectra after several hours of incubation (data not shown)but methanol exposure did have a small and immediate effecton UVVIS absorption spectra (see Figure 7)The absorptionmaximum of the Soret band shifted from 396 nm to 400 nmNotably this shift was reversible via dialysis The methanolremoval caused a 13-fold increase in sample volume Weadjusted the corresponding spectroscopic trace in Figure 7for this dilution effect

Journal of Nanotechnology 7

0

02

04

06

08

1

370 390 410 430 450

Abso

rban

ce

Wavelength (nm)

Figure 7 Absorption spectra of 9120583M CPO in 01M citrate-02Mphosphate buffer pH 42 (solid line) in 11 vv methanol and01M citrate-02M phosphate buffer pH 42 (dashed line) dialyzedsample (grey solid line) and dialyzed sample adjusted for 13-foldvolume increase (grey dotted line)

In agreement with previous studies [36] we observed adrastic decline in CPOrsquos catalytic performance after incuba-tion of CPO with organic solvents Compared to an aqueousreference sample without methanol only 57 residual activ-ity was detected after incubation with 11 vv methanol for2 hours After one day the residual activity still remainedat 57 We further discovered that the detrimental effect ofmethanol was reversible Up to 96 of the samplersquos initialactivity was recovered after dialysis Sample dilution alsoresulted in the enzymersquos recovery Decreasing the methanolcontent to 5 or 1 vv via sample dilution resulted in 90and 100 relative activity in comparison to identically dilutedsamples from the same CPO batch that were not exposed tomethanol Our finding that damage caused by methanol wasreversible in solution has implications for other studies on theuse of CPO in organic solvents or in biphasic solvent systems[36 37] Several CPO substrates which can be convertedinto products of industrial interest have high solubility innonpolar organic solvents [8]

Also if any initial damage caused by methanol exposurewas also reversible for sol-gel entrapped CPO immediatereduction in methanol content via evaporation of methanolfrom the sol solution or daily buffer exchanges during thegel maturation phase would not critically alter the finalperformance of the CPO sol-gel beads This might explainwhy the three different but parallel preparations outlinedin Section 33 resulted in virtually identical performance forthe three different CPO sol-gel bead sets On the otherhand manifestation of unrecoverable enzyme damage wouldexplain the low apparent 119896cat values determined for sol-gelentrapped CPO (see Table 1) It is conceivable that recoveryfrom damage caused by methanol exposure is less effectivefor CPO molecules entrapped within the silica sol-gel hostcompared to free CPO in solution

36 HinderedMaterial Transport in CPO Sol-Gel Beads Aftertheir first use all CPO sol-gel beads adopted a persistentyellow coloration indicating the entrapment of the productpurpurogallin Attractive intermolecular forces and physical

constraints both can delay the release of product moleculesfrom the silica nanostructure If product release becomesrate-limiting the apparent 119896cat value decreases as observedin the enzyme kinetic analysis Hindered substrate diffusionhowever was not supported by enzyme kinetic experimentsas the 119870

119872and 119870

119868values for the main substrate pyrogallol

were virtually identical for sol-gel entrapped and free CPO(see Table 1)We cannot explain why pyrogallol and purpuro-gallin would show different material transport propertiesinside the silica nanostructure Both molecules have similarfunctional groups and purpurogallin (MW 220 gmol) isonly somewhat larger than pyrogallol (MW 116 gmol) Analternative explanation would involve side-reactions formingalternate charged products or the trapping of reactive coloredintermediates

The alternative peroxidation substrate 221015840-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is particularlylarge (MW 515 gmol) and the product of the peroxidationreaction carries a positive charge [24] Deprotonated silanolgroups on the sol-gel surface can provide negative coun-tercharges The catalytic performance of CPO sol-gel beadsdropped from 126 plusmn 3 with the substrate pyrogallol to 9 plusmn04 with the substrate ABTS relative to the correspondingassay with free CPO in solution The CPO sol-gel beadsadopted the green color of the ABTS peroxidation productKadnikova and coworkers observed the formation of severalside products for the peroxidation reaction of ABTS byhorseradish peroxidase in a sol-gel matrix [38] The perox-idation reaction with ABTS and possibly other substratesincluding pyrogallol might therefore be more complex thanin solution

We further observed that preequilibration of CPO sol-gelbeadswith pyrogallol considerably improved catalytic perfor-mance For example preequilibration with 72mM pyrogallolfor 3 hours in comparison to using 35mM pyrogallol withoutpreequilibration increased the relative activity from 123 to196The strategy to preequilibrate enzyme sol-gel materialswith an excess of substrate before adding a cosubstrate wasalready successfully applied by Smith and coworkers [23] intheir study on sol-gel encapsulated horseradish peroxidaseBased on the data presented in Figure 1(a) however wewouldnot expect an increase in catalytic performance for sol-gelentrapped CPO as the pyrogallol concentration is raised from35 to 72mM In fact substrate inhibition should slightly lowerthe activity of CPOAdsorption of pyrogallolmolecules to thesol-gel surface or physical entrapment could further enhancethe local effective concentration of pyrogallol around theenzyme One key difference between the experiment leadingto Figure 1(a) and the preequilibration experiment is thetiming of adding the cosubstrate H

2O2 Manoj et al [29]

argue that substrate inhibition of CPO is not simply causedby blocking the active site of the enzymewith excess substratemolecules Instead they propose more complex substrateinhibition mechanisms that involve secondary conversionof an already formed product or competition by transientintermediates leading to alternate products Both substrateinhibition scenarios require the immediate presence of thecosubstrate H

2O2

8 Journal of Nanotechnology

Table 3 Properties of CPO sol-gel beads prepared with different casting buffers

pH of casting buffer 45 55 65BET surface area (m2g) 710 plusmn 40lowast 740 plusmn 60 470 plusmn 40Total pore volume (cm3g) 058 plusmn 002 060 plusmn 006 032 plusmn 004Average pore diameter (nm) 33 plusmn 03 32 plusmn 01 27 plusmn 01Activity (mIU) 251 plusmn 2 242 plusmn 29 161 plusmn 13Specific activity (IUmg) 157 plusmn 1 151 plusmn 18 100 plusmn 8Activity compared to free CPO () 18 17 11Cumulative leakage ()dagger 13 14 9lowastAll data are presented as mean values plusmn one standard deviation of triplicate data sets The relative activity was based on a reference assay with free CPO insolution yielding 887 plusmn 31 IUmg daggerThe cumulative leakage over ten days of maturation corresponds to the summation of the leakage data shown in Figure 8

37 Modification of Sol-Gel Procedure Using MTMS Tomodify the surface of the sol-gel material we incorporatedMTMS at molar ratios of 5 20 and 40 in the sol solutionThe addition of MTMS will introduce nonpolar methylgroups rendering the surface of the silica nanostructuremorehydrophobic [39] The casting buffer had a pH value of 60and the total CPO loading was 4 120583gmL The addition ofMTMS resulted in longer gelation times for example up to240 minutes for a molar ratio of 40 MTMS Unfortunatelythe beads prepared with MTMS were more brittle and fragilethan any of the other CPO sol-gel beads prepared in thisstudy The brittleness of the beads rendered their handlingmore challenging Regardless of the amount of incorporatedMTMS the activity was approximately 14 plusmn 1 comparedto a solution reference assay The cumulative leakage oftenexceeded 20We cannot rule out that the physical instabilityof the beads during and after a buffer exchange might havecontributed to higher apparent leakage and higher apparentactivity values In contrast to other enzymes notably lipasewhich showed interfacial activation and performed betterinsidemore hydrophobic nanostructures [40] the incorpora-tion of MTMS into the CPO sol-gel material did not improvecatalytic performance in a systematic manner

38 Modification of Sol-Gel Procedure Using More AcidicCasting Buffers The enzyme CPO is stable under acidicconditions [24] We exploited this CPO specific property andprepared CPO sol-gel beads using casting buffers with pHvalues of 45 55 and 65 All sample preparations were con-ducted in parallel with the same batches of CPO TMOS andbuffer reagents The gelation time increased with more acidiccasting buffers but the sol-gel beads remained easy to handleand transparentTheCPO loadingwas 4 120583gmL All CPO sol-gel preparations were divided into two portions One portionwas used for porosimetry studies and the other portion wasused for leakage and activity measurements (see Table 3)The properties of CPO sol-gel beads cast at pH 45 and 55are virtually identical but the CPO sol-gel beads cast at pH65 show significantly lower values in all categories Thisindicates a change in thematrix formation of the sol-gel as thecasting pHdrops to or below pH55 Overall the porosimetrydata is positively correlated with catalytic performance andunfortunately leakage All three porosimetric propertiesincluding larger average pore size BET surface area and pore

7

6

5

4

3

2

1

0

Leak

age (

)

1 2 3 4 5 6 7 8 9 10

Time (d)

Figure 8 The storage buffer of CPO sol-gel beads prepared withcasting buffers at pH 45 (dark grey bars) 55 (grey bars) and 65(light grey bars) was exchanged on a daily basis and monitored forCPO activity All samples were prepared in triplicate with eight CPOsol-gel beads per sample tube The bar height represents the meanvalue and the error bar plusmn one standard deviation

volume indicate reduced steric hindrance for material trans-port inside the sol-gel nanostructure As a consequencecatalytic performance increased Smaller average pore sizeson the other hand can aid in the retention of CPO

The dimensions of the protein CPO are 53 nm times 46 nmtimes 60 nm [19] The average pore diameters of approximately3 nm are only slightly smaller than the size of CPO Never-theless CPO remainedwell entrapped after completion of thesol-gel maturation phase Attractive electrostatic forces didnot most likely aid in the retention of CPO as the storagebuffer had a pH value of 42 which is close to the isoelectricpoint of CPO The isoelectric point of CPO from C fumagowas calculated to be approximately 40 [18 21] Isoelectricfocusing experiments on CPO from Pseudomonas pyrrociniayielded an isoelectric point of 41 [41] For all buffer condi-tions employed in our study the net charge on the surfaceof CPO is therefore either close to zero or negative

Our observation that more acidic casting buffers result ingreater porosimetry of sol-gels agrees well with several previ-ous studies [42 43] However not all enzymes will respondwell to the use of more acidic casting conditions Notablyenzymes have different pH profiles and some enzymesare inactive under acidic conditions Sol-gel entrappedcholinesterase for example showed better performance

Journal of Nanotechnology 9

in silica nanostructures prepared at pH values of 70 and 80and then 60 [44]

4 Conclusion

The enzyme CPO was successfully entrapped inside a silicananostructure prepared from the precursor TMOS with orwithout addition of the hydrophobic modifier MTMS SinceCPO is stabile in acidic buffers we further modified the sol-gel procedure by using casting buffers with pH values of 4555 60 and 65The catalytic performance of optimized CPOsol-gel beads approached 18 relative to free CPO in solutionas assessed via the pyrogallol peroxidation assay A combi-nation of factors such as enzyme leakage from the sol-gelhost insufficient recovery from inactivation caused by initialmethanol exposure hindered product release or alternatereaction pathways are most likely responsible for the declinein catalytic performance of CPOafter sol-gel entrapmentTheuse of more acidic casting buffers in the sol-gel procedureprovided themost leverage for optimization by yieldingmoreporous silica nanostructures Overall our findings are ofimportance for the optimization of other sol-gel materialsdevised for applications in biosensing or biocatalysis ordesigned for the controlled release of bioactive compounds

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Funding for this project was obtained from the ResearchCorporation for Science Advancement (Cottrell College Sci-ence Award to Monika Sommerhalter) and California StateUniversity East Bay (Faculty and Student Research Grantsto Monika Sommerhalter Selina Chan and Tuan Le and aSieber-Tombari award to Monika Sommerhalter) ProfessorDaryl Eggers San Jose State University kindly invited theauthors to perform the CD measurements in his laboratoryThe authors are also grateful to Professor AnnMcPartland forproviding detailed feedback on their paper

References

[1] B C Dave B Dunn J S Valentine and J I Zink ldquoNanocon-fined proteins and enzymes sol-gel-based biomolecular mate-rialsrdquoNanotechnology ACS Symposium Series vol 622 pp 351ndash365 1996

[2] I Gill and A Ballesteros ldquoBioencapsulation within syntheticpolymers (part 1) sol-gel encapsulated biologicalsrdquo Trends inBiotechnology vol 18 no 7 pp 282ndash296 2000

[3] D Avnir T Coradin O Lev and J Livage ldquoRecent bio-applications of sol-gel materialsrdquo Journal of Materials Chem-istry vol 16 no 11 pp 1013ndash1030 2006

[4] D R Morris and L P Hager ldquoChloroperoxidase I Isolationand properties of the crystalline glycoproteinrdquo The Journal ofBiological Chemistry vol 241 no 8 pp 1763ndash1768 1966

[5] V Yazbik and M Ansorge-Schumacher ldquoFast and efficientpurification of chloroperoxidase from C fumagordquo Process Bio-chemistry vol 45 no 2 pp 279ndash283 2010

[6] M Hofrichter and R Ullrich ldquoHeme-thiolate haloperoxidasesversatile biocatalysts with biotechnological and environmentalsignificancerdquo Applied Microbiology and Biotechnology vol 71no 3 pp 276ndash288 2006

[7] V M Dembitsky ldquoOxidation epoxidation and sulfoxidationreactions catalysed by haloperoxidasesrdquoTetrahedron vol 59 no26 pp 4701ndash4720 2003

[8] L Santhanam and J S Dordick ldquoChloroperoxidase catalyzedepoxidation of styrene in aqueous and non-aqueous mediardquoBiocatalysis and Biotransformation vol 20 no 4 pp 265ndash2742002

[9] M Ayala N R Robledo A Lopez-Munguia and R Vazquez-Duhalt ldquoSubstrate specificity and ionization potential inchloroperoxidase-catalyzed oxidation of diesel fuelrdquo Environ-mental Science and Technology vol 34 no 13 pp 2804ndash28092000

[10] R Vazquez-Duhalt M Ayala and F J Marquez-Rocha ldquoBio-catalytic chlorination of aromatic hydrocarbons by chloroper-oxidase of Caldariomyces fumagordquo Phytochemistry vol 58 no6 pp 929ndash933 2001

[11] E Terres M Montiel S Le Borgne and E Torres ldquoImmo-bilization of chloroperoxidase on mesoporous materials forthe oxidation of 46-dimethyldibenzothiophene a recalcitrantorganic sulfur compound present in petroleum fractionsrdquoBiotechnology Letters vol 30 no 1 pp 173ndash179 2008

[12] V Trevisan M Signoretto S Colonna V Pironti and GStrukul ldquoMicroencapsulated chloroperoxidase as a recyclablecatalyst for the enantioselective oxidation of sulfides withhydrogen peroxiderdquo Angewandte Chemie International Editionvol 43 no 31 pp 4097ndash4099 2004

[13] N Spreti R Germani A Incani and G Savelli ldquoStabiliza-tion of chloroperoxidase by polyethylene glycols in aqueousmedia kinetic studies and synthetic applicationsrdquoBiotechnologyProgress vol 20 no 1 pp 96ndash101 2004

[14] J-B Park and D S Clark ldquoNew reaction system for hydrocar-bon oxidation by chloroperoxidaserdquo Biotechnology and Bioengi-neering vol 94 no 1 pp 189ndash192 2006

[15] J-Z Liu and M Wang ldquoImprovement of activity and stabilityof chloroperoxidase by chemical modificationrdquo BMC Biotech-nology vol 7 no 1 article 23 2007

[16] L Zhi Y Jiang Y Wang M Hu S Li and Y Ma ldquoEffects ofadditives on the thermostability of chloroperoxidaserdquo Biotech-nology Progress vol 23 no 3 pp 729ndash733 2007

[17] T A Kadima andM A Pickard ldquoImmobilization of chloroper-oxidase on aminopropyl-glassrdquo Applied and EnvironmentalMicrobiology vol 56 no 11 pp 3473ndash3477 1990

[18] Y-J Han J T Watson G D Stucky and A Butler ldquoCatalyticactivity of mesoporous silicate-immobilized chloroperoxidaserdquoJournal ofMolecular Catalysis B Enzymatic vol 17 no 1 pp 1ndash82002

[19] J Aburto M Ayala I Bustos-Jaimes et al ldquoStability andcatalytic properties of chloroperoxidase immobilized on SBA-16mesoporousmaterialsrdquoMicroporous andMesoporousMaterialsvol 83 no 1ndash3 pp 193ndash200 2005

[20] M Hartmann and C Streb ldquoSelective oxidation of indole bychloroperoxidase immobilized on the mesoporous molecularsieve SBA-15rdquo Journal of Porous Materials vol 13 no 3-4 pp347ndash352 2006

10 Journal of Nanotechnology

[21] S Hudson J Cooney B K Hodnett and E Magner ldquoChlorop-eroxidase on periodic mesoporous organosilanes immobiliza-tion and reuserdquo Chemistry of Materials vol 19 no 8 pp 2049ndash2055 2007

[22] R A Sheldon ldquoEnzyme immobilization the quest for optimumperformancerdquo Advanced Synthesis amp Catalysis vol 349 no 8-9pp 1289ndash1307 2007

[23] K Smith N J Silvernail K R Rodgers T E Elgren M Castroand RM Parker ldquoSol-gel encapsulated horseradish peroxidasea catalytic material for peroxidationrdquo Journal of the AmericanChemical Society vol 124 no 16 pp 4247ndash4252 2002

[24] K M Manoj and L P Hager ldquoChloroperoxidase a janusenzymerdquo Biochemistry vol 47 no 9 pp 2997ndash3003 2008

[25] V M Samokyszyn and P R Ortiz de Montellano ldquoTopology ofthe chloroperoxidase active site regiospecificity of heme mod-ification by phenylhydrazine and sodium aziderdquo Biochemistryvol 30 no 50 pp 11646ndash11653 1991

[26] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[27] D K Eggers and J S Valentine ldquoMolecular confinementinfluences protein structure and enhances thermal proteinstabilityrdquo Protein Science vol 10 no 2 pp 250ndash261 2001

[28] J H Harreld T Ebina N Tsubo and G Stucky ldquoManipulationof pore size distributions in silica and ormosil gels dried underambient pressure conditionsrdquo Journal of Non-Crystalline Solidsvol 298 no 2-3 pp 241ndash251 2002

[29] K M Manoj A Baburaj B Ephraim et al ldquoExplaining theatypical reaction profiles of heme enzymes with a novel mech-anistic hypothesis and kinetic treatmentrdquo PLoS ONE vol 5 no5 Article ID e10601 2010

[30] D Jung and M Hartmann ldquoOxidation of indole with CPO andGOx immobilized on mesoporous molecular sievesrdquo CatalysisToday vol 157 no 1ndash4 pp 378ndash383 2010

[31] M L Ferrer F del Monte and D Levy ldquoA novel and simplealcohol-free sol-gel route for encapsulation of labile proteinsrdquoChemistry of Materials vol 14 no 9 pp 3619ndash3621 2002

[32] M Sundaramoorthy J Terner and T L Poulos ldquoThe crystalstructure of chloroperoxidase a heme peroxidase-cytochromeP450 functional hybridrdquo Structure vol 3 no 12 pp 1367ndash13771995

[33] X W Yi A Conesa P J Punt and L P Hager ldquoExamining therole of glutamic acid 183 in chloroperoxidase catalysisrdquo Journalof Biological Chemistry vol 278 no 16 pp 13855ndash13859 2003

[34] S R Blanke S AMartinis S G Sligar L P Hager J J Rux andJ H Dawson ldquoProbing the heme iron coordination structureof alkaline chloroperoxidaserdquo Biochemistry vol 35 no 46 pp14537ndash14543 1996

[35] B Dunn and J I Zink ldquoProbes of pore environment andmolecule-matrix interactions in sol-gel materialsrdquo Chemistry ofMaterials vol 9 no 11 pp 2280ndash2291 1997

[36] W A Loughlin and D B Hawkes ldquoEffect of organic solvents ona chloroperoxidase biotransformationrdquo Bioresource Technologyvol 71 no 2 pp 167ndash172 2000

[37] E Kiljunen and L T Kanerva ldquoChloroperoxidase-catalysedoxidation of alcohols to aldehydesrdquo Journal of Molecular Catal-ysis B Enzymatic vol 9 no 4ndash6 pp 163ndash172 2000

[38] E N Kadnikova and N M Kostic ldquoOxidation of ABTS byhydrogen peroxide catalyzed by horseradish peroxidase encap-sulated into sol-gel glass Effects of glass matrix on reactivityrdquo

Journal of Molecular Catalysis B Enzymatic vol 18 no 1ndash3 pp39ndash48 2002

[39] A Venkateswara Rao and D Haranath ldquoEffect of methyltri-methoxysilane as a synthesis component on the hydrophobicityand some physical properties of silica aerogelsrdquo Microporousand Mesoporous Materials vol 30 no 2-3 pp 267ndash273 1999

[40] M T Reetz A Zonta and J Simpelkamp ldquoEfficient immo-bilization of lipases by entrapment in hydrophobic sol-gelmaterialsrdquo Biotechnology and Bioengineering vol 49 no 5 pp527ndash534 1996

[41] W Wiesner K-H van Pee and F Lingens ldquoPurification andcharacterization of a novel bacterial non-heme chloroperox-idase from Pseudomonas pyrrociniardquo Journal of BiologicalChemistry vol 263 no 27 pp 13725ndash13732 1988

[42] Y Xi Z Liangying and W Sasa ldquoPore size and pore-sizedistribution control of porous silicardquo Sensors and Actuators BChemical vol 25 no 1-3 pp 347ndash352 1995

[43] C Lin and J A Ritter ldquoEffect of synthesis pH on the structureof carbon xerogelsrdquo Carbon vol 35 no 9 pp 1271ndash1278 1997

[44] M Altstein G Segev N Aharonson O Ben-Aziz A Tur-niansky and D Avnir ldquoSol-gel entrapped cholinesterases amicrotiter plate method for monitoring anti-cholinesterasecompoundsrdquo Journal of Agricultural and Food Chemistry vol46 no 8 pp 3318ndash3324 1998

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials

Page 2: Silica Sol-Gel Entrapment of the Enzyme Chloroperoxidase

2 Journal of Nanotechnology

that is the peroxidation of pyrogallol The effect of methanolonCPOwas also investigated sincemethanol is released fromthe precursor TMOS during the hydrolysis and condensationreactions of the sol-gel process In our attempts to optimizethe catalytic performance of sol-gel immobilized CPO weexploited the stability of CPO in acidic solutions [24] anddeviated from the pH range of 60ndash70 which is typicallyemployed in protein sol-gel immobilization procedures Theuse of more acidic solutions in the sol-gel process might alsobe beneficial for various sol-gel applications based on otherproteins that either withstand or thrive in acidic conditions

2 Materials and Methods

21 Materials CPO from C fumago was purchased fromSigma-Aldrich The turbid suspensions were centrifuged at15000 rpm for 5 minutes in an Eppendorf microcentrifugeThe concentrations of the CPO supernatants were deter-mined via the intensity of the Soret band [25] and confirmedwith protein assays [26] The CPO samples used in this studyhad concentrations in the range of 9 to 15mgmL All otherreagentswere purchased fromSigma-Aldrich or Fisher Scien-tific Aqueous solutions were prepared with deionized waterobtained from a Millipore Milli-Q device

22 Sol-Gel Entrapment A sol solution was prepared bymixing 50120583L deionized water 47 120583L 0010M hydrochloricacid and 235 120583L TMOS in a microcentrifuge tube If MTMSwas included TMOS and MTMS were premixed The molarratio ofMTMSwas either 5 20 or 40of the totalMTMSandTMOS precursor mixtureThe sol solution was sonicated in aBranson 1510 sonicator bath filled with ice water for 30 min-utes and either used immediately thereafter or stored for upto a week at 4∘C In amicrocentrifuge tube 140 120583L sol 480 120583Lcasting buffer and 500120583L diluted CPO solution were mixedThe casting buffers were prepared by mixing 01M citric acidand 02M dibasic sodium phosphate to reach pH values of45 55 60 or 65 CPO was diluted in 20mM potassiumphosphate-potassium hydroxide buffer pH 60 Small beadsof 50120583L volume were prepared by pipetting the mixtureonto parafilm sheets As the pH value of the casting bufferdecreased the gelation time increased from approximately 15minutes (pH 65) to 240min (pH 45)The CPO sol-gel beadswere transferred into glass tubes in sets of three to eight beadsand covered with a storage buffer composed of 21mL deion-izedwater and 032mL 01M citric acid-02Mdibasic sodiumphosphate buffer pH 42 This storage buffer was typicallyexchangedwith a newportion of storage buffer after one hourand thereafter each day for seven or ten days The storagetemperature was 4∘C

23 Activity Assays Activity assays were typically conductedin 01M citric acid-02M dibasic sodium phosphate bufferpH 42 with 27 or 86mM H

2O2 35mM pyrogallol and

02 120583gmL CPO CPO was added last to start the reactionThe assay volume was 3mL Control reactions without theenzyme were performed in parallel The hydrogen peroxideand pyrogallol solutions were prepared fresh on the dayof the measurements and pyrogallol was kept in the dark

The absorbance increase at 420 nm was monitored every 3seconds for 2 minutes using a NanoDrop 2000c (ThermoFisher Scientific) with cuvette option A molar absorptivityvalue of 2640Mminus1 cmminus1 for the product purpurogallin wasused to calculate the enzyme activity [24] One InternationalUnit (IU) corresponds to the formation of one micromolepurpurogallin in oneminute at room temperature andpH42

For enzyme kinetic studies the concentrations of thesubstrates pyrogallol and H

2O2were varied The substrate

inhibition model described by (1) was used to fit the depen-dence of the initial reaction velocity V

0 on the substrate

concentration [119878] The fit parameters are the Michaelis-Menten constant 119870

119872 the substrate inhibition constant

119870

119868 and the maximum initial reaction velocity Vmax The

parameter Vmax depends on the turnover number 119896cat and theenzyme concentration [119864] The fit was carried out with theprogram EnzFitter from Biosoft

V0=

Vmax sdot [119878]

119870

119872+ [119878] + [119878]

2119870

119868

with Vmax = 119896cat sdot [119864] (1)

To detect how much CPO leaked from sol-gel beadsactivity assays were conducted with the storage buffers thatwere removed and replaced during the maturation phase ofthe sol-gel beads These assays were conducted in a similarmanner as described above with the following exception theassay was started with the addition of H

2O2as no extra CPO

was addedActivity assays with CPO sol-gel beads were performed

with longer time intervals of 20 or 30 seconds for a total dura-tion of 5 minutes A Milton Roy Spectronic 20D instrumentwas used to monitor the absorbance at 420 nm Agitation ofthe reaction solution and the CPO sol-gel beads via inversionor pipetting was crucial to ensure good mixing in betweenabsorbance recordings Control reactions with sol-gel beadsthat did not contain CPO confirmed that the sol-gel beadsthemselves do not catalyze the peroxidation of pyrogallol

24 Spectroscopic Studies (CD andUVVIS) CD spectra wererecorded with an AVIV Model 215 circular dichroism spec-trometer at 25∘CThe temperature was controlled with a ther-mostat We employed the sample preparation method devel-oped by Eggers andValentine [27] A buffer of low concentra-tion (2mM sodium phosphate pH 70) was chosen to mini-mize total sample absorbance in the UV region To achieveefficient gelation despite the low concentration of the buffera pH value of 70 was selected A sol solution was preparedby sonicating 737mL TMOS 092mL dH

2O and 088mL

001M hydrochloric acid for 20 minutes in an ice-water bathA portion of 175mL of this sol-gel solution was gently mixedwith 2625mL CPO solution containing 12120583MCPO in 2mMsodium phosphate pH 70 and cast into a plastic cassetteof 1mm thickness The cassettes were purchased from LifeTechnologies For the first four days the storage buffer ontop of the sol-gel sheet was exchanged daily For completematuration inside the more enclosed cassettes the CPO sol-gel sheets were stored for onemonth at 4∘C Prior to the actualCD measurements the cassette housing the CPO sol-gel wasopened A small piece was cut out with a razor blade and

Journal of Nanotechnology 3

transferred into a CD quartz cuvette of 2mm thickness Theremaining space was filled with 2mM sodium phosphatebuffer pH 70

UVVIS spectra were recorded using a UV mini 1240Shimadzu single beam spectrophotometer The CPO sol-gelmaterial was prepared as described in Section 22 Howeverinstead of casting beads the solution was poured into 1mmthick plastic cassettes and a higher CPO concentration yield-ing 16mgmL CPO in the final sol-gel sheet was used TheCPO sol-gel sheet was stored in the cassette for one month at4∘C A rectangular piece was cut out and placed onto one wallof a plastic cuvette of 10mm thicknessThe CPO sol-gel sheetremained attached to the plastic wall after the cuvette wasfilled with 50mMpotassiumphosphate buffer pH 60 To testthe effect of changing the pH value of buffers surrounding theCPO sol-gel sheets the molarity of the potassium phosphatebuffers at various pH values was increased to 05M

25 Influence of Methanol on CPOrsquos Catalytic Activity andSpectroscopic Properties CPO dissolved at concentrations ofup to 9 120583M in 01M citrate-02M phosphate buffer pH 42was incubated with 11 vv methanol After specific timeintervals sample aliquots were removed to record UVVISor CD spectra or to measure the enzymatic activity viathe pyrogallol peroxidation assay Reference samples withoutmethanol were prepared in parallel To test whether anyeffects caused by methanol exposure might be reversiblemethanol treated samples were dialyzed for three hours ina Pierce Slide-A-Lyzer (10000 MWCO) against 1 L of 001Mcitrate-002M phosphate buffer pH 42 at room temperature

26 Porosimetry Measurements All porosimetry measure-ments were conducted with an ASAP 2020 physisorptionanalyzer from Micromeritics using nitrogen gas To preparethe samples the solvent exchange procedure described byHarreld and coworkers was used [28]The sol-gel beads (typ-ically 8) were placed in sim4mL acetone overnightThe acetonewas exchanged each day for three days For the final two daysn-pentane was used instead of acetone N-pentane and ace-tone are miscible with each other but n-pentane evaporatesmore readily than acetone This technique yields dry beadswhile minimizing the risk of pore collapse The beads wereplaced in the sample degas station of the physisorption ana-lyzer for approximately 8 hours at 37∘C until constant pres-sure was reached Nitrogen adsorption and desorption curveswere recorded and analyzed with the Brunauer-Emmett-Teller (BET) algorithm Proper performance of the instru-ment was confirmed with a silica-alumina standard providedby Micromeritics

3 Results and Discussion

31 Peroxidation of Pyrogallol Catalyzed by Free and Sol-GelEntrapped CPO The catalytic activity of CPO was assessedby monitoring the peroxidation reaction of pyrogallol Todetermine optimal reaction conditions we varied the pHvalue of the assay buffer and the concentrations of pyrogalloland hydrogen peroxide Our observations for free CPO agreewell with previous studies [24 29] Acidic conditions with

pH values in the range of 35 to 45 and pyrogallol concentra-tions at approximately 35mM resulted in optimum catalyticperformance Very high pyrogallol concentrations gave riseto substrate inactivation but the enzyme was even moresensitive towards inactivation by the cosubstrate H

2O2 The

H2O2concentration should not exceed 10mM The enzyme

kinetic data obtained for sol-gel entrapped CPO displayedsimilar features albeit at much lower specific activity Notablysol-gel entrapment did not abolish the detrimental effect ofH2O2 All data displayed in Figure 1 was thus fitted with a

substrate inhibition model (see (1))The enzyme kinetic parameters for the peroxidation of

pyrogallol catalyzed by free and sol-gel entrapped CPO aresummarized in Table 1 Significantly smaller Vmax and thus119896cat values were obtained for sol-gel entrapped CPO com-pared to free CPOThe parameters119870

119898and119870

119868did not change

significantly in response to sol-gel entrapment for either sub-strate The poor quality of the fit shown in Figure 1(b) for thedata on sol-gel entrapped CPOmight be caused by large datapoint variations and the limitations of the substrate inhibitionmodel Manoj et al [29] demonstrated that the substrateinhibition model used here (see (1)) can yield acceptable fitsfor individual substrate variations but global fit parameterswere shown to be unattainable [29] For example the119870

119872and

119870

119868values of one substrate depended on the concentration of

the other cosubstrate All kinetic parameters determined hereare thus only apparent values and the parameters for sol-gelentrapped CPO are further influenced by additional factorsFor example 119896cat was calculated based on the assumption thatall entrapped CPO molecules are able to participate in thecatalytic process This will not be the case for CPOmoleculesthat are entrapped in closed silica pores or are otherwisepermanently shielded and for CPO molecules with compro-mised functionality due to enzyme damage The decrease in119896cat (and also Vmax)might thus signify an apparent decrease inaccessible andor active CPO molecules upon sol-gel entrap-ment Notably hindered product release can also cause adecrease in 119896catThe 119896cat value is either determined by the rateof catalytic turnover or the rate of product release dependingon which of the two steps is slower and thus rate-limitingThe silica nanostructure does not seem to impose anysignificant diffusional constraints on themolecules pyrogalloland possiblyH

2O2 If these substratemolecules were encoun-

tering substantial diffusional barriers an increase in theirapparent119870

119872and119870

119868values would be expected

32 Entrapment and Catalytic Performance of CPO in Sol-GelBeads To further assess the entrapment and catalytic perfor-mance of the immobilized enzyme different amounts of CPOwere immobilized inside sol-gel beads corresponding to finalconcentrations of 40120583gmL 8 120583gmL and 4 120583gmL per totalsol-gel solution The storage buffer was exchanged to removemethanol originating from the hydrolysis and condensationsteps of the sol-gel formation CPO activity was measured inthese exchanged buffer samples and compared to a referencesample with free CPO in solution to quantify enzyme leakage(see Figure 2) Higher enzyme loading resulted in more

4 Journal of Nanotechnology

0

500

1000

1500

0 20 40 60 80 100 120 140

Activ

ity o

f fre

e CPO

(IU

mg)

Pyrogallol (mM)

0

50

100

150

Activ

ity o

f sol

-gel

CPO

(IU

mg)

(a)

0 20 40 60 80 100H2O2 (mM)

2500

2000

Activ

ity o

f fre

e CPO

(IU

mg)

1500

1000

500

0 0

50

100

150

200

250

Activ

ity o

f sol

-gel

CPO

(IU

mg)

(b)

Figure 1 Peroxidation of pyrogallol catalyzed by free CPO (open squares 0575 120583g CPO in 3mL total assay volume) and sol-gel entrappedCPO (grey circles 0575120583g CPO entrapped in three sol-gel beads placed in 3mL total assay volume) The CPO sol-gel beads were preparedfrom TMOS with a pH 60 casting buffer and matured for one week with a buffer exchange on every other day The activity assays wereperformed in 01M citric acid-02M dibasic sodium phosphate buffer pH 42 with constant concentrations of either 27mM H

2O2(a) or

35mM pyrogallol (b) All data were measured in triplicate and are displayed as mean values plusmn one standard deviation The parameters of thecurve fits are summarized in Table 1

Table 1 Enzyme kinetic parameters for the CPO catalyzed peroxidation of pyrogallollowast

119870

119872(mM) 119870

119868(mM) Vmax (IUmg) 119896cat (1sec)

PyrogallolFree CPO (1198772 = 0983) 11 plusmn 10 160 plusmn 20 2000 plusmn 80 1400 plusmn 60Sol-gel entrapped CPO (1198772 = 0847) 93 plusmn 26 190 plusmn 74 100 plusmn 12 70 plusmn 84

H2O2

Free CPO (1198772 = 0921) 17 plusmn 62 64 plusmn 22 9100 plusmn 2500 6370 plusmn 1750Sol-gel entrapped CPO (1198772 = 0657) 27 plusmn 39 61 plusmn 86 680 plusmn 830 480 plusmn 580

lowastEnzyme kinetic parameters were obtained by fitting the data shown in Figure 1(a) (pyrogallol) and Figure 1(b) (H2O2) to a substrate inhibition model (see(1)) All parameters are presented as value of the fit plusmn one standard error of the fit

0

1

2

3

4

5

6

7

Leak

age (

)

Time1d1h 2d 3d 4d 6d

Figure 2Three sets of CPO sol-gel beads with CPO concentrationsof 40 120583gmL (dark grey bars) 8 120583gmL (grey bars) and 4 120583gmL(light grey bars) were aged for one week The storage buffer wasreplaced in the time intervals indicated on the graph and tested forCPO leakage Each set was replicated three times and containedthree beads per sample tube The specific activity of the referencesample with free CPO in solution was 1436plusmn39 IUmgThe columnheights represent the mean value and the error bars represent plusmn onestandard deviation

enzyme leakage As the sol-gel material matured and moreconnections formed within the silica mesh enzyme leakagedeclined

Table 2 contains the results of the activity measurementswith the CPO sol-gel beads and summarizes the cumulativeleakage over the first six days The activity measurementswere performed with CPO sol-gel beads that were seven daysold Each assay comprised 01M citric acid-02M dibasicsodium phosphate buffer pH 42 27mM H

2O2 and 35mM

pyrogallol The CPO sol-gel beads with the highest CPOloading of 40 120583g CPO per mL sol-gel yielded the highestabsolute activity values of 249 plusmn 33mIU A comparisonamong specific activity values reported per mg of initiallyloaded CPO however clearly showed that the CPO sol-gelbeads with a lower enzyme content of 8 or 4120583gmL perfor-med better At a higher loading of CPOmore CPOmoleculesmight be obstructed by either other enzyme molecules orthe silica nanostructure Also some of these CPO moleculesmight be entrapped in closed pores

Another set of three tubes each containing three beadsloaded with 4120583gmL CPO was aged for one week and testedfor reusability (see Figure 3) The CPO sol-gel beads can bereused up to three times in a convenient manner by replacingthe liquid phase composed of buffer and product moleculeswith new buffer and substrate However a further decline incatalytic performance was apparent We also noticed that allCPO sol-gel beads adopted the yellow-orange color of theproduct purpurogallin after the first use Gentle washing with

Journal of Nanotechnology 5

Table 2 Catalytic performance of sol-gel beads loaded with different CPO amounts

CPO loading of sol-gel beads (120583gmL) 40 8 4Activity (mIU)lowast 249 plusmn 33 138 plusmn 18 76 plusmn 4Specific activity (IUmg)lowast 41 plusmn 6 115 plusmn 15 127 plusmn 6Relative activity compared to free CPO () 29 80 880Cumulative leakage for six days ()dagger 15 12 7lowastAbsolute activity values in mIU (times10minus3 International Units) and specific activity values per mg initially loaded CPO are presented as mean values plusmn onestandard deviation All sample sets were prepared in triplicate The relative activity was based on a reference assay with free CPO in solution The referencevalue was 1436 plusmn 39 IUmg daggerThe cumulative leakage over six days of maturation corresponds to the summation of the leakage data shown in Figure 2

0

50

100

150

200

First use

Activ

ity (I

Um

g )

Time1d 3d 4d 5d 11d 12d

Figure 3 Reusability test for CPO sol-gel beads loaded with4 120583gmL CPOThree sets each with three beads per tube were usedin this test The column heights represent the mean specific activityvalue and the error bars representplusmn one standard deviationThe firstmeasurement was taken after the beads were aged for one week withdaily buffer exchanges The number of days that passed before thenext reuse is listed on the 119909-axis

the storage buffer diminished the coloration only slightlyThus clogging of the sol-gel nanostructure with productmolecules was one factor that hampered the reusability of theCPO sol-gel beads Furthermore the peroxidation reactionscatalyzed by CPO are prone to substrate inhibition (seeFigure 1) Trapped pyrogallol and H

2O2molecules could

therefore interfere with effective reuse Jung and Hartmanndemonstrated that in situ generation of hydrogen peroxidevia coimmobilization of glucose oxidase can improve thereusability of cross-linked CPO molecules entrapped inmesoporous molecular sieves [30]

The specific activity of three independently prepared setsof sol-gel beads all containing sim4 120583gmL CPO was different70 plusmn 15 IUmg (Figure 1) 127 plusmn 6 IUmg (Table 2) and171 plusmn 14 IUmg (Figure 3) The CPO sol-gel materials usedfor generating the data presented in Table 2 and Figure 3were prepared from the same CPO vial with a free CPOreference value of 1436 plusmn 39 IUmg The reference value forthe first CPO vial was only 1280 plusmn 80 IUmg However manyother experimental parameters such as the exact sol-gelcomposition the quality of all starting materials humiditytemperature and duration of sol-gel drying phase can alsoinfluence the properties of the final sol-gel material There-fore all CPO sol-gel beads that are compared within onetable or graph were prepared on the same day with the samereagent batches in parallel

33Modification of Sol-Gel Procedure with respect toMethanolRelease Next we tested whether minor modifications in thesol-gel procedure that influence the retention of methanolreleased from the sol-gel precursor TMOS would result inany significant changes On the same day three differentsets of CPO sol-gel beads all containing 4 120583gmL CPO wereprepared in triplicateThe first set was prepared with a proce-dure that facilitates methanol release by using an open vesselwhile sonicating the sol solution and performing daily bufferexchanges during the first week of sol-gel maturation Thesecond and third sets were prepared using a closed sonicationvessel and no buffer exchange was performed for the thirdset Despite these modifications all three sets prepared onthe same day yielded virtually identical specific activity valuesof 167 plusmn 18 IUmg 164 plusmn 10 IUmg and 178 plusmn 21 IUmgrespectively A more vigorous procedure presented by Ferreret al [31] involves rotavaporization of the sol solution prior toaddition of the buffered enzyme solution In our laboratorythis method was not successful as sol solidification started toset in too rapidly to achieve consistent gelation

Overall the catalytic performance of the best set of sol-gel beads was only 125plusmn15 in comparison to the referenceassay with free CPO in solution Possible reasons for thedecline in catalytic performance upon entrapment include (1)loss of enzyme due to leakage from the sol-gel matrix (2)damage to the enzyme caused by the entrapment procedureor (3) hindered substrate or product diffusion within the sol-gel nanostructure As mentioned above enzyme leakage didoccur but it can only account for a small loss of approximately10 In the experiments described below we tested for thetwo remaining possible reasons for the decline in catalyticperformance of the sol-gel entrappedCPOand also examinedthe effect of methanol on CPO

34 CD and UVVIS Spectroscopy with Sol-Gel EntrappedCPO To monitor possible enzyme damage we exploited thefact that sol-gels prepared from TMOS are transparent Cir-cular dichroism and visible absorbance spectra of free CPOin solution and CPO in sol-gel entrapped form are shown inFigures 4 and 5 respectively The spectra of free CPO andsol-gel entrapped CPO are virtually identical to each otherMinor changes in intensity of the spectroscopic signals aremost likely due to scattering effects from the sol-gel surfaceor the shrinkage of the sol-gel material during thematurationprocess Shrinkage slightly raises the concentration of thesample but also decreases the spectroscopic path length TheCD spectra are typical for a protein with high alpha helical

6 Journal of Nanotechnology

minus20

minus15

minus10

minus5

0

5

200 210 220 230 240 250 260Wavelength (nm)

CD si

gnal

(mde

g)

Figure 4 CD spectra of sol-gel entrapped CPO (black squares)and free CPO (white squares) The CPO concentration was 7120583Min the silica sol-gel sheet of sim1mm thickness The solution samplecontaining 7 120583M CPO in 2mM sodium phosphate buffer pH 70was measured in a cuvette of 1mm path length

0

01

02

03

04

05

300 400 500 600 700 800 900

Abso

rban

ce

Wavelength (nm)

Figure 5 Absorbance spectra of sol-gel entrapped CPO (solid line)and free CPO (dashed line) The silica sol-gel sheet was sim1mmthick and contained 16mgmL CPO The sheet was placed onthe wall of a 10mm wide plastic cuvette and immersed in 50mMpotassium phosphate buffer pH 60 The solution reference samplewas prepared from the same CPO stock via dilution to 016mgmL(4 120583M) with 50mM potassium phosphate buffer pH 60 in a 10mmthick plastic cuvette

content This finding agrees with the X-ray protein structureof CPO [32] and previously determined CD data [15 33]

The visible absorbance spectra shown in Figure 5 aretypical for the active form of CPO with a five-coordinateiron center in the heme chromophore [34] As the pH valueincreases above pH 70 CPO is inactivated the iron centerin the heme group becomes six-coordinate and the Soretband shifts to a longer wavelength [34] This spectroscopictransition can also be observed in CPO sol-gel sheets despitethe entrapment of the enzyme inside the silica matrixFigure 6 displays the absorption spectra of CPO sol-gelsheets immersed in 05M phosphate buffers at pH values of60 80 and 100 In comparison to solution spectra morealkaline conditions are necessary to achieve the Soret peakcharacteristic for sixfold coordinated heme iron centers It isconceivable that the silanol groups in the sol-gel impose anadditional buffering effect According toDunn and Zink [35]

0005

01015

02025

03035

04045

300 320 340 360 380 400 420 440 460 480 500

Abso

rban

ce

Wavelength (nm)

Figure 6 Absorbance spectra of sol-gel entrapped CPO immersedin 05M potassium phosphate buffer with pH values of 60 (solidline) 80 (dashed line) and 100 (dotted line)The silica sol-gel sheetsof 1mm thickness loaded with 16mgmL CPO were placed on thewall of plastic cuvette with a path length of 10mm

the pH can be up to one pH unit lower inside a sol-gel porethan in the surrounding aqueous buffer

The low apparent 119896cat values for sol-gel entrapped CPO(see Table 1) indicate that a significant number of entrappedCPO molecules were unable to catalyze the peroxidation ofpyrogallol in an effective manner CD and visible absorptionspectra however did not indicate any enzyme damage Itshould be noted that both spectroscopic methods can onlyaddress specific features of the enzymeThese features are theoverall secondary structure of the protein the electronic con-figuration of the active site chromophore and the ability torearrange the coordination sphere of the heme iron center inresponse to an external change in pH value

35 Influence of Methanol on the Spectroscopic Features andActivity of Free CPO Methanol is released from the sol-gelprecursor TMOS If we assume complete hydrolysis of TMOSand no evaporation of methanol the protein is exposed toa methanol concentration of 11 vv as the buffered enzymesolution and the sol are mixed and pipetted onto the parafilmfor gelation and drying The transfer into storage bufferand the subsequent exchange of buffer drastically lower themethanol content during the maturation phase of the sol-gelAfter the first buffer exchange the methanol concentration isonly 003 vv

To study the influence of methanol on the spectroscopicfeatures of CPO the enzyme was incubated with 11 vvmethanol andUVVIS andCD spectrawere recordedWedidnot detect a change in the spectroscopic features of the CDspectra after several hours of incubation (data not shown)but methanol exposure did have a small and immediate effecton UVVIS absorption spectra (see Figure 7)The absorptionmaximum of the Soret band shifted from 396 nm to 400 nmNotably this shift was reversible via dialysis The methanolremoval caused a 13-fold increase in sample volume Weadjusted the corresponding spectroscopic trace in Figure 7for this dilution effect

Journal of Nanotechnology 7

0

02

04

06

08

1

370 390 410 430 450

Abso

rban

ce

Wavelength (nm)

Figure 7 Absorption spectra of 9120583M CPO in 01M citrate-02Mphosphate buffer pH 42 (solid line) in 11 vv methanol and01M citrate-02M phosphate buffer pH 42 (dashed line) dialyzedsample (grey solid line) and dialyzed sample adjusted for 13-foldvolume increase (grey dotted line)

In agreement with previous studies [36] we observed adrastic decline in CPOrsquos catalytic performance after incuba-tion of CPO with organic solvents Compared to an aqueousreference sample without methanol only 57 residual activ-ity was detected after incubation with 11 vv methanol for2 hours After one day the residual activity still remainedat 57 We further discovered that the detrimental effect ofmethanol was reversible Up to 96 of the samplersquos initialactivity was recovered after dialysis Sample dilution alsoresulted in the enzymersquos recovery Decreasing the methanolcontent to 5 or 1 vv via sample dilution resulted in 90and 100 relative activity in comparison to identically dilutedsamples from the same CPO batch that were not exposed tomethanol Our finding that damage caused by methanol wasreversible in solution has implications for other studies on theuse of CPO in organic solvents or in biphasic solvent systems[36 37] Several CPO substrates which can be convertedinto products of industrial interest have high solubility innonpolar organic solvents [8]

Also if any initial damage caused by methanol exposurewas also reversible for sol-gel entrapped CPO immediatereduction in methanol content via evaporation of methanolfrom the sol solution or daily buffer exchanges during thegel maturation phase would not critically alter the finalperformance of the CPO sol-gel beads This might explainwhy the three different but parallel preparations outlinedin Section 33 resulted in virtually identical performance forthe three different CPO sol-gel bead sets On the otherhand manifestation of unrecoverable enzyme damage wouldexplain the low apparent 119896cat values determined for sol-gelentrapped CPO (see Table 1) It is conceivable that recoveryfrom damage caused by methanol exposure is less effectivefor CPO molecules entrapped within the silica sol-gel hostcompared to free CPO in solution

36 HinderedMaterial Transport in CPO Sol-Gel Beads Aftertheir first use all CPO sol-gel beads adopted a persistentyellow coloration indicating the entrapment of the productpurpurogallin Attractive intermolecular forces and physical

constraints both can delay the release of product moleculesfrom the silica nanostructure If product release becomesrate-limiting the apparent 119896cat value decreases as observedin the enzyme kinetic analysis Hindered substrate diffusionhowever was not supported by enzyme kinetic experimentsas the 119870

119872and 119870

119868values for the main substrate pyrogallol

were virtually identical for sol-gel entrapped and free CPO(see Table 1)We cannot explain why pyrogallol and purpuro-gallin would show different material transport propertiesinside the silica nanostructure Both molecules have similarfunctional groups and purpurogallin (MW 220 gmol) isonly somewhat larger than pyrogallol (MW 116 gmol) Analternative explanation would involve side-reactions formingalternate charged products or the trapping of reactive coloredintermediates

The alternative peroxidation substrate 221015840-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is particularlylarge (MW 515 gmol) and the product of the peroxidationreaction carries a positive charge [24] Deprotonated silanolgroups on the sol-gel surface can provide negative coun-tercharges The catalytic performance of CPO sol-gel beadsdropped from 126 plusmn 3 with the substrate pyrogallol to 9 plusmn04 with the substrate ABTS relative to the correspondingassay with free CPO in solution The CPO sol-gel beadsadopted the green color of the ABTS peroxidation productKadnikova and coworkers observed the formation of severalside products for the peroxidation reaction of ABTS byhorseradish peroxidase in a sol-gel matrix [38] The perox-idation reaction with ABTS and possibly other substratesincluding pyrogallol might therefore be more complex thanin solution

We further observed that preequilibration of CPO sol-gelbeadswith pyrogallol considerably improved catalytic perfor-mance For example preequilibration with 72mM pyrogallolfor 3 hours in comparison to using 35mM pyrogallol withoutpreequilibration increased the relative activity from 123 to196The strategy to preequilibrate enzyme sol-gel materialswith an excess of substrate before adding a cosubstrate wasalready successfully applied by Smith and coworkers [23] intheir study on sol-gel encapsulated horseradish peroxidaseBased on the data presented in Figure 1(a) however wewouldnot expect an increase in catalytic performance for sol-gelentrapped CPO as the pyrogallol concentration is raised from35 to 72mM In fact substrate inhibition should slightly lowerthe activity of CPOAdsorption of pyrogallolmolecules to thesol-gel surface or physical entrapment could further enhancethe local effective concentration of pyrogallol around theenzyme One key difference between the experiment leadingto Figure 1(a) and the preequilibration experiment is thetiming of adding the cosubstrate H

2O2 Manoj et al [29]

argue that substrate inhibition of CPO is not simply causedby blocking the active site of the enzymewith excess substratemolecules Instead they propose more complex substrateinhibition mechanisms that involve secondary conversionof an already formed product or competition by transientintermediates leading to alternate products Both substrateinhibition scenarios require the immediate presence of thecosubstrate H

2O2

8 Journal of Nanotechnology

Table 3 Properties of CPO sol-gel beads prepared with different casting buffers

pH of casting buffer 45 55 65BET surface area (m2g) 710 plusmn 40lowast 740 plusmn 60 470 plusmn 40Total pore volume (cm3g) 058 plusmn 002 060 plusmn 006 032 plusmn 004Average pore diameter (nm) 33 plusmn 03 32 plusmn 01 27 plusmn 01Activity (mIU) 251 plusmn 2 242 plusmn 29 161 plusmn 13Specific activity (IUmg) 157 plusmn 1 151 plusmn 18 100 plusmn 8Activity compared to free CPO () 18 17 11Cumulative leakage ()dagger 13 14 9lowastAll data are presented as mean values plusmn one standard deviation of triplicate data sets The relative activity was based on a reference assay with free CPO insolution yielding 887 plusmn 31 IUmg daggerThe cumulative leakage over ten days of maturation corresponds to the summation of the leakage data shown in Figure 8

37 Modification of Sol-Gel Procedure Using MTMS Tomodify the surface of the sol-gel material we incorporatedMTMS at molar ratios of 5 20 and 40 in the sol solutionThe addition of MTMS will introduce nonpolar methylgroups rendering the surface of the silica nanostructuremorehydrophobic [39] The casting buffer had a pH value of 60and the total CPO loading was 4 120583gmL The addition ofMTMS resulted in longer gelation times for example up to240 minutes for a molar ratio of 40 MTMS Unfortunatelythe beads prepared with MTMS were more brittle and fragilethan any of the other CPO sol-gel beads prepared in thisstudy The brittleness of the beads rendered their handlingmore challenging Regardless of the amount of incorporatedMTMS the activity was approximately 14 plusmn 1 comparedto a solution reference assay The cumulative leakage oftenexceeded 20We cannot rule out that the physical instabilityof the beads during and after a buffer exchange might havecontributed to higher apparent leakage and higher apparentactivity values In contrast to other enzymes notably lipasewhich showed interfacial activation and performed betterinsidemore hydrophobic nanostructures [40] the incorpora-tion of MTMS into the CPO sol-gel material did not improvecatalytic performance in a systematic manner

38 Modification of Sol-Gel Procedure Using More AcidicCasting Buffers The enzyme CPO is stable under acidicconditions [24] We exploited this CPO specific property andprepared CPO sol-gel beads using casting buffers with pHvalues of 45 55 and 65 All sample preparations were con-ducted in parallel with the same batches of CPO TMOS andbuffer reagents The gelation time increased with more acidiccasting buffers but the sol-gel beads remained easy to handleand transparentTheCPO loadingwas 4 120583gmL All CPO sol-gel preparations were divided into two portions One portionwas used for porosimetry studies and the other portion wasused for leakage and activity measurements (see Table 3)The properties of CPO sol-gel beads cast at pH 45 and 55are virtually identical but the CPO sol-gel beads cast at pH65 show significantly lower values in all categories Thisindicates a change in thematrix formation of the sol-gel as thecasting pHdrops to or below pH55 Overall the porosimetrydata is positively correlated with catalytic performance andunfortunately leakage All three porosimetric propertiesincluding larger average pore size BET surface area and pore

7

6

5

4

3

2

1

0

Leak

age (

)

1 2 3 4 5 6 7 8 9 10

Time (d)

Figure 8 The storage buffer of CPO sol-gel beads prepared withcasting buffers at pH 45 (dark grey bars) 55 (grey bars) and 65(light grey bars) was exchanged on a daily basis and monitored forCPO activity All samples were prepared in triplicate with eight CPOsol-gel beads per sample tube The bar height represents the meanvalue and the error bar plusmn one standard deviation

volume indicate reduced steric hindrance for material trans-port inside the sol-gel nanostructure As a consequencecatalytic performance increased Smaller average pore sizeson the other hand can aid in the retention of CPO

The dimensions of the protein CPO are 53 nm times 46 nmtimes 60 nm [19] The average pore diameters of approximately3 nm are only slightly smaller than the size of CPO Never-theless CPO remainedwell entrapped after completion of thesol-gel maturation phase Attractive electrostatic forces didnot most likely aid in the retention of CPO as the storagebuffer had a pH value of 42 which is close to the isoelectricpoint of CPO The isoelectric point of CPO from C fumagowas calculated to be approximately 40 [18 21] Isoelectricfocusing experiments on CPO from Pseudomonas pyrrociniayielded an isoelectric point of 41 [41] For all buffer condi-tions employed in our study the net charge on the surfaceof CPO is therefore either close to zero or negative

Our observation that more acidic casting buffers result ingreater porosimetry of sol-gels agrees well with several previ-ous studies [42 43] However not all enzymes will respondwell to the use of more acidic casting conditions Notablyenzymes have different pH profiles and some enzymesare inactive under acidic conditions Sol-gel entrappedcholinesterase for example showed better performance

Journal of Nanotechnology 9

in silica nanostructures prepared at pH values of 70 and 80and then 60 [44]

4 Conclusion

The enzyme CPO was successfully entrapped inside a silicananostructure prepared from the precursor TMOS with orwithout addition of the hydrophobic modifier MTMS SinceCPO is stabile in acidic buffers we further modified the sol-gel procedure by using casting buffers with pH values of 4555 60 and 65The catalytic performance of optimized CPOsol-gel beads approached 18 relative to free CPO in solutionas assessed via the pyrogallol peroxidation assay A combi-nation of factors such as enzyme leakage from the sol-gelhost insufficient recovery from inactivation caused by initialmethanol exposure hindered product release or alternatereaction pathways are most likely responsible for the declinein catalytic performance of CPOafter sol-gel entrapmentTheuse of more acidic casting buffers in the sol-gel procedureprovided themost leverage for optimization by yieldingmoreporous silica nanostructures Overall our findings are ofimportance for the optimization of other sol-gel materialsdevised for applications in biosensing or biocatalysis ordesigned for the controlled release of bioactive compounds

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Funding for this project was obtained from the ResearchCorporation for Science Advancement (Cottrell College Sci-ence Award to Monika Sommerhalter) and California StateUniversity East Bay (Faculty and Student Research Grantsto Monika Sommerhalter Selina Chan and Tuan Le and aSieber-Tombari award to Monika Sommerhalter) ProfessorDaryl Eggers San Jose State University kindly invited theauthors to perform the CD measurements in his laboratoryThe authors are also grateful to Professor AnnMcPartland forproviding detailed feedback on their paper

References

[1] B C Dave B Dunn J S Valentine and J I Zink ldquoNanocon-fined proteins and enzymes sol-gel-based biomolecular mate-rialsrdquoNanotechnology ACS Symposium Series vol 622 pp 351ndash365 1996

[2] I Gill and A Ballesteros ldquoBioencapsulation within syntheticpolymers (part 1) sol-gel encapsulated biologicalsrdquo Trends inBiotechnology vol 18 no 7 pp 282ndash296 2000

[3] D Avnir T Coradin O Lev and J Livage ldquoRecent bio-applications of sol-gel materialsrdquo Journal of Materials Chem-istry vol 16 no 11 pp 1013ndash1030 2006

[4] D R Morris and L P Hager ldquoChloroperoxidase I Isolationand properties of the crystalline glycoproteinrdquo The Journal ofBiological Chemistry vol 241 no 8 pp 1763ndash1768 1966

[5] V Yazbik and M Ansorge-Schumacher ldquoFast and efficientpurification of chloroperoxidase from C fumagordquo Process Bio-chemistry vol 45 no 2 pp 279ndash283 2010

[6] M Hofrichter and R Ullrich ldquoHeme-thiolate haloperoxidasesversatile biocatalysts with biotechnological and environmentalsignificancerdquo Applied Microbiology and Biotechnology vol 71no 3 pp 276ndash288 2006

[7] V M Dembitsky ldquoOxidation epoxidation and sulfoxidationreactions catalysed by haloperoxidasesrdquoTetrahedron vol 59 no26 pp 4701ndash4720 2003

[8] L Santhanam and J S Dordick ldquoChloroperoxidase catalyzedepoxidation of styrene in aqueous and non-aqueous mediardquoBiocatalysis and Biotransformation vol 20 no 4 pp 265ndash2742002

[9] M Ayala N R Robledo A Lopez-Munguia and R Vazquez-Duhalt ldquoSubstrate specificity and ionization potential inchloroperoxidase-catalyzed oxidation of diesel fuelrdquo Environ-mental Science and Technology vol 34 no 13 pp 2804ndash28092000

[10] R Vazquez-Duhalt M Ayala and F J Marquez-Rocha ldquoBio-catalytic chlorination of aromatic hydrocarbons by chloroper-oxidase of Caldariomyces fumagordquo Phytochemistry vol 58 no6 pp 929ndash933 2001

[11] E Terres M Montiel S Le Borgne and E Torres ldquoImmo-bilization of chloroperoxidase on mesoporous materials forthe oxidation of 46-dimethyldibenzothiophene a recalcitrantorganic sulfur compound present in petroleum fractionsrdquoBiotechnology Letters vol 30 no 1 pp 173ndash179 2008

[12] V Trevisan M Signoretto S Colonna V Pironti and GStrukul ldquoMicroencapsulated chloroperoxidase as a recyclablecatalyst for the enantioselective oxidation of sulfides withhydrogen peroxiderdquo Angewandte Chemie International Editionvol 43 no 31 pp 4097ndash4099 2004

[13] N Spreti R Germani A Incani and G Savelli ldquoStabiliza-tion of chloroperoxidase by polyethylene glycols in aqueousmedia kinetic studies and synthetic applicationsrdquoBiotechnologyProgress vol 20 no 1 pp 96ndash101 2004

[14] J-B Park and D S Clark ldquoNew reaction system for hydrocar-bon oxidation by chloroperoxidaserdquo Biotechnology and Bioengi-neering vol 94 no 1 pp 189ndash192 2006

[15] J-Z Liu and M Wang ldquoImprovement of activity and stabilityof chloroperoxidase by chemical modificationrdquo BMC Biotech-nology vol 7 no 1 article 23 2007

[16] L Zhi Y Jiang Y Wang M Hu S Li and Y Ma ldquoEffects ofadditives on the thermostability of chloroperoxidaserdquo Biotech-nology Progress vol 23 no 3 pp 729ndash733 2007

[17] T A Kadima andM A Pickard ldquoImmobilization of chloroper-oxidase on aminopropyl-glassrdquo Applied and EnvironmentalMicrobiology vol 56 no 11 pp 3473ndash3477 1990

[18] Y-J Han J T Watson G D Stucky and A Butler ldquoCatalyticactivity of mesoporous silicate-immobilized chloroperoxidaserdquoJournal ofMolecular Catalysis B Enzymatic vol 17 no 1 pp 1ndash82002

[19] J Aburto M Ayala I Bustos-Jaimes et al ldquoStability andcatalytic properties of chloroperoxidase immobilized on SBA-16mesoporousmaterialsrdquoMicroporous andMesoporousMaterialsvol 83 no 1ndash3 pp 193ndash200 2005

[20] M Hartmann and C Streb ldquoSelective oxidation of indole bychloroperoxidase immobilized on the mesoporous molecularsieve SBA-15rdquo Journal of Porous Materials vol 13 no 3-4 pp347ndash352 2006

10 Journal of Nanotechnology

[21] S Hudson J Cooney B K Hodnett and E Magner ldquoChlorop-eroxidase on periodic mesoporous organosilanes immobiliza-tion and reuserdquo Chemistry of Materials vol 19 no 8 pp 2049ndash2055 2007

[22] R A Sheldon ldquoEnzyme immobilization the quest for optimumperformancerdquo Advanced Synthesis amp Catalysis vol 349 no 8-9pp 1289ndash1307 2007

[23] K Smith N J Silvernail K R Rodgers T E Elgren M Castroand RM Parker ldquoSol-gel encapsulated horseradish peroxidasea catalytic material for peroxidationrdquo Journal of the AmericanChemical Society vol 124 no 16 pp 4247ndash4252 2002

[24] K M Manoj and L P Hager ldquoChloroperoxidase a janusenzymerdquo Biochemistry vol 47 no 9 pp 2997ndash3003 2008

[25] V M Samokyszyn and P R Ortiz de Montellano ldquoTopology ofthe chloroperoxidase active site regiospecificity of heme mod-ification by phenylhydrazine and sodium aziderdquo Biochemistryvol 30 no 50 pp 11646ndash11653 1991

[26] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[27] D K Eggers and J S Valentine ldquoMolecular confinementinfluences protein structure and enhances thermal proteinstabilityrdquo Protein Science vol 10 no 2 pp 250ndash261 2001

[28] J H Harreld T Ebina N Tsubo and G Stucky ldquoManipulationof pore size distributions in silica and ormosil gels dried underambient pressure conditionsrdquo Journal of Non-Crystalline Solidsvol 298 no 2-3 pp 241ndash251 2002

[29] K M Manoj A Baburaj B Ephraim et al ldquoExplaining theatypical reaction profiles of heme enzymes with a novel mech-anistic hypothesis and kinetic treatmentrdquo PLoS ONE vol 5 no5 Article ID e10601 2010

[30] D Jung and M Hartmann ldquoOxidation of indole with CPO andGOx immobilized on mesoporous molecular sievesrdquo CatalysisToday vol 157 no 1ndash4 pp 378ndash383 2010

[31] M L Ferrer F del Monte and D Levy ldquoA novel and simplealcohol-free sol-gel route for encapsulation of labile proteinsrdquoChemistry of Materials vol 14 no 9 pp 3619ndash3621 2002

[32] M Sundaramoorthy J Terner and T L Poulos ldquoThe crystalstructure of chloroperoxidase a heme peroxidase-cytochromeP450 functional hybridrdquo Structure vol 3 no 12 pp 1367ndash13771995

[33] X W Yi A Conesa P J Punt and L P Hager ldquoExamining therole of glutamic acid 183 in chloroperoxidase catalysisrdquo Journalof Biological Chemistry vol 278 no 16 pp 13855ndash13859 2003

[34] S R Blanke S AMartinis S G Sligar L P Hager J J Rux andJ H Dawson ldquoProbing the heme iron coordination structureof alkaline chloroperoxidaserdquo Biochemistry vol 35 no 46 pp14537ndash14543 1996

[35] B Dunn and J I Zink ldquoProbes of pore environment andmolecule-matrix interactions in sol-gel materialsrdquo Chemistry ofMaterials vol 9 no 11 pp 2280ndash2291 1997

[36] W A Loughlin and D B Hawkes ldquoEffect of organic solvents ona chloroperoxidase biotransformationrdquo Bioresource Technologyvol 71 no 2 pp 167ndash172 2000

[37] E Kiljunen and L T Kanerva ldquoChloroperoxidase-catalysedoxidation of alcohols to aldehydesrdquo Journal of Molecular Catal-ysis B Enzymatic vol 9 no 4ndash6 pp 163ndash172 2000

[38] E N Kadnikova and N M Kostic ldquoOxidation of ABTS byhydrogen peroxide catalyzed by horseradish peroxidase encap-sulated into sol-gel glass Effects of glass matrix on reactivityrdquo

Journal of Molecular Catalysis B Enzymatic vol 18 no 1ndash3 pp39ndash48 2002

[39] A Venkateswara Rao and D Haranath ldquoEffect of methyltri-methoxysilane as a synthesis component on the hydrophobicityand some physical properties of silica aerogelsrdquo Microporousand Mesoporous Materials vol 30 no 2-3 pp 267ndash273 1999

[40] M T Reetz A Zonta and J Simpelkamp ldquoEfficient immo-bilization of lipases by entrapment in hydrophobic sol-gelmaterialsrdquo Biotechnology and Bioengineering vol 49 no 5 pp527ndash534 1996

[41] W Wiesner K-H van Pee and F Lingens ldquoPurification andcharacterization of a novel bacterial non-heme chloroperox-idase from Pseudomonas pyrrociniardquo Journal of BiologicalChemistry vol 263 no 27 pp 13725ndash13732 1988

[42] Y Xi Z Liangying and W Sasa ldquoPore size and pore-sizedistribution control of porous silicardquo Sensors and Actuators BChemical vol 25 no 1-3 pp 347ndash352 1995

[43] C Lin and J A Ritter ldquoEffect of synthesis pH on the structureof carbon xerogelsrdquo Carbon vol 35 no 9 pp 1271ndash1278 1997

[44] M Altstein G Segev N Aharonson O Ben-Aziz A Tur-niansky and D Avnir ldquoSol-gel entrapped cholinesterases amicrotiter plate method for monitoring anti-cholinesterasecompoundsrdquo Journal of Agricultural and Food Chemistry vol46 no 8 pp 3318ndash3324 1998

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials

Page 3: Silica Sol-Gel Entrapment of the Enzyme Chloroperoxidase

Journal of Nanotechnology 3

transferred into a CD quartz cuvette of 2mm thickness Theremaining space was filled with 2mM sodium phosphatebuffer pH 70

UVVIS spectra were recorded using a UV mini 1240Shimadzu single beam spectrophotometer The CPO sol-gelmaterial was prepared as described in Section 22 Howeverinstead of casting beads the solution was poured into 1mmthick plastic cassettes and a higher CPO concentration yield-ing 16mgmL CPO in the final sol-gel sheet was used TheCPO sol-gel sheet was stored in the cassette for one month at4∘C A rectangular piece was cut out and placed onto one wallof a plastic cuvette of 10mm thicknessThe CPO sol-gel sheetremained attached to the plastic wall after the cuvette wasfilled with 50mMpotassiumphosphate buffer pH 60 To testthe effect of changing the pH value of buffers surrounding theCPO sol-gel sheets the molarity of the potassium phosphatebuffers at various pH values was increased to 05M

25 Influence of Methanol on CPOrsquos Catalytic Activity andSpectroscopic Properties CPO dissolved at concentrations ofup to 9 120583M in 01M citrate-02M phosphate buffer pH 42was incubated with 11 vv methanol After specific timeintervals sample aliquots were removed to record UVVISor CD spectra or to measure the enzymatic activity viathe pyrogallol peroxidation assay Reference samples withoutmethanol were prepared in parallel To test whether anyeffects caused by methanol exposure might be reversiblemethanol treated samples were dialyzed for three hours ina Pierce Slide-A-Lyzer (10000 MWCO) against 1 L of 001Mcitrate-002M phosphate buffer pH 42 at room temperature

26 Porosimetry Measurements All porosimetry measure-ments were conducted with an ASAP 2020 physisorptionanalyzer from Micromeritics using nitrogen gas To preparethe samples the solvent exchange procedure described byHarreld and coworkers was used [28]The sol-gel beads (typ-ically 8) were placed in sim4mL acetone overnightThe acetonewas exchanged each day for three days For the final two daysn-pentane was used instead of acetone N-pentane and ace-tone are miscible with each other but n-pentane evaporatesmore readily than acetone This technique yields dry beadswhile minimizing the risk of pore collapse The beads wereplaced in the sample degas station of the physisorption ana-lyzer for approximately 8 hours at 37∘C until constant pres-sure was reached Nitrogen adsorption and desorption curveswere recorded and analyzed with the Brunauer-Emmett-Teller (BET) algorithm Proper performance of the instru-ment was confirmed with a silica-alumina standard providedby Micromeritics

3 Results and Discussion

31 Peroxidation of Pyrogallol Catalyzed by Free and Sol-GelEntrapped CPO The catalytic activity of CPO was assessedby monitoring the peroxidation reaction of pyrogallol Todetermine optimal reaction conditions we varied the pHvalue of the assay buffer and the concentrations of pyrogalloland hydrogen peroxide Our observations for free CPO agreewell with previous studies [24 29] Acidic conditions with

pH values in the range of 35 to 45 and pyrogallol concentra-tions at approximately 35mM resulted in optimum catalyticperformance Very high pyrogallol concentrations gave riseto substrate inactivation but the enzyme was even moresensitive towards inactivation by the cosubstrate H

2O2 The

H2O2concentration should not exceed 10mM The enzyme

kinetic data obtained for sol-gel entrapped CPO displayedsimilar features albeit at much lower specific activity Notablysol-gel entrapment did not abolish the detrimental effect ofH2O2 All data displayed in Figure 1 was thus fitted with a

substrate inhibition model (see (1))The enzyme kinetic parameters for the peroxidation of

pyrogallol catalyzed by free and sol-gel entrapped CPO aresummarized in Table 1 Significantly smaller Vmax and thus119896cat values were obtained for sol-gel entrapped CPO com-pared to free CPOThe parameters119870

119898and119870

119868did not change

significantly in response to sol-gel entrapment for either sub-strate The poor quality of the fit shown in Figure 1(b) for thedata on sol-gel entrapped CPOmight be caused by large datapoint variations and the limitations of the substrate inhibitionmodel Manoj et al [29] demonstrated that the substrateinhibition model used here (see (1)) can yield acceptable fitsfor individual substrate variations but global fit parameterswere shown to be unattainable [29] For example the119870

119872and

119870

119868values of one substrate depended on the concentration of

the other cosubstrate All kinetic parameters determined hereare thus only apparent values and the parameters for sol-gelentrapped CPO are further influenced by additional factorsFor example 119896cat was calculated based on the assumption thatall entrapped CPO molecules are able to participate in thecatalytic process This will not be the case for CPOmoleculesthat are entrapped in closed silica pores or are otherwisepermanently shielded and for CPO molecules with compro-mised functionality due to enzyme damage The decrease in119896cat (and also Vmax)might thus signify an apparent decrease inaccessible andor active CPO molecules upon sol-gel entrap-ment Notably hindered product release can also cause adecrease in 119896catThe 119896cat value is either determined by the rateof catalytic turnover or the rate of product release dependingon which of the two steps is slower and thus rate-limitingThe silica nanostructure does not seem to impose anysignificant diffusional constraints on themolecules pyrogalloland possiblyH

2O2 If these substratemolecules were encoun-

tering substantial diffusional barriers an increase in theirapparent119870

119872and119870

119868values would be expected

32 Entrapment and Catalytic Performance of CPO in Sol-GelBeads To further assess the entrapment and catalytic perfor-mance of the immobilized enzyme different amounts of CPOwere immobilized inside sol-gel beads corresponding to finalconcentrations of 40120583gmL 8 120583gmL and 4 120583gmL per totalsol-gel solution The storage buffer was exchanged to removemethanol originating from the hydrolysis and condensationsteps of the sol-gel formation CPO activity was measured inthese exchanged buffer samples and compared to a referencesample with free CPO in solution to quantify enzyme leakage(see Figure 2) Higher enzyme loading resulted in more

4 Journal of Nanotechnology

0

500

1000

1500

0 20 40 60 80 100 120 140

Activ

ity o

f fre

e CPO

(IU

mg)

Pyrogallol (mM)

0

50

100

150

Activ

ity o

f sol

-gel

CPO

(IU

mg)

(a)

0 20 40 60 80 100H2O2 (mM)

2500

2000

Activ

ity o

f fre

e CPO

(IU

mg)

1500

1000

500

0 0

50

100

150

200

250

Activ

ity o

f sol

-gel

CPO

(IU

mg)

(b)

Figure 1 Peroxidation of pyrogallol catalyzed by free CPO (open squares 0575 120583g CPO in 3mL total assay volume) and sol-gel entrappedCPO (grey circles 0575120583g CPO entrapped in three sol-gel beads placed in 3mL total assay volume) The CPO sol-gel beads were preparedfrom TMOS with a pH 60 casting buffer and matured for one week with a buffer exchange on every other day The activity assays wereperformed in 01M citric acid-02M dibasic sodium phosphate buffer pH 42 with constant concentrations of either 27mM H

2O2(a) or

35mM pyrogallol (b) All data were measured in triplicate and are displayed as mean values plusmn one standard deviation The parameters of thecurve fits are summarized in Table 1

Table 1 Enzyme kinetic parameters for the CPO catalyzed peroxidation of pyrogallollowast

119870

119872(mM) 119870

119868(mM) Vmax (IUmg) 119896cat (1sec)

PyrogallolFree CPO (1198772 = 0983) 11 plusmn 10 160 plusmn 20 2000 plusmn 80 1400 plusmn 60Sol-gel entrapped CPO (1198772 = 0847) 93 plusmn 26 190 plusmn 74 100 plusmn 12 70 plusmn 84

H2O2

Free CPO (1198772 = 0921) 17 plusmn 62 64 plusmn 22 9100 plusmn 2500 6370 plusmn 1750Sol-gel entrapped CPO (1198772 = 0657) 27 plusmn 39 61 plusmn 86 680 plusmn 830 480 plusmn 580

lowastEnzyme kinetic parameters were obtained by fitting the data shown in Figure 1(a) (pyrogallol) and Figure 1(b) (H2O2) to a substrate inhibition model (see(1)) All parameters are presented as value of the fit plusmn one standard error of the fit

0

1

2

3

4

5

6

7

Leak

age (

)

Time1d1h 2d 3d 4d 6d

Figure 2Three sets of CPO sol-gel beads with CPO concentrationsof 40 120583gmL (dark grey bars) 8 120583gmL (grey bars) and 4 120583gmL(light grey bars) were aged for one week The storage buffer wasreplaced in the time intervals indicated on the graph and tested forCPO leakage Each set was replicated three times and containedthree beads per sample tube The specific activity of the referencesample with free CPO in solution was 1436plusmn39 IUmgThe columnheights represent the mean value and the error bars represent plusmn onestandard deviation

enzyme leakage As the sol-gel material matured and moreconnections formed within the silica mesh enzyme leakagedeclined

Table 2 contains the results of the activity measurementswith the CPO sol-gel beads and summarizes the cumulativeleakage over the first six days The activity measurementswere performed with CPO sol-gel beads that were seven daysold Each assay comprised 01M citric acid-02M dibasicsodium phosphate buffer pH 42 27mM H

2O2 and 35mM

pyrogallol The CPO sol-gel beads with the highest CPOloading of 40 120583g CPO per mL sol-gel yielded the highestabsolute activity values of 249 plusmn 33mIU A comparisonamong specific activity values reported per mg of initiallyloaded CPO however clearly showed that the CPO sol-gelbeads with a lower enzyme content of 8 or 4120583gmL perfor-med better At a higher loading of CPOmore CPOmoleculesmight be obstructed by either other enzyme molecules orthe silica nanostructure Also some of these CPO moleculesmight be entrapped in closed pores

Another set of three tubes each containing three beadsloaded with 4120583gmL CPO was aged for one week and testedfor reusability (see Figure 3) The CPO sol-gel beads can bereused up to three times in a convenient manner by replacingthe liquid phase composed of buffer and product moleculeswith new buffer and substrate However a further decline incatalytic performance was apparent We also noticed that allCPO sol-gel beads adopted the yellow-orange color of theproduct purpurogallin after the first use Gentle washing with

Journal of Nanotechnology 5

Table 2 Catalytic performance of sol-gel beads loaded with different CPO amounts

CPO loading of sol-gel beads (120583gmL) 40 8 4Activity (mIU)lowast 249 plusmn 33 138 plusmn 18 76 plusmn 4Specific activity (IUmg)lowast 41 plusmn 6 115 plusmn 15 127 plusmn 6Relative activity compared to free CPO () 29 80 880Cumulative leakage for six days ()dagger 15 12 7lowastAbsolute activity values in mIU (times10minus3 International Units) and specific activity values per mg initially loaded CPO are presented as mean values plusmn onestandard deviation All sample sets were prepared in triplicate The relative activity was based on a reference assay with free CPO in solution The referencevalue was 1436 plusmn 39 IUmg daggerThe cumulative leakage over six days of maturation corresponds to the summation of the leakage data shown in Figure 2

0

50

100

150

200

First use

Activ

ity (I

Um

g )

Time1d 3d 4d 5d 11d 12d

Figure 3 Reusability test for CPO sol-gel beads loaded with4 120583gmL CPOThree sets each with three beads per tube were usedin this test The column heights represent the mean specific activityvalue and the error bars representplusmn one standard deviationThe firstmeasurement was taken after the beads were aged for one week withdaily buffer exchanges The number of days that passed before thenext reuse is listed on the 119909-axis

the storage buffer diminished the coloration only slightlyThus clogging of the sol-gel nanostructure with productmolecules was one factor that hampered the reusability of theCPO sol-gel beads Furthermore the peroxidation reactionscatalyzed by CPO are prone to substrate inhibition (seeFigure 1) Trapped pyrogallol and H

2O2molecules could

therefore interfere with effective reuse Jung and Hartmanndemonstrated that in situ generation of hydrogen peroxidevia coimmobilization of glucose oxidase can improve thereusability of cross-linked CPO molecules entrapped inmesoporous molecular sieves [30]

The specific activity of three independently prepared setsof sol-gel beads all containing sim4 120583gmL CPO was different70 plusmn 15 IUmg (Figure 1) 127 plusmn 6 IUmg (Table 2) and171 plusmn 14 IUmg (Figure 3) The CPO sol-gel materials usedfor generating the data presented in Table 2 and Figure 3were prepared from the same CPO vial with a free CPOreference value of 1436 plusmn 39 IUmg The reference value forthe first CPO vial was only 1280 plusmn 80 IUmg However manyother experimental parameters such as the exact sol-gelcomposition the quality of all starting materials humiditytemperature and duration of sol-gel drying phase can alsoinfluence the properties of the final sol-gel material There-fore all CPO sol-gel beads that are compared within onetable or graph were prepared on the same day with the samereagent batches in parallel

33Modification of Sol-Gel Procedure with respect toMethanolRelease Next we tested whether minor modifications in thesol-gel procedure that influence the retention of methanolreleased from the sol-gel precursor TMOS would result inany significant changes On the same day three differentsets of CPO sol-gel beads all containing 4 120583gmL CPO wereprepared in triplicateThe first set was prepared with a proce-dure that facilitates methanol release by using an open vesselwhile sonicating the sol solution and performing daily bufferexchanges during the first week of sol-gel maturation Thesecond and third sets were prepared using a closed sonicationvessel and no buffer exchange was performed for the thirdset Despite these modifications all three sets prepared onthe same day yielded virtually identical specific activity valuesof 167 plusmn 18 IUmg 164 plusmn 10 IUmg and 178 plusmn 21 IUmgrespectively A more vigorous procedure presented by Ferreret al [31] involves rotavaporization of the sol solution prior toaddition of the buffered enzyme solution In our laboratorythis method was not successful as sol solidification started toset in too rapidly to achieve consistent gelation

Overall the catalytic performance of the best set of sol-gel beads was only 125plusmn15 in comparison to the referenceassay with free CPO in solution Possible reasons for thedecline in catalytic performance upon entrapment include (1)loss of enzyme due to leakage from the sol-gel matrix (2)damage to the enzyme caused by the entrapment procedureor (3) hindered substrate or product diffusion within the sol-gel nanostructure As mentioned above enzyme leakage didoccur but it can only account for a small loss of approximately10 In the experiments described below we tested for thetwo remaining possible reasons for the decline in catalyticperformance of the sol-gel entrappedCPOand also examinedthe effect of methanol on CPO

34 CD and UVVIS Spectroscopy with Sol-Gel EntrappedCPO To monitor possible enzyme damage we exploited thefact that sol-gels prepared from TMOS are transparent Cir-cular dichroism and visible absorbance spectra of free CPOin solution and CPO in sol-gel entrapped form are shown inFigures 4 and 5 respectively The spectra of free CPO andsol-gel entrapped CPO are virtually identical to each otherMinor changes in intensity of the spectroscopic signals aremost likely due to scattering effects from the sol-gel surfaceor the shrinkage of the sol-gel material during thematurationprocess Shrinkage slightly raises the concentration of thesample but also decreases the spectroscopic path length TheCD spectra are typical for a protein with high alpha helical

6 Journal of Nanotechnology

minus20

minus15

minus10

minus5

0

5

200 210 220 230 240 250 260Wavelength (nm)

CD si

gnal

(mde

g)

Figure 4 CD spectra of sol-gel entrapped CPO (black squares)and free CPO (white squares) The CPO concentration was 7120583Min the silica sol-gel sheet of sim1mm thickness The solution samplecontaining 7 120583M CPO in 2mM sodium phosphate buffer pH 70was measured in a cuvette of 1mm path length

0

01

02

03

04

05

300 400 500 600 700 800 900

Abso

rban

ce

Wavelength (nm)

Figure 5 Absorbance spectra of sol-gel entrapped CPO (solid line)and free CPO (dashed line) The silica sol-gel sheet was sim1mmthick and contained 16mgmL CPO The sheet was placed onthe wall of a 10mm wide plastic cuvette and immersed in 50mMpotassium phosphate buffer pH 60 The solution reference samplewas prepared from the same CPO stock via dilution to 016mgmL(4 120583M) with 50mM potassium phosphate buffer pH 60 in a 10mmthick plastic cuvette

content This finding agrees with the X-ray protein structureof CPO [32] and previously determined CD data [15 33]

The visible absorbance spectra shown in Figure 5 aretypical for the active form of CPO with a five-coordinateiron center in the heme chromophore [34] As the pH valueincreases above pH 70 CPO is inactivated the iron centerin the heme group becomes six-coordinate and the Soretband shifts to a longer wavelength [34] This spectroscopictransition can also be observed in CPO sol-gel sheets despitethe entrapment of the enzyme inside the silica matrixFigure 6 displays the absorption spectra of CPO sol-gelsheets immersed in 05M phosphate buffers at pH values of60 80 and 100 In comparison to solution spectra morealkaline conditions are necessary to achieve the Soret peakcharacteristic for sixfold coordinated heme iron centers It isconceivable that the silanol groups in the sol-gel impose anadditional buffering effect According toDunn and Zink [35]

0005

01015

02025

03035

04045

300 320 340 360 380 400 420 440 460 480 500

Abso

rban

ce

Wavelength (nm)

Figure 6 Absorbance spectra of sol-gel entrapped CPO immersedin 05M potassium phosphate buffer with pH values of 60 (solidline) 80 (dashed line) and 100 (dotted line)The silica sol-gel sheetsof 1mm thickness loaded with 16mgmL CPO were placed on thewall of plastic cuvette with a path length of 10mm

the pH can be up to one pH unit lower inside a sol-gel porethan in the surrounding aqueous buffer

The low apparent 119896cat values for sol-gel entrapped CPO(see Table 1) indicate that a significant number of entrappedCPO molecules were unable to catalyze the peroxidation ofpyrogallol in an effective manner CD and visible absorptionspectra however did not indicate any enzyme damage Itshould be noted that both spectroscopic methods can onlyaddress specific features of the enzymeThese features are theoverall secondary structure of the protein the electronic con-figuration of the active site chromophore and the ability torearrange the coordination sphere of the heme iron center inresponse to an external change in pH value

35 Influence of Methanol on the Spectroscopic Features andActivity of Free CPO Methanol is released from the sol-gelprecursor TMOS If we assume complete hydrolysis of TMOSand no evaporation of methanol the protein is exposed toa methanol concentration of 11 vv as the buffered enzymesolution and the sol are mixed and pipetted onto the parafilmfor gelation and drying The transfer into storage bufferand the subsequent exchange of buffer drastically lower themethanol content during the maturation phase of the sol-gelAfter the first buffer exchange the methanol concentration isonly 003 vv

To study the influence of methanol on the spectroscopicfeatures of CPO the enzyme was incubated with 11 vvmethanol andUVVIS andCD spectrawere recordedWedidnot detect a change in the spectroscopic features of the CDspectra after several hours of incubation (data not shown)but methanol exposure did have a small and immediate effecton UVVIS absorption spectra (see Figure 7)The absorptionmaximum of the Soret band shifted from 396 nm to 400 nmNotably this shift was reversible via dialysis The methanolremoval caused a 13-fold increase in sample volume Weadjusted the corresponding spectroscopic trace in Figure 7for this dilution effect

Journal of Nanotechnology 7

0

02

04

06

08

1

370 390 410 430 450

Abso

rban

ce

Wavelength (nm)

Figure 7 Absorption spectra of 9120583M CPO in 01M citrate-02Mphosphate buffer pH 42 (solid line) in 11 vv methanol and01M citrate-02M phosphate buffer pH 42 (dashed line) dialyzedsample (grey solid line) and dialyzed sample adjusted for 13-foldvolume increase (grey dotted line)

In agreement with previous studies [36] we observed adrastic decline in CPOrsquos catalytic performance after incuba-tion of CPO with organic solvents Compared to an aqueousreference sample without methanol only 57 residual activ-ity was detected after incubation with 11 vv methanol for2 hours After one day the residual activity still remainedat 57 We further discovered that the detrimental effect ofmethanol was reversible Up to 96 of the samplersquos initialactivity was recovered after dialysis Sample dilution alsoresulted in the enzymersquos recovery Decreasing the methanolcontent to 5 or 1 vv via sample dilution resulted in 90and 100 relative activity in comparison to identically dilutedsamples from the same CPO batch that were not exposed tomethanol Our finding that damage caused by methanol wasreversible in solution has implications for other studies on theuse of CPO in organic solvents or in biphasic solvent systems[36 37] Several CPO substrates which can be convertedinto products of industrial interest have high solubility innonpolar organic solvents [8]

Also if any initial damage caused by methanol exposurewas also reversible for sol-gel entrapped CPO immediatereduction in methanol content via evaporation of methanolfrom the sol solution or daily buffer exchanges during thegel maturation phase would not critically alter the finalperformance of the CPO sol-gel beads This might explainwhy the three different but parallel preparations outlinedin Section 33 resulted in virtually identical performance forthe three different CPO sol-gel bead sets On the otherhand manifestation of unrecoverable enzyme damage wouldexplain the low apparent 119896cat values determined for sol-gelentrapped CPO (see Table 1) It is conceivable that recoveryfrom damage caused by methanol exposure is less effectivefor CPO molecules entrapped within the silica sol-gel hostcompared to free CPO in solution

36 HinderedMaterial Transport in CPO Sol-Gel Beads Aftertheir first use all CPO sol-gel beads adopted a persistentyellow coloration indicating the entrapment of the productpurpurogallin Attractive intermolecular forces and physical

constraints both can delay the release of product moleculesfrom the silica nanostructure If product release becomesrate-limiting the apparent 119896cat value decreases as observedin the enzyme kinetic analysis Hindered substrate diffusionhowever was not supported by enzyme kinetic experimentsas the 119870

119872and 119870

119868values for the main substrate pyrogallol

were virtually identical for sol-gel entrapped and free CPO(see Table 1)We cannot explain why pyrogallol and purpuro-gallin would show different material transport propertiesinside the silica nanostructure Both molecules have similarfunctional groups and purpurogallin (MW 220 gmol) isonly somewhat larger than pyrogallol (MW 116 gmol) Analternative explanation would involve side-reactions formingalternate charged products or the trapping of reactive coloredintermediates

The alternative peroxidation substrate 221015840-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is particularlylarge (MW 515 gmol) and the product of the peroxidationreaction carries a positive charge [24] Deprotonated silanolgroups on the sol-gel surface can provide negative coun-tercharges The catalytic performance of CPO sol-gel beadsdropped from 126 plusmn 3 with the substrate pyrogallol to 9 plusmn04 with the substrate ABTS relative to the correspondingassay with free CPO in solution The CPO sol-gel beadsadopted the green color of the ABTS peroxidation productKadnikova and coworkers observed the formation of severalside products for the peroxidation reaction of ABTS byhorseradish peroxidase in a sol-gel matrix [38] The perox-idation reaction with ABTS and possibly other substratesincluding pyrogallol might therefore be more complex thanin solution

We further observed that preequilibration of CPO sol-gelbeadswith pyrogallol considerably improved catalytic perfor-mance For example preequilibration with 72mM pyrogallolfor 3 hours in comparison to using 35mM pyrogallol withoutpreequilibration increased the relative activity from 123 to196The strategy to preequilibrate enzyme sol-gel materialswith an excess of substrate before adding a cosubstrate wasalready successfully applied by Smith and coworkers [23] intheir study on sol-gel encapsulated horseradish peroxidaseBased on the data presented in Figure 1(a) however wewouldnot expect an increase in catalytic performance for sol-gelentrapped CPO as the pyrogallol concentration is raised from35 to 72mM In fact substrate inhibition should slightly lowerthe activity of CPOAdsorption of pyrogallolmolecules to thesol-gel surface or physical entrapment could further enhancethe local effective concentration of pyrogallol around theenzyme One key difference between the experiment leadingto Figure 1(a) and the preequilibration experiment is thetiming of adding the cosubstrate H

2O2 Manoj et al [29]

argue that substrate inhibition of CPO is not simply causedby blocking the active site of the enzymewith excess substratemolecules Instead they propose more complex substrateinhibition mechanisms that involve secondary conversionof an already formed product or competition by transientintermediates leading to alternate products Both substrateinhibition scenarios require the immediate presence of thecosubstrate H

2O2

8 Journal of Nanotechnology

Table 3 Properties of CPO sol-gel beads prepared with different casting buffers

pH of casting buffer 45 55 65BET surface area (m2g) 710 plusmn 40lowast 740 plusmn 60 470 plusmn 40Total pore volume (cm3g) 058 plusmn 002 060 plusmn 006 032 plusmn 004Average pore diameter (nm) 33 plusmn 03 32 plusmn 01 27 plusmn 01Activity (mIU) 251 plusmn 2 242 plusmn 29 161 plusmn 13Specific activity (IUmg) 157 plusmn 1 151 plusmn 18 100 plusmn 8Activity compared to free CPO () 18 17 11Cumulative leakage ()dagger 13 14 9lowastAll data are presented as mean values plusmn one standard deviation of triplicate data sets The relative activity was based on a reference assay with free CPO insolution yielding 887 plusmn 31 IUmg daggerThe cumulative leakage over ten days of maturation corresponds to the summation of the leakage data shown in Figure 8

37 Modification of Sol-Gel Procedure Using MTMS Tomodify the surface of the sol-gel material we incorporatedMTMS at molar ratios of 5 20 and 40 in the sol solutionThe addition of MTMS will introduce nonpolar methylgroups rendering the surface of the silica nanostructuremorehydrophobic [39] The casting buffer had a pH value of 60and the total CPO loading was 4 120583gmL The addition ofMTMS resulted in longer gelation times for example up to240 minutes for a molar ratio of 40 MTMS Unfortunatelythe beads prepared with MTMS were more brittle and fragilethan any of the other CPO sol-gel beads prepared in thisstudy The brittleness of the beads rendered their handlingmore challenging Regardless of the amount of incorporatedMTMS the activity was approximately 14 plusmn 1 comparedto a solution reference assay The cumulative leakage oftenexceeded 20We cannot rule out that the physical instabilityof the beads during and after a buffer exchange might havecontributed to higher apparent leakage and higher apparentactivity values In contrast to other enzymes notably lipasewhich showed interfacial activation and performed betterinsidemore hydrophobic nanostructures [40] the incorpora-tion of MTMS into the CPO sol-gel material did not improvecatalytic performance in a systematic manner

38 Modification of Sol-Gel Procedure Using More AcidicCasting Buffers The enzyme CPO is stable under acidicconditions [24] We exploited this CPO specific property andprepared CPO sol-gel beads using casting buffers with pHvalues of 45 55 and 65 All sample preparations were con-ducted in parallel with the same batches of CPO TMOS andbuffer reagents The gelation time increased with more acidiccasting buffers but the sol-gel beads remained easy to handleand transparentTheCPO loadingwas 4 120583gmL All CPO sol-gel preparations were divided into two portions One portionwas used for porosimetry studies and the other portion wasused for leakage and activity measurements (see Table 3)The properties of CPO sol-gel beads cast at pH 45 and 55are virtually identical but the CPO sol-gel beads cast at pH65 show significantly lower values in all categories Thisindicates a change in thematrix formation of the sol-gel as thecasting pHdrops to or below pH55 Overall the porosimetrydata is positively correlated with catalytic performance andunfortunately leakage All three porosimetric propertiesincluding larger average pore size BET surface area and pore

7

6

5

4

3

2

1

0

Leak

age (

)

1 2 3 4 5 6 7 8 9 10

Time (d)

Figure 8 The storage buffer of CPO sol-gel beads prepared withcasting buffers at pH 45 (dark grey bars) 55 (grey bars) and 65(light grey bars) was exchanged on a daily basis and monitored forCPO activity All samples were prepared in triplicate with eight CPOsol-gel beads per sample tube The bar height represents the meanvalue and the error bar plusmn one standard deviation

volume indicate reduced steric hindrance for material trans-port inside the sol-gel nanostructure As a consequencecatalytic performance increased Smaller average pore sizeson the other hand can aid in the retention of CPO

The dimensions of the protein CPO are 53 nm times 46 nmtimes 60 nm [19] The average pore diameters of approximately3 nm are only slightly smaller than the size of CPO Never-theless CPO remainedwell entrapped after completion of thesol-gel maturation phase Attractive electrostatic forces didnot most likely aid in the retention of CPO as the storagebuffer had a pH value of 42 which is close to the isoelectricpoint of CPO The isoelectric point of CPO from C fumagowas calculated to be approximately 40 [18 21] Isoelectricfocusing experiments on CPO from Pseudomonas pyrrociniayielded an isoelectric point of 41 [41] For all buffer condi-tions employed in our study the net charge on the surfaceof CPO is therefore either close to zero or negative

Our observation that more acidic casting buffers result ingreater porosimetry of sol-gels agrees well with several previ-ous studies [42 43] However not all enzymes will respondwell to the use of more acidic casting conditions Notablyenzymes have different pH profiles and some enzymesare inactive under acidic conditions Sol-gel entrappedcholinesterase for example showed better performance

Journal of Nanotechnology 9

in silica nanostructures prepared at pH values of 70 and 80and then 60 [44]

4 Conclusion

The enzyme CPO was successfully entrapped inside a silicananostructure prepared from the precursor TMOS with orwithout addition of the hydrophobic modifier MTMS SinceCPO is stabile in acidic buffers we further modified the sol-gel procedure by using casting buffers with pH values of 4555 60 and 65The catalytic performance of optimized CPOsol-gel beads approached 18 relative to free CPO in solutionas assessed via the pyrogallol peroxidation assay A combi-nation of factors such as enzyme leakage from the sol-gelhost insufficient recovery from inactivation caused by initialmethanol exposure hindered product release or alternatereaction pathways are most likely responsible for the declinein catalytic performance of CPOafter sol-gel entrapmentTheuse of more acidic casting buffers in the sol-gel procedureprovided themost leverage for optimization by yieldingmoreporous silica nanostructures Overall our findings are ofimportance for the optimization of other sol-gel materialsdevised for applications in biosensing or biocatalysis ordesigned for the controlled release of bioactive compounds

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Funding for this project was obtained from the ResearchCorporation for Science Advancement (Cottrell College Sci-ence Award to Monika Sommerhalter) and California StateUniversity East Bay (Faculty and Student Research Grantsto Monika Sommerhalter Selina Chan and Tuan Le and aSieber-Tombari award to Monika Sommerhalter) ProfessorDaryl Eggers San Jose State University kindly invited theauthors to perform the CD measurements in his laboratoryThe authors are also grateful to Professor AnnMcPartland forproviding detailed feedback on their paper

References

[1] B C Dave B Dunn J S Valentine and J I Zink ldquoNanocon-fined proteins and enzymes sol-gel-based biomolecular mate-rialsrdquoNanotechnology ACS Symposium Series vol 622 pp 351ndash365 1996

[2] I Gill and A Ballesteros ldquoBioencapsulation within syntheticpolymers (part 1) sol-gel encapsulated biologicalsrdquo Trends inBiotechnology vol 18 no 7 pp 282ndash296 2000

[3] D Avnir T Coradin O Lev and J Livage ldquoRecent bio-applications of sol-gel materialsrdquo Journal of Materials Chem-istry vol 16 no 11 pp 1013ndash1030 2006

[4] D R Morris and L P Hager ldquoChloroperoxidase I Isolationand properties of the crystalline glycoproteinrdquo The Journal ofBiological Chemistry vol 241 no 8 pp 1763ndash1768 1966

[5] V Yazbik and M Ansorge-Schumacher ldquoFast and efficientpurification of chloroperoxidase from C fumagordquo Process Bio-chemistry vol 45 no 2 pp 279ndash283 2010

[6] M Hofrichter and R Ullrich ldquoHeme-thiolate haloperoxidasesversatile biocatalysts with biotechnological and environmentalsignificancerdquo Applied Microbiology and Biotechnology vol 71no 3 pp 276ndash288 2006

[7] V M Dembitsky ldquoOxidation epoxidation and sulfoxidationreactions catalysed by haloperoxidasesrdquoTetrahedron vol 59 no26 pp 4701ndash4720 2003

[8] L Santhanam and J S Dordick ldquoChloroperoxidase catalyzedepoxidation of styrene in aqueous and non-aqueous mediardquoBiocatalysis and Biotransformation vol 20 no 4 pp 265ndash2742002

[9] M Ayala N R Robledo A Lopez-Munguia and R Vazquez-Duhalt ldquoSubstrate specificity and ionization potential inchloroperoxidase-catalyzed oxidation of diesel fuelrdquo Environ-mental Science and Technology vol 34 no 13 pp 2804ndash28092000

[10] R Vazquez-Duhalt M Ayala and F J Marquez-Rocha ldquoBio-catalytic chlorination of aromatic hydrocarbons by chloroper-oxidase of Caldariomyces fumagordquo Phytochemistry vol 58 no6 pp 929ndash933 2001

[11] E Terres M Montiel S Le Borgne and E Torres ldquoImmo-bilization of chloroperoxidase on mesoporous materials forthe oxidation of 46-dimethyldibenzothiophene a recalcitrantorganic sulfur compound present in petroleum fractionsrdquoBiotechnology Letters vol 30 no 1 pp 173ndash179 2008

[12] V Trevisan M Signoretto S Colonna V Pironti and GStrukul ldquoMicroencapsulated chloroperoxidase as a recyclablecatalyst for the enantioselective oxidation of sulfides withhydrogen peroxiderdquo Angewandte Chemie International Editionvol 43 no 31 pp 4097ndash4099 2004

[13] N Spreti R Germani A Incani and G Savelli ldquoStabiliza-tion of chloroperoxidase by polyethylene glycols in aqueousmedia kinetic studies and synthetic applicationsrdquoBiotechnologyProgress vol 20 no 1 pp 96ndash101 2004

[14] J-B Park and D S Clark ldquoNew reaction system for hydrocar-bon oxidation by chloroperoxidaserdquo Biotechnology and Bioengi-neering vol 94 no 1 pp 189ndash192 2006

[15] J-Z Liu and M Wang ldquoImprovement of activity and stabilityof chloroperoxidase by chemical modificationrdquo BMC Biotech-nology vol 7 no 1 article 23 2007

[16] L Zhi Y Jiang Y Wang M Hu S Li and Y Ma ldquoEffects ofadditives on the thermostability of chloroperoxidaserdquo Biotech-nology Progress vol 23 no 3 pp 729ndash733 2007

[17] T A Kadima andM A Pickard ldquoImmobilization of chloroper-oxidase on aminopropyl-glassrdquo Applied and EnvironmentalMicrobiology vol 56 no 11 pp 3473ndash3477 1990

[18] Y-J Han J T Watson G D Stucky and A Butler ldquoCatalyticactivity of mesoporous silicate-immobilized chloroperoxidaserdquoJournal ofMolecular Catalysis B Enzymatic vol 17 no 1 pp 1ndash82002

[19] J Aburto M Ayala I Bustos-Jaimes et al ldquoStability andcatalytic properties of chloroperoxidase immobilized on SBA-16mesoporousmaterialsrdquoMicroporous andMesoporousMaterialsvol 83 no 1ndash3 pp 193ndash200 2005

[20] M Hartmann and C Streb ldquoSelective oxidation of indole bychloroperoxidase immobilized on the mesoporous molecularsieve SBA-15rdquo Journal of Porous Materials vol 13 no 3-4 pp347ndash352 2006

10 Journal of Nanotechnology

[21] S Hudson J Cooney B K Hodnett and E Magner ldquoChlorop-eroxidase on periodic mesoporous organosilanes immobiliza-tion and reuserdquo Chemistry of Materials vol 19 no 8 pp 2049ndash2055 2007

[22] R A Sheldon ldquoEnzyme immobilization the quest for optimumperformancerdquo Advanced Synthesis amp Catalysis vol 349 no 8-9pp 1289ndash1307 2007

[23] K Smith N J Silvernail K R Rodgers T E Elgren M Castroand RM Parker ldquoSol-gel encapsulated horseradish peroxidasea catalytic material for peroxidationrdquo Journal of the AmericanChemical Society vol 124 no 16 pp 4247ndash4252 2002

[24] K M Manoj and L P Hager ldquoChloroperoxidase a janusenzymerdquo Biochemistry vol 47 no 9 pp 2997ndash3003 2008

[25] V M Samokyszyn and P R Ortiz de Montellano ldquoTopology ofthe chloroperoxidase active site regiospecificity of heme mod-ification by phenylhydrazine and sodium aziderdquo Biochemistryvol 30 no 50 pp 11646ndash11653 1991

[26] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[27] D K Eggers and J S Valentine ldquoMolecular confinementinfluences protein structure and enhances thermal proteinstabilityrdquo Protein Science vol 10 no 2 pp 250ndash261 2001

[28] J H Harreld T Ebina N Tsubo and G Stucky ldquoManipulationof pore size distributions in silica and ormosil gels dried underambient pressure conditionsrdquo Journal of Non-Crystalline Solidsvol 298 no 2-3 pp 241ndash251 2002

[29] K M Manoj A Baburaj B Ephraim et al ldquoExplaining theatypical reaction profiles of heme enzymes with a novel mech-anistic hypothesis and kinetic treatmentrdquo PLoS ONE vol 5 no5 Article ID e10601 2010

[30] D Jung and M Hartmann ldquoOxidation of indole with CPO andGOx immobilized on mesoporous molecular sievesrdquo CatalysisToday vol 157 no 1ndash4 pp 378ndash383 2010

[31] M L Ferrer F del Monte and D Levy ldquoA novel and simplealcohol-free sol-gel route for encapsulation of labile proteinsrdquoChemistry of Materials vol 14 no 9 pp 3619ndash3621 2002

[32] M Sundaramoorthy J Terner and T L Poulos ldquoThe crystalstructure of chloroperoxidase a heme peroxidase-cytochromeP450 functional hybridrdquo Structure vol 3 no 12 pp 1367ndash13771995

[33] X W Yi A Conesa P J Punt and L P Hager ldquoExamining therole of glutamic acid 183 in chloroperoxidase catalysisrdquo Journalof Biological Chemistry vol 278 no 16 pp 13855ndash13859 2003

[34] S R Blanke S AMartinis S G Sligar L P Hager J J Rux andJ H Dawson ldquoProbing the heme iron coordination structureof alkaline chloroperoxidaserdquo Biochemistry vol 35 no 46 pp14537ndash14543 1996

[35] B Dunn and J I Zink ldquoProbes of pore environment andmolecule-matrix interactions in sol-gel materialsrdquo Chemistry ofMaterials vol 9 no 11 pp 2280ndash2291 1997

[36] W A Loughlin and D B Hawkes ldquoEffect of organic solvents ona chloroperoxidase biotransformationrdquo Bioresource Technologyvol 71 no 2 pp 167ndash172 2000

[37] E Kiljunen and L T Kanerva ldquoChloroperoxidase-catalysedoxidation of alcohols to aldehydesrdquo Journal of Molecular Catal-ysis B Enzymatic vol 9 no 4ndash6 pp 163ndash172 2000

[38] E N Kadnikova and N M Kostic ldquoOxidation of ABTS byhydrogen peroxide catalyzed by horseradish peroxidase encap-sulated into sol-gel glass Effects of glass matrix on reactivityrdquo

Journal of Molecular Catalysis B Enzymatic vol 18 no 1ndash3 pp39ndash48 2002

[39] A Venkateswara Rao and D Haranath ldquoEffect of methyltri-methoxysilane as a synthesis component on the hydrophobicityand some physical properties of silica aerogelsrdquo Microporousand Mesoporous Materials vol 30 no 2-3 pp 267ndash273 1999

[40] M T Reetz A Zonta and J Simpelkamp ldquoEfficient immo-bilization of lipases by entrapment in hydrophobic sol-gelmaterialsrdquo Biotechnology and Bioengineering vol 49 no 5 pp527ndash534 1996

[41] W Wiesner K-H van Pee and F Lingens ldquoPurification andcharacterization of a novel bacterial non-heme chloroperox-idase from Pseudomonas pyrrociniardquo Journal of BiologicalChemistry vol 263 no 27 pp 13725ndash13732 1988

[42] Y Xi Z Liangying and W Sasa ldquoPore size and pore-sizedistribution control of porous silicardquo Sensors and Actuators BChemical vol 25 no 1-3 pp 347ndash352 1995

[43] C Lin and J A Ritter ldquoEffect of synthesis pH on the structureof carbon xerogelsrdquo Carbon vol 35 no 9 pp 1271ndash1278 1997

[44] M Altstein G Segev N Aharonson O Ben-Aziz A Tur-niansky and D Avnir ldquoSol-gel entrapped cholinesterases amicrotiter plate method for monitoring anti-cholinesterasecompoundsrdquo Journal of Agricultural and Food Chemistry vol46 no 8 pp 3318ndash3324 1998

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials

Page 4: Silica Sol-Gel Entrapment of the Enzyme Chloroperoxidase

4 Journal of Nanotechnology

0

500

1000

1500

0 20 40 60 80 100 120 140

Activ

ity o

f fre

e CPO

(IU

mg)

Pyrogallol (mM)

0

50

100

150

Activ

ity o

f sol

-gel

CPO

(IU

mg)

(a)

0 20 40 60 80 100H2O2 (mM)

2500

2000

Activ

ity o

f fre

e CPO

(IU

mg)

1500

1000

500

0 0

50

100

150

200

250

Activ

ity o

f sol

-gel

CPO

(IU

mg)

(b)

Figure 1 Peroxidation of pyrogallol catalyzed by free CPO (open squares 0575 120583g CPO in 3mL total assay volume) and sol-gel entrappedCPO (grey circles 0575120583g CPO entrapped in three sol-gel beads placed in 3mL total assay volume) The CPO sol-gel beads were preparedfrom TMOS with a pH 60 casting buffer and matured for one week with a buffer exchange on every other day The activity assays wereperformed in 01M citric acid-02M dibasic sodium phosphate buffer pH 42 with constant concentrations of either 27mM H

2O2(a) or

35mM pyrogallol (b) All data were measured in triplicate and are displayed as mean values plusmn one standard deviation The parameters of thecurve fits are summarized in Table 1

Table 1 Enzyme kinetic parameters for the CPO catalyzed peroxidation of pyrogallollowast

119870

119872(mM) 119870

119868(mM) Vmax (IUmg) 119896cat (1sec)

PyrogallolFree CPO (1198772 = 0983) 11 plusmn 10 160 plusmn 20 2000 plusmn 80 1400 plusmn 60Sol-gel entrapped CPO (1198772 = 0847) 93 plusmn 26 190 plusmn 74 100 plusmn 12 70 plusmn 84

H2O2

Free CPO (1198772 = 0921) 17 plusmn 62 64 plusmn 22 9100 plusmn 2500 6370 plusmn 1750Sol-gel entrapped CPO (1198772 = 0657) 27 plusmn 39 61 plusmn 86 680 plusmn 830 480 plusmn 580

lowastEnzyme kinetic parameters were obtained by fitting the data shown in Figure 1(a) (pyrogallol) and Figure 1(b) (H2O2) to a substrate inhibition model (see(1)) All parameters are presented as value of the fit plusmn one standard error of the fit

0

1

2

3

4

5

6

7

Leak

age (

)

Time1d1h 2d 3d 4d 6d

Figure 2Three sets of CPO sol-gel beads with CPO concentrationsof 40 120583gmL (dark grey bars) 8 120583gmL (grey bars) and 4 120583gmL(light grey bars) were aged for one week The storage buffer wasreplaced in the time intervals indicated on the graph and tested forCPO leakage Each set was replicated three times and containedthree beads per sample tube The specific activity of the referencesample with free CPO in solution was 1436plusmn39 IUmgThe columnheights represent the mean value and the error bars represent plusmn onestandard deviation

enzyme leakage As the sol-gel material matured and moreconnections formed within the silica mesh enzyme leakagedeclined

Table 2 contains the results of the activity measurementswith the CPO sol-gel beads and summarizes the cumulativeleakage over the first six days The activity measurementswere performed with CPO sol-gel beads that were seven daysold Each assay comprised 01M citric acid-02M dibasicsodium phosphate buffer pH 42 27mM H

2O2 and 35mM

pyrogallol The CPO sol-gel beads with the highest CPOloading of 40 120583g CPO per mL sol-gel yielded the highestabsolute activity values of 249 plusmn 33mIU A comparisonamong specific activity values reported per mg of initiallyloaded CPO however clearly showed that the CPO sol-gelbeads with a lower enzyme content of 8 or 4120583gmL perfor-med better At a higher loading of CPOmore CPOmoleculesmight be obstructed by either other enzyme molecules orthe silica nanostructure Also some of these CPO moleculesmight be entrapped in closed pores

Another set of three tubes each containing three beadsloaded with 4120583gmL CPO was aged for one week and testedfor reusability (see Figure 3) The CPO sol-gel beads can bereused up to three times in a convenient manner by replacingthe liquid phase composed of buffer and product moleculeswith new buffer and substrate However a further decline incatalytic performance was apparent We also noticed that allCPO sol-gel beads adopted the yellow-orange color of theproduct purpurogallin after the first use Gentle washing with

Journal of Nanotechnology 5

Table 2 Catalytic performance of sol-gel beads loaded with different CPO amounts

CPO loading of sol-gel beads (120583gmL) 40 8 4Activity (mIU)lowast 249 plusmn 33 138 plusmn 18 76 plusmn 4Specific activity (IUmg)lowast 41 plusmn 6 115 plusmn 15 127 plusmn 6Relative activity compared to free CPO () 29 80 880Cumulative leakage for six days ()dagger 15 12 7lowastAbsolute activity values in mIU (times10minus3 International Units) and specific activity values per mg initially loaded CPO are presented as mean values plusmn onestandard deviation All sample sets were prepared in triplicate The relative activity was based on a reference assay with free CPO in solution The referencevalue was 1436 plusmn 39 IUmg daggerThe cumulative leakage over six days of maturation corresponds to the summation of the leakage data shown in Figure 2

0

50

100

150

200

First use

Activ

ity (I

Um

g )

Time1d 3d 4d 5d 11d 12d

Figure 3 Reusability test for CPO sol-gel beads loaded with4 120583gmL CPOThree sets each with three beads per tube were usedin this test The column heights represent the mean specific activityvalue and the error bars representplusmn one standard deviationThe firstmeasurement was taken after the beads were aged for one week withdaily buffer exchanges The number of days that passed before thenext reuse is listed on the 119909-axis

the storage buffer diminished the coloration only slightlyThus clogging of the sol-gel nanostructure with productmolecules was one factor that hampered the reusability of theCPO sol-gel beads Furthermore the peroxidation reactionscatalyzed by CPO are prone to substrate inhibition (seeFigure 1) Trapped pyrogallol and H

2O2molecules could

therefore interfere with effective reuse Jung and Hartmanndemonstrated that in situ generation of hydrogen peroxidevia coimmobilization of glucose oxidase can improve thereusability of cross-linked CPO molecules entrapped inmesoporous molecular sieves [30]

The specific activity of three independently prepared setsof sol-gel beads all containing sim4 120583gmL CPO was different70 plusmn 15 IUmg (Figure 1) 127 plusmn 6 IUmg (Table 2) and171 plusmn 14 IUmg (Figure 3) The CPO sol-gel materials usedfor generating the data presented in Table 2 and Figure 3were prepared from the same CPO vial with a free CPOreference value of 1436 plusmn 39 IUmg The reference value forthe first CPO vial was only 1280 plusmn 80 IUmg However manyother experimental parameters such as the exact sol-gelcomposition the quality of all starting materials humiditytemperature and duration of sol-gel drying phase can alsoinfluence the properties of the final sol-gel material There-fore all CPO sol-gel beads that are compared within onetable or graph were prepared on the same day with the samereagent batches in parallel

33Modification of Sol-Gel Procedure with respect toMethanolRelease Next we tested whether minor modifications in thesol-gel procedure that influence the retention of methanolreleased from the sol-gel precursor TMOS would result inany significant changes On the same day three differentsets of CPO sol-gel beads all containing 4 120583gmL CPO wereprepared in triplicateThe first set was prepared with a proce-dure that facilitates methanol release by using an open vesselwhile sonicating the sol solution and performing daily bufferexchanges during the first week of sol-gel maturation Thesecond and third sets were prepared using a closed sonicationvessel and no buffer exchange was performed for the thirdset Despite these modifications all three sets prepared onthe same day yielded virtually identical specific activity valuesof 167 plusmn 18 IUmg 164 plusmn 10 IUmg and 178 plusmn 21 IUmgrespectively A more vigorous procedure presented by Ferreret al [31] involves rotavaporization of the sol solution prior toaddition of the buffered enzyme solution In our laboratorythis method was not successful as sol solidification started toset in too rapidly to achieve consistent gelation

Overall the catalytic performance of the best set of sol-gel beads was only 125plusmn15 in comparison to the referenceassay with free CPO in solution Possible reasons for thedecline in catalytic performance upon entrapment include (1)loss of enzyme due to leakage from the sol-gel matrix (2)damage to the enzyme caused by the entrapment procedureor (3) hindered substrate or product diffusion within the sol-gel nanostructure As mentioned above enzyme leakage didoccur but it can only account for a small loss of approximately10 In the experiments described below we tested for thetwo remaining possible reasons for the decline in catalyticperformance of the sol-gel entrappedCPOand also examinedthe effect of methanol on CPO

34 CD and UVVIS Spectroscopy with Sol-Gel EntrappedCPO To monitor possible enzyme damage we exploited thefact that sol-gels prepared from TMOS are transparent Cir-cular dichroism and visible absorbance spectra of free CPOin solution and CPO in sol-gel entrapped form are shown inFigures 4 and 5 respectively The spectra of free CPO andsol-gel entrapped CPO are virtually identical to each otherMinor changes in intensity of the spectroscopic signals aremost likely due to scattering effects from the sol-gel surfaceor the shrinkage of the sol-gel material during thematurationprocess Shrinkage slightly raises the concentration of thesample but also decreases the spectroscopic path length TheCD spectra are typical for a protein with high alpha helical

6 Journal of Nanotechnology

minus20

minus15

minus10

minus5

0

5

200 210 220 230 240 250 260Wavelength (nm)

CD si

gnal

(mde

g)

Figure 4 CD spectra of sol-gel entrapped CPO (black squares)and free CPO (white squares) The CPO concentration was 7120583Min the silica sol-gel sheet of sim1mm thickness The solution samplecontaining 7 120583M CPO in 2mM sodium phosphate buffer pH 70was measured in a cuvette of 1mm path length

0

01

02

03

04

05

300 400 500 600 700 800 900

Abso

rban

ce

Wavelength (nm)

Figure 5 Absorbance spectra of sol-gel entrapped CPO (solid line)and free CPO (dashed line) The silica sol-gel sheet was sim1mmthick and contained 16mgmL CPO The sheet was placed onthe wall of a 10mm wide plastic cuvette and immersed in 50mMpotassium phosphate buffer pH 60 The solution reference samplewas prepared from the same CPO stock via dilution to 016mgmL(4 120583M) with 50mM potassium phosphate buffer pH 60 in a 10mmthick plastic cuvette

content This finding agrees with the X-ray protein structureof CPO [32] and previously determined CD data [15 33]

The visible absorbance spectra shown in Figure 5 aretypical for the active form of CPO with a five-coordinateiron center in the heme chromophore [34] As the pH valueincreases above pH 70 CPO is inactivated the iron centerin the heme group becomes six-coordinate and the Soretband shifts to a longer wavelength [34] This spectroscopictransition can also be observed in CPO sol-gel sheets despitethe entrapment of the enzyme inside the silica matrixFigure 6 displays the absorption spectra of CPO sol-gelsheets immersed in 05M phosphate buffers at pH values of60 80 and 100 In comparison to solution spectra morealkaline conditions are necessary to achieve the Soret peakcharacteristic for sixfold coordinated heme iron centers It isconceivable that the silanol groups in the sol-gel impose anadditional buffering effect According toDunn and Zink [35]

0005

01015

02025

03035

04045

300 320 340 360 380 400 420 440 460 480 500

Abso

rban

ce

Wavelength (nm)

Figure 6 Absorbance spectra of sol-gel entrapped CPO immersedin 05M potassium phosphate buffer with pH values of 60 (solidline) 80 (dashed line) and 100 (dotted line)The silica sol-gel sheetsof 1mm thickness loaded with 16mgmL CPO were placed on thewall of plastic cuvette with a path length of 10mm

the pH can be up to one pH unit lower inside a sol-gel porethan in the surrounding aqueous buffer

The low apparent 119896cat values for sol-gel entrapped CPO(see Table 1) indicate that a significant number of entrappedCPO molecules were unable to catalyze the peroxidation ofpyrogallol in an effective manner CD and visible absorptionspectra however did not indicate any enzyme damage Itshould be noted that both spectroscopic methods can onlyaddress specific features of the enzymeThese features are theoverall secondary structure of the protein the electronic con-figuration of the active site chromophore and the ability torearrange the coordination sphere of the heme iron center inresponse to an external change in pH value

35 Influence of Methanol on the Spectroscopic Features andActivity of Free CPO Methanol is released from the sol-gelprecursor TMOS If we assume complete hydrolysis of TMOSand no evaporation of methanol the protein is exposed toa methanol concentration of 11 vv as the buffered enzymesolution and the sol are mixed and pipetted onto the parafilmfor gelation and drying The transfer into storage bufferand the subsequent exchange of buffer drastically lower themethanol content during the maturation phase of the sol-gelAfter the first buffer exchange the methanol concentration isonly 003 vv

To study the influence of methanol on the spectroscopicfeatures of CPO the enzyme was incubated with 11 vvmethanol andUVVIS andCD spectrawere recordedWedidnot detect a change in the spectroscopic features of the CDspectra after several hours of incubation (data not shown)but methanol exposure did have a small and immediate effecton UVVIS absorption spectra (see Figure 7)The absorptionmaximum of the Soret band shifted from 396 nm to 400 nmNotably this shift was reversible via dialysis The methanolremoval caused a 13-fold increase in sample volume Weadjusted the corresponding spectroscopic trace in Figure 7for this dilution effect

Journal of Nanotechnology 7

0

02

04

06

08

1

370 390 410 430 450

Abso

rban

ce

Wavelength (nm)

Figure 7 Absorption spectra of 9120583M CPO in 01M citrate-02Mphosphate buffer pH 42 (solid line) in 11 vv methanol and01M citrate-02M phosphate buffer pH 42 (dashed line) dialyzedsample (grey solid line) and dialyzed sample adjusted for 13-foldvolume increase (grey dotted line)

In agreement with previous studies [36] we observed adrastic decline in CPOrsquos catalytic performance after incuba-tion of CPO with organic solvents Compared to an aqueousreference sample without methanol only 57 residual activ-ity was detected after incubation with 11 vv methanol for2 hours After one day the residual activity still remainedat 57 We further discovered that the detrimental effect ofmethanol was reversible Up to 96 of the samplersquos initialactivity was recovered after dialysis Sample dilution alsoresulted in the enzymersquos recovery Decreasing the methanolcontent to 5 or 1 vv via sample dilution resulted in 90and 100 relative activity in comparison to identically dilutedsamples from the same CPO batch that were not exposed tomethanol Our finding that damage caused by methanol wasreversible in solution has implications for other studies on theuse of CPO in organic solvents or in biphasic solvent systems[36 37] Several CPO substrates which can be convertedinto products of industrial interest have high solubility innonpolar organic solvents [8]

Also if any initial damage caused by methanol exposurewas also reversible for sol-gel entrapped CPO immediatereduction in methanol content via evaporation of methanolfrom the sol solution or daily buffer exchanges during thegel maturation phase would not critically alter the finalperformance of the CPO sol-gel beads This might explainwhy the three different but parallel preparations outlinedin Section 33 resulted in virtually identical performance forthe three different CPO sol-gel bead sets On the otherhand manifestation of unrecoverable enzyme damage wouldexplain the low apparent 119896cat values determined for sol-gelentrapped CPO (see Table 1) It is conceivable that recoveryfrom damage caused by methanol exposure is less effectivefor CPO molecules entrapped within the silica sol-gel hostcompared to free CPO in solution

36 HinderedMaterial Transport in CPO Sol-Gel Beads Aftertheir first use all CPO sol-gel beads adopted a persistentyellow coloration indicating the entrapment of the productpurpurogallin Attractive intermolecular forces and physical

constraints both can delay the release of product moleculesfrom the silica nanostructure If product release becomesrate-limiting the apparent 119896cat value decreases as observedin the enzyme kinetic analysis Hindered substrate diffusionhowever was not supported by enzyme kinetic experimentsas the 119870

119872and 119870

119868values for the main substrate pyrogallol

were virtually identical for sol-gel entrapped and free CPO(see Table 1)We cannot explain why pyrogallol and purpuro-gallin would show different material transport propertiesinside the silica nanostructure Both molecules have similarfunctional groups and purpurogallin (MW 220 gmol) isonly somewhat larger than pyrogallol (MW 116 gmol) Analternative explanation would involve side-reactions formingalternate charged products or the trapping of reactive coloredintermediates

The alternative peroxidation substrate 221015840-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is particularlylarge (MW 515 gmol) and the product of the peroxidationreaction carries a positive charge [24] Deprotonated silanolgroups on the sol-gel surface can provide negative coun-tercharges The catalytic performance of CPO sol-gel beadsdropped from 126 plusmn 3 with the substrate pyrogallol to 9 plusmn04 with the substrate ABTS relative to the correspondingassay with free CPO in solution The CPO sol-gel beadsadopted the green color of the ABTS peroxidation productKadnikova and coworkers observed the formation of severalside products for the peroxidation reaction of ABTS byhorseradish peroxidase in a sol-gel matrix [38] The perox-idation reaction with ABTS and possibly other substratesincluding pyrogallol might therefore be more complex thanin solution

We further observed that preequilibration of CPO sol-gelbeadswith pyrogallol considerably improved catalytic perfor-mance For example preequilibration with 72mM pyrogallolfor 3 hours in comparison to using 35mM pyrogallol withoutpreequilibration increased the relative activity from 123 to196The strategy to preequilibrate enzyme sol-gel materialswith an excess of substrate before adding a cosubstrate wasalready successfully applied by Smith and coworkers [23] intheir study on sol-gel encapsulated horseradish peroxidaseBased on the data presented in Figure 1(a) however wewouldnot expect an increase in catalytic performance for sol-gelentrapped CPO as the pyrogallol concentration is raised from35 to 72mM In fact substrate inhibition should slightly lowerthe activity of CPOAdsorption of pyrogallolmolecules to thesol-gel surface or physical entrapment could further enhancethe local effective concentration of pyrogallol around theenzyme One key difference between the experiment leadingto Figure 1(a) and the preequilibration experiment is thetiming of adding the cosubstrate H

2O2 Manoj et al [29]

argue that substrate inhibition of CPO is not simply causedby blocking the active site of the enzymewith excess substratemolecules Instead they propose more complex substrateinhibition mechanisms that involve secondary conversionof an already formed product or competition by transientintermediates leading to alternate products Both substrateinhibition scenarios require the immediate presence of thecosubstrate H

2O2

8 Journal of Nanotechnology

Table 3 Properties of CPO sol-gel beads prepared with different casting buffers

pH of casting buffer 45 55 65BET surface area (m2g) 710 plusmn 40lowast 740 plusmn 60 470 plusmn 40Total pore volume (cm3g) 058 plusmn 002 060 plusmn 006 032 plusmn 004Average pore diameter (nm) 33 plusmn 03 32 plusmn 01 27 plusmn 01Activity (mIU) 251 plusmn 2 242 plusmn 29 161 plusmn 13Specific activity (IUmg) 157 plusmn 1 151 plusmn 18 100 plusmn 8Activity compared to free CPO () 18 17 11Cumulative leakage ()dagger 13 14 9lowastAll data are presented as mean values plusmn one standard deviation of triplicate data sets The relative activity was based on a reference assay with free CPO insolution yielding 887 plusmn 31 IUmg daggerThe cumulative leakage over ten days of maturation corresponds to the summation of the leakage data shown in Figure 8

37 Modification of Sol-Gel Procedure Using MTMS Tomodify the surface of the sol-gel material we incorporatedMTMS at molar ratios of 5 20 and 40 in the sol solutionThe addition of MTMS will introduce nonpolar methylgroups rendering the surface of the silica nanostructuremorehydrophobic [39] The casting buffer had a pH value of 60and the total CPO loading was 4 120583gmL The addition ofMTMS resulted in longer gelation times for example up to240 minutes for a molar ratio of 40 MTMS Unfortunatelythe beads prepared with MTMS were more brittle and fragilethan any of the other CPO sol-gel beads prepared in thisstudy The brittleness of the beads rendered their handlingmore challenging Regardless of the amount of incorporatedMTMS the activity was approximately 14 plusmn 1 comparedto a solution reference assay The cumulative leakage oftenexceeded 20We cannot rule out that the physical instabilityof the beads during and after a buffer exchange might havecontributed to higher apparent leakage and higher apparentactivity values In contrast to other enzymes notably lipasewhich showed interfacial activation and performed betterinsidemore hydrophobic nanostructures [40] the incorpora-tion of MTMS into the CPO sol-gel material did not improvecatalytic performance in a systematic manner

38 Modification of Sol-Gel Procedure Using More AcidicCasting Buffers The enzyme CPO is stable under acidicconditions [24] We exploited this CPO specific property andprepared CPO sol-gel beads using casting buffers with pHvalues of 45 55 and 65 All sample preparations were con-ducted in parallel with the same batches of CPO TMOS andbuffer reagents The gelation time increased with more acidiccasting buffers but the sol-gel beads remained easy to handleand transparentTheCPO loadingwas 4 120583gmL All CPO sol-gel preparations were divided into two portions One portionwas used for porosimetry studies and the other portion wasused for leakage and activity measurements (see Table 3)The properties of CPO sol-gel beads cast at pH 45 and 55are virtually identical but the CPO sol-gel beads cast at pH65 show significantly lower values in all categories Thisindicates a change in thematrix formation of the sol-gel as thecasting pHdrops to or below pH55 Overall the porosimetrydata is positively correlated with catalytic performance andunfortunately leakage All three porosimetric propertiesincluding larger average pore size BET surface area and pore

7

6

5

4

3

2

1

0

Leak

age (

)

1 2 3 4 5 6 7 8 9 10

Time (d)

Figure 8 The storage buffer of CPO sol-gel beads prepared withcasting buffers at pH 45 (dark grey bars) 55 (grey bars) and 65(light grey bars) was exchanged on a daily basis and monitored forCPO activity All samples were prepared in triplicate with eight CPOsol-gel beads per sample tube The bar height represents the meanvalue and the error bar plusmn one standard deviation

volume indicate reduced steric hindrance for material trans-port inside the sol-gel nanostructure As a consequencecatalytic performance increased Smaller average pore sizeson the other hand can aid in the retention of CPO

The dimensions of the protein CPO are 53 nm times 46 nmtimes 60 nm [19] The average pore diameters of approximately3 nm are only slightly smaller than the size of CPO Never-theless CPO remainedwell entrapped after completion of thesol-gel maturation phase Attractive electrostatic forces didnot most likely aid in the retention of CPO as the storagebuffer had a pH value of 42 which is close to the isoelectricpoint of CPO The isoelectric point of CPO from C fumagowas calculated to be approximately 40 [18 21] Isoelectricfocusing experiments on CPO from Pseudomonas pyrrociniayielded an isoelectric point of 41 [41] For all buffer condi-tions employed in our study the net charge on the surfaceof CPO is therefore either close to zero or negative

Our observation that more acidic casting buffers result ingreater porosimetry of sol-gels agrees well with several previ-ous studies [42 43] However not all enzymes will respondwell to the use of more acidic casting conditions Notablyenzymes have different pH profiles and some enzymesare inactive under acidic conditions Sol-gel entrappedcholinesterase for example showed better performance

Journal of Nanotechnology 9

in silica nanostructures prepared at pH values of 70 and 80and then 60 [44]

4 Conclusion

The enzyme CPO was successfully entrapped inside a silicananostructure prepared from the precursor TMOS with orwithout addition of the hydrophobic modifier MTMS SinceCPO is stabile in acidic buffers we further modified the sol-gel procedure by using casting buffers with pH values of 4555 60 and 65The catalytic performance of optimized CPOsol-gel beads approached 18 relative to free CPO in solutionas assessed via the pyrogallol peroxidation assay A combi-nation of factors such as enzyme leakage from the sol-gelhost insufficient recovery from inactivation caused by initialmethanol exposure hindered product release or alternatereaction pathways are most likely responsible for the declinein catalytic performance of CPOafter sol-gel entrapmentTheuse of more acidic casting buffers in the sol-gel procedureprovided themost leverage for optimization by yieldingmoreporous silica nanostructures Overall our findings are ofimportance for the optimization of other sol-gel materialsdevised for applications in biosensing or biocatalysis ordesigned for the controlled release of bioactive compounds

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Funding for this project was obtained from the ResearchCorporation for Science Advancement (Cottrell College Sci-ence Award to Monika Sommerhalter) and California StateUniversity East Bay (Faculty and Student Research Grantsto Monika Sommerhalter Selina Chan and Tuan Le and aSieber-Tombari award to Monika Sommerhalter) ProfessorDaryl Eggers San Jose State University kindly invited theauthors to perform the CD measurements in his laboratoryThe authors are also grateful to Professor AnnMcPartland forproviding detailed feedback on their paper

References

[1] B C Dave B Dunn J S Valentine and J I Zink ldquoNanocon-fined proteins and enzymes sol-gel-based biomolecular mate-rialsrdquoNanotechnology ACS Symposium Series vol 622 pp 351ndash365 1996

[2] I Gill and A Ballesteros ldquoBioencapsulation within syntheticpolymers (part 1) sol-gel encapsulated biologicalsrdquo Trends inBiotechnology vol 18 no 7 pp 282ndash296 2000

[3] D Avnir T Coradin O Lev and J Livage ldquoRecent bio-applications of sol-gel materialsrdquo Journal of Materials Chem-istry vol 16 no 11 pp 1013ndash1030 2006

[4] D R Morris and L P Hager ldquoChloroperoxidase I Isolationand properties of the crystalline glycoproteinrdquo The Journal ofBiological Chemistry vol 241 no 8 pp 1763ndash1768 1966

[5] V Yazbik and M Ansorge-Schumacher ldquoFast and efficientpurification of chloroperoxidase from C fumagordquo Process Bio-chemistry vol 45 no 2 pp 279ndash283 2010

[6] M Hofrichter and R Ullrich ldquoHeme-thiolate haloperoxidasesversatile biocatalysts with biotechnological and environmentalsignificancerdquo Applied Microbiology and Biotechnology vol 71no 3 pp 276ndash288 2006

[7] V M Dembitsky ldquoOxidation epoxidation and sulfoxidationreactions catalysed by haloperoxidasesrdquoTetrahedron vol 59 no26 pp 4701ndash4720 2003

[8] L Santhanam and J S Dordick ldquoChloroperoxidase catalyzedepoxidation of styrene in aqueous and non-aqueous mediardquoBiocatalysis and Biotransformation vol 20 no 4 pp 265ndash2742002

[9] M Ayala N R Robledo A Lopez-Munguia and R Vazquez-Duhalt ldquoSubstrate specificity and ionization potential inchloroperoxidase-catalyzed oxidation of diesel fuelrdquo Environ-mental Science and Technology vol 34 no 13 pp 2804ndash28092000

[10] R Vazquez-Duhalt M Ayala and F J Marquez-Rocha ldquoBio-catalytic chlorination of aromatic hydrocarbons by chloroper-oxidase of Caldariomyces fumagordquo Phytochemistry vol 58 no6 pp 929ndash933 2001

[11] E Terres M Montiel S Le Borgne and E Torres ldquoImmo-bilization of chloroperoxidase on mesoporous materials forthe oxidation of 46-dimethyldibenzothiophene a recalcitrantorganic sulfur compound present in petroleum fractionsrdquoBiotechnology Letters vol 30 no 1 pp 173ndash179 2008

[12] V Trevisan M Signoretto S Colonna V Pironti and GStrukul ldquoMicroencapsulated chloroperoxidase as a recyclablecatalyst for the enantioselective oxidation of sulfides withhydrogen peroxiderdquo Angewandte Chemie International Editionvol 43 no 31 pp 4097ndash4099 2004

[13] N Spreti R Germani A Incani and G Savelli ldquoStabiliza-tion of chloroperoxidase by polyethylene glycols in aqueousmedia kinetic studies and synthetic applicationsrdquoBiotechnologyProgress vol 20 no 1 pp 96ndash101 2004

[14] J-B Park and D S Clark ldquoNew reaction system for hydrocar-bon oxidation by chloroperoxidaserdquo Biotechnology and Bioengi-neering vol 94 no 1 pp 189ndash192 2006

[15] J-Z Liu and M Wang ldquoImprovement of activity and stabilityof chloroperoxidase by chemical modificationrdquo BMC Biotech-nology vol 7 no 1 article 23 2007

[16] L Zhi Y Jiang Y Wang M Hu S Li and Y Ma ldquoEffects ofadditives on the thermostability of chloroperoxidaserdquo Biotech-nology Progress vol 23 no 3 pp 729ndash733 2007

[17] T A Kadima andM A Pickard ldquoImmobilization of chloroper-oxidase on aminopropyl-glassrdquo Applied and EnvironmentalMicrobiology vol 56 no 11 pp 3473ndash3477 1990

[18] Y-J Han J T Watson G D Stucky and A Butler ldquoCatalyticactivity of mesoporous silicate-immobilized chloroperoxidaserdquoJournal ofMolecular Catalysis B Enzymatic vol 17 no 1 pp 1ndash82002

[19] J Aburto M Ayala I Bustos-Jaimes et al ldquoStability andcatalytic properties of chloroperoxidase immobilized on SBA-16mesoporousmaterialsrdquoMicroporous andMesoporousMaterialsvol 83 no 1ndash3 pp 193ndash200 2005

[20] M Hartmann and C Streb ldquoSelective oxidation of indole bychloroperoxidase immobilized on the mesoporous molecularsieve SBA-15rdquo Journal of Porous Materials vol 13 no 3-4 pp347ndash352 2006

10 Journal of Nanotechnology

[21] S Hudson J Cooney B K Hodnett and E Magner ldquoChlorop-eroxidase on periodic mesoporous organosilanes immobiliza-tion and reuserdquo Chemistry of Materials vol 19 no 8 pp 2049ndash2055 2007

[22] R A Sheldon ldquoEnzyme immobilization the quest for optimumperformancerdquo Advanced Synthesis amp Catalysis vol 349 no 8-9pp 1289ndash1307 2007

[23] K Smith N J Silvernail K R Rodgers T E Elgren M Castroand RM Parker ldquoSol-gel encapsulated horseradish peroxidasea catalytic material for peroxidationrdquo Journal of the AmericanChemical Society vol 124 no 16 pp 4247ndash4252 2002

[24] K M Manoj and L P Hager ldquoChloroperoxidase a janusenzymerdquo Biochemistry vol 47 no 9 pp 2997ndash3003 2008

[25] V M Samokyszyn and P R Ortiz de Montellano ldquoTopology ofthe chloroperoxidase active site regiospecificity of heme mod-ification by phenylhydrazine and sodium aziderdquo Biochemistryvol 30 no 50 pp 11646ndash11653 1991

[26] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[27] D K Eggers and J S Valentine ldquoMolecular confinementinfluences protein structure and enhances thermal proteinstabilityrdquo Protein Science vol 10 no 2 pp 250ndash261 2001

[28] J H Harreld T Ebina N Tsubo and G Stucky ldquoManipulationof pore size distributions in silica and ormosil gels dried underambient pressure conditionsrdquo Journal of Non-Crystalline Solidsvol 298 no 2-3 pp 241ndash251 2002

[29] K M Manoj A Baburaj B Ephraim et al ldquoExplaining theatypical reaction profiles of heme enzymes with a novel mech-anistic hypothesis and kinetic treatmentrdquo PLoS ONE vol 5 no5 Article ID e10601 2010

[30] D Jung and M Hartmann ldquoOxidation of indole with CPO andGOx immobilized on mesoporous molecular sievesrdquo CatalysisToday vol 157 no 1ndash4 pp 378ndash383 2010

[31] M L Ferrer F del Monte and D Levy ldquoA novel and simplealcohol-free sol-gel route for encapsulation of labile proteinsrdquoChemistry of Materials vol 14 no 9 pp 3619ndash3621 2002

[32] M Sundaramoorthy J Terner and T L Poulos ldquoThe crystalstructure of chloroperoxidase a heme peroxidase-cytochromeP450 functional hybridrdquo Structure vol 3 no 12 pp 1367ndash13771995

[33] X W Yi A Conesa P J Punt and L P Hager ldquoExamining therole of glutamic acid 183 in chloroperoxidase catalysisrdquo Journalof Biological Chemistry vol 278 no 16 pp 13855ndash13859 2003

[34] S R Blanke S AMartinis S G Sligar L P Hager J J Rux andJ H Dawson ldquoProbing the heme iron coordination structureof alkaline chloroperoxidaserdquo Biochemistry vol 35 no 46 pp14537ndash14543 1996

[35] B Dunn and J I Zink ldquoProbes of pore environment andmolecule-matrix interactions in sol-gel materialsrdquo Chemistry ofMaterials vol 9 no 11 pp 2280ndash2291 1997

[36] W A Loughlin and D B Hawkes ldquoEffect of organic solvents ona chloroperoxidase biotransformationrdquo Bioresource Technologyvol 71 no 2 pp 167ndash172 2000

[37] E Kiljunen and L T Kanerva ldquoChloroperoxidase-catalysedoxidation of alcohols to aldehydesrdquo Journal of Molecular Catal-ysis B Enzymatic vol 9 no 4ndash6 pp 163ndash172 2000

[38] E N Kadnikova and N M Kostic ldquoOxidation of ABTS byhydrogen peroxide catalyzed by horseradish peroxidase encap-sulated into sol-gel glass Effects of glass matrix on reactivityrdquo

Journal of Molecular Catalysis B Enzymatic vol 18 no 1ndash3 pp39ndash48 2002

[39] A Venkateswara Rao and D Haranath ldquoEffect of methyltri-methoxysilane as a synthesis component on the hydrophobicityand some physical properties of silica aerogelsrdquo Microporousand Mesoporous Materials vol 30 no 2-3 pp 267ndash273 1999

[40] M T Reetz A Zonta and J Simpelkamp ldquoEfficient immo-bilization of lipases by entrapment in hydrophobic sol-gelmaterialsrdquo Biotechnology and Bioengineering vol 49 no 5 pp527ndash534 1996

[41] W Wiesner K-H van Pee and F Lingens ldquoPurification andcharacterization of a novel bacterial non-heme chloroperox-idase from Pseudomonas pyrrociniardquo Journal of BiologicalChemistry vol 263 no 27 pp 13725ndash13732 1988

[42] Y Xi Z Liangying and W Sasa ldquoPore size and pore-sizedistribution control of porous silicardquo Sensors and Actuators BChemical vol 25 no 1-3 pp 347ndash352 1995

[43] C Lin and J A Ritter ldquoEffect of synthesis pH on the structureof carbon xerogelsrdquo Carbon vol 35 no 9 pp 1271ndash1278 1997

[44] M Altstein G Segev N Aharonson O Ben-Aziz A Tur-niansky and D Avnir ldquoSol-gel entrapped cholinesterases amicrotiter plate method for monitoring anti-cholinesterasecompoundsrdquo Journal of Agricultural and Food Chemistry vol46 no 8 pp 3318ndash3324 1998

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials

Page 5: Silica Sol-Gel Entrapment of the Enzyme Chloroperoxidase

Journal of Nanotechnology 5

Table 2 Catalytic performance of sol-gel beads loaded with different CPO amounts

CPO loading of sol-gel beads (120583gmL) 40 8 4Activity (mIU)lowast 249 plusmn 33 138 plusmn 18 76 plusmn 4Specific activity (IUmg)lowast 41 plusmn 6 115 plusmn 15 127 plusmn 6Relative activity compared to free CPO () 29 80 880Cumulative leakage for six days ()dagger 15 12 7lowastAbsolute activity values in mIU (times10minus3 International Units) and specific activity values per mg initially loaded CPO are presented as mean values plusmn onestandard deviation All sample sets were prepared in triplicate The relative activity was based on a reference assay with free CPO in solution The referencevalue was 1436 plusmn 39 IUmg daggerThe cumulative leakage over six days of maturation corresponds to the summation of the leakage data shown in Figure 2

0

50

100

150

200

First use

Activ

ity (I

Um

g )

Time1d 3d 4d 5d 11d 12d

Figure 3 Reusability test for CPO sol-gel beads loaded with4 120583gmL CPOThree sets each with three beads per tube were usedin this test The column heights represent the mean specific activityvalue and the error bars representplusmn one standard deviationThe firstmeasurement was taken after the beads were aged for one week withdaily buffer exchanges The number of days that passed before thenext reuse is listed on the 119909-axis

the storage buffer diminished the coloration only slightlyThus clogging of the sol-gel nanostructure with productmolecules was one factor that hampered the reusability of theCPO sol-gel beads Furthermore the peroxidation reactionscatalyzed by CPO are prone to substrate inhibition (seeFigure 1) Trapped pyrogallol and H

2O2molecules could

therefore interfere with effective reuse Jung and Hartmanndemonstrated that in situ generation of hydrogen peroxidevia coimmobilization of glucose oxidase can improve thereusability of cross-linked CPO molecules entrapped inmesoporous molecular sieves [30]

The specific activity of three independently prepared setsof sol-gel beads all containing sim4 120583gmL CPO was different70 plusmn 15 IUmg (Figure 1) 127 plusmn 6 IUmg (Table 2) and171 plusmn 14 IUmg (Figure 3) The CPO sol-gel materials usedfor generating the data presented in Table 2 and Figure 3were prepared from the same CPO vial with a free CPOreference value of 1436 plusmn 39 IUmg The reference value forthe first CPO vial was only 1280 plusmn 80 IUmg However manyother experimental parameters such as the exact sol-gelcomposition the quality of all starting materials humiditytemperature and duration of sol-gel drying phase can alsoinfluence the properties of the final sol-gel material There-fore all CPO sol-gel beads that are compared within onetable or graph were prepared on the same day with the samereagent batches in parallel

33Modification of Sol-Gel Procedure with respect toMethanolRelease Next we tested whether minor modifications in thesol-gel procedure that influence the retention of methanolreleased from the sol-gel precursor TMOS would result inany significant changes On the same day three differentsets of CPO sol-gel beads all containing 4 120583gmL CPO wereprepared in triplicateThe first set was prepared with a proce-dure that facilitates methanol release by using an open vesselwhile sonicating the sol solution and performing daily bufferexchanges during the first week of sol-gel maturation Thesecond and third sets were prepared using a closed sonicationvessel and no buffer exchange was performed for the thirdset Despite these modifications all three sets prepared onthe same day yielded virtually identical specific activity valuesof 167 plusmn 18 IUmg 164 plusmn 10 IUmg and 178 plusmn 21 IUmgrespectively A more vigorous procedure presented by Ferreret al [31] involves rotavaporization of the sol solution prior toaddition of the buffered enzyme solution In our laboratorythis method was not successful as sol solidification started toset in too rapidly to achieve consistent gelation

Overall the catalytic performance of the best set of sol-gel beads was only 125plusmn15 in comparison to the referenceassay with free CPO in solution Possible reasons for thedecline in catalytic performance upon entrapment include (1)loss of enzyme due to leakage from the sol-gel matrix (2)damage to the enzyme caused by the entrapment procedureor (3) hindered substrate or product diffusion within the sol-gel nanostructure As mentioned above enzyme leakage didoccur but it can only account for a small loss of approximately10 In the experiments described below we tested for thetwo remaining possible reasons for the decline in catalyticperformance of the sol-gel entrappedCPOand also examinedthe effect of methanol on CPO

34 CD and UVVIS Spectroscopy with Sol-Gel EntrappedCPO To monitor possible enzyme damage we exploited thefact that sol-gels prepared from TMOS are transparent Cir-cular dichroism and visible absorbance spectra of free CPOin solution and CPO in sol-gel entrapped form are shown inFigures 4 and 5 respectively The spectra of free CPO andsol-gel entrapped CPO are virtually identical to each otherMinor changes in intensity of the spectroscopic signals aremost likely due to scattering effects from the sol-gel surfaceor the shrinkage of the sol-gel material during thematurationprocess Shrinkage slightly raises the concentration of thesample but also decreases the spectroscopic path length TheCD spectra are typical for a protein with high alpha helical

6 Journal of Nanotechnology

minus20

minus15

minus10

minus5

0

5

200 210 220 230 240 250 260Wavelength (nm)

CD si

gnal

(mde

g)

Figure 4 CD spectra of sol-gel entrapped CPO (black squares)and free CPO (white squares) The CPO concentration was 7120583Min the silica sol-gel sheet of sim1mm thickness The solution samplecontaining 7 120583M CPO in 2mM sodium phosphate buffer pH 70was measured in a cuvette of 1mm path length

0

01

02

03

04

05

300 400 500 600 700 800 900

Abso

rban

ce

Wavelength (nm)

Figure 5 Absorbance spectra of sol-gel entrapped CPO (solid line)and free CPO (dashed line) The silica sol-gel sheet was sim1mmthick and contained 16mgmL CPO The sheet was placed onthe wall of a 10mm wide plastic cuvette and immersed in 50mMpotassium phosphate buffer pH 60 The solution reference samplewas prepared from the same CPO stock via dilution to 016mgmL(4 120583M) with 50mM potassium phosphate buffer pH 60 in a 10mmthick plastic cuvette

content This finding agrees with the X-ray protein structureof CPO [32] and previously determined CD data [15 33]

The visible absorbance spectra shown in Figure 5 aretypical for the active form of CPO with a five-coordinateiron center in the heme chromophore [34] As the pH valueincreases above pH 70 CPO is inactivated the iron centerin the heme group becomes six-coordinate and the Soretband shifts to a longer wavelength [34] This spectroscopictransition can also be observed in CPO sol-gel sheets despitethe entrapment of the enzyme inside the silica matrixFigure 6 displays the absorption spectra of CPO sol-gelsheets immersed in 05M phosphate buffers at pH values of60 80 and 100 In comparison to solution spectra morealkaline conditions are necessary to achieve the Soret peakcharacteristic for sixfold coordinated heme iron centers It isconceivable that the silanol groups in the sol-gel impose anadditional buffering effect According toDunn and Zink [35]

0005

01015

02025

03035

04045

300 320 340 360 380 400 420 440 460 480 500

Abso

rban

ce

Wavelength (nm)

Figure 6 Absorbance spectra of sol-gel entrapped CPO immersedin 05M potassium phosphate buffer with pH values of 60 (solidline) 80 (dashed line) and 100 (dotted line)The silica sol-gel sheetsof 1mm thickness loaded with 16mgmL CPO were placed on thewall of plastic cuvette with a path length of 10mm

the pH can be up to one pH unit lower inside a sol-gel porethan in the surrounding aqueous buffer

The low apparent 119896cat values for sol-gel entrapped CPO(see Table 1) indicate that a significant number of entrappedCPO molecules were unable to catalyze the peroxidation ofpyrogallol in an effective manner CD and visible absorptionspectra however did not indicate any enzyme damage Itshould be noted that both spectroscopic methods can onlyaddress specific features of the enzymeThese features are theoverall secondary structure of the protein the electronic con-figuration of the active site chromophore and the ability torearrange the coordination sphere of the heme iron center inresponse to an external change in pH value

35 Influence of Methanol on the Spectroscopic Features andActivity of Free CPO Methanol is released from the sol-gelprecursor TMOS If we assume complete hydrolysis of TMOSand no evaporation of methanol the protein is exposed toa methanol concentration of 11 vv as the buffered enzymesolution and the sol are mixed and pipetted onto the parafilmfor gelation and drying The transfer into storage bufferand the subsequent exchange of buffer drastically lower themethanol content during the maturation phase of the sol-gelAfter the first buffer exchange the methanol concentration isonly 003 vv

To study the influence of methanol on the spectroscopicfeatures of CPO the enzyme was incubated with 11 vvmethanol andUVVIS andCD spectrawere recordedWedidnot detect a change in the spectroscopic features of the CDspectra after several hours of incubation (data not shown)but methanol exposure did have a small and immediate effecton UVVIS absorption spectra (see Figure 7)The absorptionmaximum of the Soret band shifted from 396 nm to 400 nmNotably this shift was reversible via dialysis The methanolremoval caused a 13-fold increase in sample volume Weadjusted the corresponding spectroscopic trace in Figure 7for this dilution effect

Journal of Nanotechnology 7

0

02

04

06

08

1

370 390 410 430 450

Abso

rban

ce

Wavelength (nm)

Figure 7 Absorption spectra of 9120583M CPO in 01M citrate-02Mphosphate buffer pH 42 (solid line) in 11 vv methanol and01M citrate-02M phosphate buffer pH 42 (dashed line) dialyzedsample (grey solid line) and dialyzed sample adjusted for 13-foldvolume increase (grey dotted line)

In agreement with previous studies [36] we observed adrastic decline in CPOrsquos catalytic performance after incuba-tion of CPO with organic solvents Compared to an aqueousreference sample without methanol only 57 residual activ-ity was detected after incubation with 11 vv methanol for2 hours After one day the residual activity still remainedat 57 We further discovered that the detrimental effect ofmethanol was reversible Up to 96 of the samplersquos initialactivity was recovered after dialysis Sample dilution alsoresulted in the enzymersquos recovery Decreasing the methanolcontent to 5 or 1 vv via sample dilution resulted in 90and 100 relative activity in comparison to identically dilutedsamples from the same CPO batch that were not exposed tomethanol Our finding that damage caused by methanol wasreversible in solution has implications for other studies on theuse of CPO in organic solvents or in biphasic solvent systems[36 37] Several CPO substrates which can be convertedinto products of industrial interest have high solubility innonpolar organic solvents [8]

Also if any initial damage caused by methanol exposurewas also reversible for sol-gel entrapped CPO immediatereduction in methanol content via evaporation of methanolfrom the sol solution or daily buffer exchanges during thegel maturation phase would not critically alter the finalperformance of the CPO sol-gel beads This might explainwhy the three different but parallel preparations outlinedin Section 33 resulted in virtually identical performance forthe three different CPO sol-gel bead sets On the otherhand manifestation of unrecoverable enzyme damage wouldexplain the low apparent 119896cat values determined for sol-gelentrapped CPO (see Table 1) It is conceivable that recoveryfrom damage caused by methanol exposure is less effectivefor CPO molecules entrapped within the silica sol-gel hostcompared to free CPO in solution

36 HinderedMaterial Transport in CPO Sol-Gel Beads Aftertheir first use all CPO sol-gel beads adopted a persistentyellow coloration indicating the entrapment of the productpurpurogallin Attractive intermolecular forces and physical

constraints both can delay the release of product moleculesfrom the silica nanostructure If product release becomesrate-limiting the apparent 119896cat value decreases as observedin the enzyme kinetic analysis Hindered substrate diffusionhowever was not supported by enzyme kinetic experimentsas the 119870

119872and 119870

119868values for the main substrate pyrogallol

were virtually identical for sol-gel entrapped and free CPO(see Table 1)We cannot explain why pyrogallol and purpuro-gallin would show different material transport propertiesinside the silica nanostructure Both molecules have similarfunctional groups and purpurogallin (MW 220 gmol) isonly somewhat larger than pyrogallol (MW 116 gmol) Analternative explanation would involve side-reactions formingalternate charged products or the trapping of reactive coloredintermediates

The alternative peroxidation substrate 221015840-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is particularlylarge (MW 515 gmol) and the product of the peroxidationreaction carries a positive charge [24] Deprotonated silanolgroups on the sol-gel surface can provide negative coun-tercharges The catalytic performance of CPO sol-gel beadsdropped from 126 plusmn 3 with the substrate pyrogallol to 9 plusmn04 with the substrate ABTS relative to the correspondingassay with free CPO in solution The CPO sol-gel beadsadopted the green color of the ABTS peroxidation productKadnikova and coworkers observed the formation of severalside products for the peroxidation reaction of ABTS byhorseradish peroxidase in a sol-gel matrix [38] The perox-idation reaction with ABTS and possibly other substratesincluding pyrogallol might therefore be more complex thanin solution

We further observed that preequilibration of CPO sol-gelbeadswith pyrogallol considerably improved catalytic perfor-mance For example preequilibration with 72mM pyrogallolfor 3 hours in comparison to using 35mM pyrogallol withoutpreequilibration increased the relative activity from 123 to196The strategy to preequilibrate enzyme sol-gel materialswith an excess of substrate before adding a cosubstrate wasalready successfully applied by Smith and coworkers [23] intheir study on sol-gel encapsulated horseradish peroxidaseBased on the data presented in Figure 1(a) however wewouldnot expect an increase in catalytic performance for sol-gelentrapped CPO as the pyrogallol concentration is raised from35 to 72mM In fact substrate inhibition should slightly lowerthe activity of CPOAdsorption of pyrogallolmolecules to thesol-gel surface or physical entrapment could further enhancethe local effective concentration of pyrogallol around theenzyme One key difference between the experiment leadingto Figure 1(a) and the preequilibration experiment is thetiming of adding the cosubstrate H

2O2 Manoj et al [29]

argue that substrate inhibition of CPO is not simply causedby blocking the active site of the enzymewith excess substratemolecules Instead they propose more complex substrateinhibition mechanisms that involve secondary conversionof an already formed product or competition by transientintermediates leading to alternate products Both substrateinhibition scenarios require the immediate presence of thecosubstrate H

2O2

8 Journal of Nanotechnology

Table 3 Properties of CPO sol-gel beads prepared with different casting buffers

pH of casting buffer 45 55 65BET surface area (m2g) 710 plusmn 40lowast 740 plusmn 60 470 plusmn 40Total pore volume (cm3g) 058 plusmn 002 060 plusmn 006 032 plusmn 004Average pore diameter (nm) 33 plusmn 03 32 plusmn 01 27 plusmn 01Activity (mIU) 251 plusmn 2 242 plusmn 29 161 plusmn 13Specific activity (IUmg) 157 plusmn 1 151 plusmn 18 100 plusmn 8Activity compared to free CPO () 18 17 11Cumulative leakage ()dagger 13 14 9lowastAll data are presented as mean values plusmn one standard deviation of triplicate data sets The relative activity was based on a reference assay with free CPO insolution yielding 887 plusmn 31 IUmg daggerThe cumulative leakage over ten days of maturation corresponds to the summation of the leakage data shown in Figure 8

37 Modification of Sol-Gel Procedure Using MTMS Tomodify the surface of the sol-gel material we incorporatedMTMS at molar ratios of 5 20 and 40 in the sol solutionThe addition of MTMS will introduce nonpolar methylgroups rendering the surface of the silica nanostructuremorehydrophobic [39] The casting buffer had a pH value of 60and the total CPO loading was 4 120583gmL The addition ofMTMS resulted in longer gelation times for example up to240 minutes for a molar ratio of 40 MTMS Unfortunatelythe beads prepared with MTMS were more brittle and fragilethan any of the other CPO sol-gel beads prepared in thisstudy The brittleness of the beads rendered their handlingmore challenging Regardless of the amount of incorporatedMTMS the activity was approximately 14 plusmn 1 comparedto a solution reference assay The cumulative leakage oftenexceeded 20We cannot rule out that the physical instabilityof the beads during and after a buffer exchange might havecontributed to higher apparent leakage and higher apparentactivity values In contrast to other enzymes notably lipasewhich showed interfacial activation and performed betterinsidemore hydrophobic nanostructures [40] the incorpora-tion of MTMS into the CPO sol-gel material did not improvecatalytic performance in a systematic manner

38 Modification of Sol-Gel Procedure Using More AcidicCasting Buffers The enzyme CPO is stable under acidicconditions [24] We exploited this CPO specific property andprepared CPO sol-gel beads using casting buffers with pHvalues of 45 55 and 65 All sample preparations were con-ducted in parallel with the same batches of CPO TMOS andbuffer reagents The gelation time increased with more acidiccasting buffers but the sol-gel beads remained easy to handleand transparentTheCPO loadingwas 4 120583gmL All CPO sol-gel preparations were divided into two portions One portionwas used for porosimetry studies and the other portion wasused for leakage and activity measurements (see Table 3)The properties of CPO sol-gel beads cast at pH 45 and 55are virtually identical but the CPO sol-gel beads cast at pH65 show significantly lower values in all categories Thisindicates a change in thematrix formation of the sol-gel as thecasting pHdrops to or below pH55 Overall the porosimetrydata is positively correlated with catalytic performance andunfortunately leakage All three porosimetric propertiesincluding larger average pore size BET surface area and pore

7

6

5

4

3

2

1

0

Leak

age (

)

1 2 3 4 5 6 7 8 9 10

Time (d)

Figure 8 The storage buffer of CPO sol-gel beads prepared withcasting buffers at pH 45 (dark grey bars) 55 (grey bars) and 65(light grey bars) was exchanged on a daily basis and monitored forCPO activity All samples were prepared in triplicate with eight CPOsol-gel beads per sample tube The bar height represents the meanvalue and the error bar plusmn one standard deviation

volume indicate reduced steric hindrance for material trans-port inside the sol-gel nanostructure As a consequencecatalytic performance increased Smaller average pore sizeson the other hand can aid in the retention of CPO

The dimensions of the protein CPO are 53 nm times 46 nmtimes 60 nm [19] The average pore diameters of approximately3 nm are only slightly smaller than the size of CPO Never-theless CPO remainedwell entrapped after completion of thesol-gel maturation phase Attractive electrostatic forces didnot most likely aid in the retention of CPO as the storagebuffer had a pH value of 42 which is close to the isoelectricpoint of CPO The isoelectric point of CPO from C fumagowas calculated to be approximately 40 [18 21] Isoelectricfocusing experiments on CPO from Pseudomonas pyrrociniayielded an isoelectric point of 41 [41] For all buffer condi-tions employed in our study the net charge on the surfaceof CPO is therefore either close to zero or negative

Our observation that more acidic casting buffers result ingreater porosimetry of sol-gels agrees well with several previ-ous studies [42 43] However not all enzymes will respondwell to the use of more acidic casting conditions Notablyenzymes have different pH profiles and some enzymesare inactive under acidic conditions Sol-gel entrappedcholinesterase for example showed better performance

Journal of Nanotechnology 9

in silica nanostructures prepared at pH values of 70 and 80and then 60 [44]

4 Conclusion

The enzyme CPO was successfully entrapped inside a silicananostructure prepared from the precursor TMOS with orwithout addition of the hydrophobic modifier MTMS SinceCPO is stabile in acidic buffers we further modified the sol-gel procedure by using casting buffers with pH values of 4555 60 and 65The catalytic performance of optimized CPOsol-gel beads approached 18 relative to free CPO in solutionas assessed via the pyrogallol peroxidation assay A combi-nation of factors such as enzyme leakage from the sol-gelhost insufficient recovery from inactivation caused by initialmethanol exposure hindered product release or alternatereaction pathways are most likely responsible for the declinein catalytic performance of CPOafter sol-gel entrapmentTheuse of more acidic casting buffers in the sol-gel procedureprovided themost leverage for optimization by yieldingmoreporous silica nanostructures Overall our findings are ofimportance for the optimization of other sol-gel materialsdevised for applications in biosensing or biocatalysis ordesigned for the controlled release of bioactive compounds

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Funding for this project was obtained from the ResearchCorporation for Science Advancement (Cottrell College Sci-ence Award to Monika Sommerhalter) and California StateUniversity East Bay (Faculty and Student Research Grantsto Monika Sommerhalter Selina Chan and Tuan Le and aSieber-Tombari award to Monika Sommerhalter) ProfessorDaryl Eggers San Jose State University kindly invited theauthors to perform the CD measurements in his laboratoryThe authors are also grateful to Professor AnnMcPartland forproviding detailed feedback on their paper

References

[1] B C Dave B Dunn J S Valentine and J I Zink ldquoNanocon-fined proteins and enzymes sol-gel-based biomolecular mate-rialsrdquoNanotechnology ACS Symposium Series vol 622 pp 351ndash365 1996

[2] I Gill and A Ballesteros ldquoBioencapsulation within syntheticpolymers (part 1) sol-gel encapsulated biologicalsrdquo Trends inBiotechnology vol 18 no 7 pp 282ndash296 2000

[3] D Avnir T Coradin O Lev and J Livage ldquoRecent bio-applications of sol-gel materialsrdquo Journal of Materials Chem-istry vol 16 no 11 pp 1013ndash1030 2006

[4] D R Morris and L P Hager ldquoChloroperoxidase I Isolationand properties of the crystalline glycoproteinrdquo The Journal ofBiological Chemistry vol 241 no 8 pp 1763ndash1768 1966

[5] V Yazbik and M Ansorge-Schumacher ldquoFast and efficientpurification of chloroperoxidase from C fumagordquo Process Bio-chemistry vol 45 no 2 pp 279ndash283 2010

[6] M Hofrichter and R Ullrich ldquoHeme-thiolate haloperoxidasesversatile biocatalysts with biotechnological and environmentalsignificancerdquo Applied Microbiology and Biotechnology vol 71no 3 pp 276ndash288 2006

[7] V M Dembitsky ldquoOxidation epoxidation and sulfoxidationreactions catalysed by haloperoxidasesrdquoTetrahedron vol 59 no26 pp 4701ndash4720 2003

[8] L Santhanam and J S Dordick ldquoChloroperoxidase catalyzedepoxidation of styrene in aqueous and non-aqueous mediardquoBiocatalysis and Biotransformation vol 20 no 4 pp 265ndash2742002

[9] M Ayala N R Robledo A Lopez-Munguia and R Vazquez-Duhalt ldquoSubstrate specificity and ionization potential inchloroperoxidase-catalyzed oxidation of diesel fuelrdquo Environ-mental Science and Technology vol 34 no 13 pp 2804ndash28092000

[10] R Vazquez-Duhalt M Ayala and F J Marquez-Rocha ldquoBio-catalytic chlorination of aromatic hydrocarbons by chloroper-oxidase of Caldariomyces fumagordquo Phytochemistry vol 58 no6 pp 929ndash933 2001

[11] E Terres M Montiel S Le Borgne and E Torres ldquoImmo-bilization of chloroperoxidase on mesoporous materials forthe oxidation of 46-dimethyldibenzothiophene a recalcitrantorganic sulfur compound present in petroleum fractionsrdquoBiotechnology Letters vol 30 no 1 pp 173ndash179 2008

[12] V Trevisan M Signoretto S Colonna V Pironti and GStrukul ldquoMicroencapsulated chloroperoxidase as a recyclablecatalyst for the enantioselective oxidation of sulfides withhydrogen peroxiderdquo Angewandte Chemie International Editionvol 43 no 31 pp 4097ndash4099 2004

[13] N Spreti R Germani A Incani and G Savelli ldquoStabiliza-tion of chloroperoxidase by polyethylene glycols in aqueousmedia kinetic studies and synthetic applicationsrdquoBiotechnologyProgress vol 20 no 1 pp 96ndash101 2004

[14] J-B Park and D S Clark ldquoNew reaction system for hydrocar-bon oxidation by chloroperoxidaserdquo Biotechnology and Bioengi-neering vol 94 no 1 pp 189ndash192 2006

[15] J-Z Liu and M Wang ldquoImprovement of activity and stabilityof chloroperoxidase by chemical modificationrdquo BMC Biotech-nology vol 7 no 1 article 23 2007

[16] L Zhi Y Jiang Y Wang M Hu S Li and Y Ma ldquoEffects ofadditives on the thermostability of chloroperoxidaserdquo Biotech-nology Progress vol 23 no 3 pp 729ndash733 2007

[17] T A Kadima andM A Pickard ldquoImmobilization of chloroper-oxidase on aminopropyl-glassrdquo Applied and EnvironmentalMicrobiology vol 56 no 11 pp 3473ndash3477 1990

[18] Y-J Han J T Watson G D Stucky and A Butler ldquoCatalyticactivity of mesoporous silicate-immobilized chloroperoxidaserdquoJournal ofMolecular Catalysis B Enzymatic vol 17 no 1 pp 1ndash82002

[19] J Aburto M Ayala I Bustos-Jaimes et al ldquoStability andcatalytic properties of chloroperoxidase immobilized on SBA-16mesoporousmaterialsrdquoMicroporous andMesoporousMaterialsvol 83 no 1ndash3 pp 193ndash200 2005

[20] M Hartmann and C Streb ldquoSelective oxidation of indole bychloroperoxidase immobilized on the mesoporous molecularsieve SBA-15rdquo Journal of Porous Materials vol 13 no 3-4 pp347ndash352 2006

10 Journal of Nanotechnology

[21] S Hudson J Cooney B K Hodnett and E Magner ldquoChlorop-eroxidase on periodic mesoporous organosilanes immobiliza-tion and reuserdquo Chemistry of Materials vol 19 no 8 pp 2049ndash2055 2007

[22] R A Sheldon ldquoEnzyme immobilization the quest for optimumperformancerdquo Advanced Synthesis amp Catalysis vol 349 no 8-9pp 1289ndash1307 2007

[23] K Smith N J Silvernail K R Rodgers T E Elgren M Castroand RM Parker ldquoSol-gel encapsulated horseradish peroxidasea catalytic material for peroxidationrdquo Journal of the AmericanChemical Society vol 124 no 16 pp 4247ndash4252 2002

[24] K M Manoj and L P Hager ldquoChloroperoxidase a janusenzymerdquo Biochemistry vol 47 no 9 pp 2997ndash3003 2008

[25] V M Samokyszyn and P R Ortiz de Montellano ldquoTopology ofthe chloroperoxidase active site regiospecificity of heme mod-ification by phenylhydrazine and sodium aziderdquo Biochemistryvol 30 no 50 pp 11646ndash11653 1991

[26] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[27] D K Eggers and J S Valentine ldquoMolecular confinementinfluences protein structure and enhances thermal proteinstabilityrdquo Protein Science vol 10 no 2 pp 250ndash261 2001

[28] J H Harreld T Ebina N Tsubo and G Stucky ldquoManipulationof pore size distributions in silica and ormosil gels dried underambient pressure conditionsrdquo Journal of Non-Crystalline Solidsvol 298 no 2-3 pp 241ndash251 2002

[29] K M Manoj A Baburaj B Ephraim et al ldquoExplaining theatypical reaction profiles of heme enzymes with a novel mech-anistic hypothesis and kinetic treatmentrdquo PLoS ONE vol 5 no5 Article ID e10601 2010

[30] D Jung and M Hartmann ldquoOxidation of indole with CPO andGOx immobilized on mesoporous molecular sievesrdquo CatalysisToday vol 157 no 1ndash4 pp 378ndash383 2010

[31] M L Ferrer F del Monte and D Levy ldquoA novel and simplealcohol-free sol-gel route for encapsulation of labile proteinsrdquoChemistry of Materials vol 14 no 9 pp 3619ndash3621 2002

[32] M Sundaramoorthy J Terner and T L Poulos ldquoThe crystalstructure of chloroperoxidase a heme peroxidase-cytochromeP450 functional hybridrdquo Structure vol 3 no 12 pp 1367ndash13771995

[33] X W Yi A Conesa P J Punt and L P Hager ldquoExamining therole of glutamic acid 183 in chloroperoxidase catalysisrdquo Journalof Biological Chemistry vol 278 no 16 pp 13855ndash13859 2003

[34] S R Blanke S AMartinis S G Sligar L P Hager J J Rux andJ H Dawson ldquoProbing the heme iron coordination structureof alkaline chloroperoxidaserdquo Biochemistry vol 35 no 46 pp14537ndash14543 1996

[35] B Dunn and J I Zink ldquoProbes of pore environment andmolecule-matrix interactions in sol-gel materialsrdquo Chemistry ofMaterials vol 9 no 11 pp 2280ndash2291 1997

[36] W A Loughlin and D B Hawkes ldquoEffect of organic solvents ona chloroperoxidase biotransformationrdquo Bioresource Technologyvol 71 no 2 pp 167ndash172 2000

[37] E Kiljunen and L T Kanerva ldquoChloroperoxidase-catalysedoxidation of alcohols to aldehydesrdquo Journal of Molecular Catal-ysis B Enzymatic vol 9 no 4ndash6 pp 163ndash172 2000

[38] E N Kadnikova and N M Kostic ldquoOxidation of ABTS byhydrogen peroxide catalyzed by horseradish peroxidase encap-sulated into sol-gel glass Effects of glass matrix on reactivityrdquo

Journal of Molecular Catalysis B Enzymatic vol 18 no 1ndash3 pp39ndash48 2002

[39] A Venkateswara Rao and D Haranath ldquoEffect of methyltri-methoxysilane as a synthesis component on the hydrophobicityand some physical properties of silica aerogelsrdquo Microporousand Mesoporous Materials vol 30 no 2-3 pp 267ndash273 1999

[40] M T Reetz A Zonta and J Simpelkamp ldquoEfficient immo-bilization of lipases by entrapment in hydrophobic sol-gelmaterialsrdquo Biotechnology and Bioengineering vol 49 no 5 pp527ndash534 1996

[41] W Wiesner K-H van Pee and F Lingens ldquoPurification andcharacterization of a novel bacterial non-heme chloroperox-idase from Pseudomonas pyrrociniardquo Journal of BiologicalChemistry vol 263 no 27 pp 13725ndash13732 1988

[42] Y Xi Z Liangying and W Sasa ldquoPore size and pore-sizedistribution control of porous silicardquo Sensors and Actuators BChemical vol 25 no 1-3 pp 347ndash352 1995

[43] C Lin and J A Ritter ldquoEffect of synthesis pH on the structureof carbon xerogelsrdquo Carbon vol 35 no 9 pp 1271ndash1278 1997

[44] M Altstein G Segev N Aharonson O Ben-Aziz A Tur-niansky and D Avnir ldquoSol-gel entrapped cholinesterases amicrotiter plate method for monitoring anti-cholinesterasecompoundsrdquo Journal of Agricultural and Food Chemistry vol46 no 8 pp 3318ndash3324 1998

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials

Page 6: Silica Sol-Gel Entrapment of the Enzyme Chloroperoxidase

6 Journal of Nanotechnology

minus20

minus15

minus10

minus5

0

5

200 210 220 230 240 250 260Wavelength (nm)

CD si

gnal

(mde

g)

Figure 4 CD spectra of sol-gel entrapped CPO (black squares)and free CPO (white squares) The CPO concentration was 7120583Min the silica sol-gel sheet of sim1mm thickness The solution samplecontaining 7 120583M CPO in 2mM sodium phosphate buffer pH 70was measured in a cuvette of 1mm path length

0

01

02

03

04

05

300 400 500 600 700 800 900

Abso

rban

ce

Wavelength (nm)

Figure 5 Absorbance spectra of sol-gel entrapped CPO (solid line)and free CPO (dashed line) The silica sol-gel sheet was sim1mmthick and contained 16mgmL CPO The sheet was placed onthe wall of a 10mm wide plastic cuvette and immersed in 50mMpotassium phosphate buffer pH 60 The solution reference samplewas prepared from the same CPO stock via dilution to 016mgmL(4 120583M) with 50mM potassium phosphate buffer pH 60 in a 10mmthick plastic cuvette

content This finding agrees with the X-ray protein structureof CPO [32] and previously determined CD data [15 33]

The visible absorbance spectra shown in Figure 5 aretypical for the active form of CPO with a five-coordinateiron center in the heme chromophore [34] As the pH valueincreases above pH 70 CPO is inactivated the iron centerin the heme group becomes six-coordinate and the Soretband shifts to a longer wavelength [34] This spectroscopictransition can also be observed in CPO sol-gel sheets despitethe entrapment of the enzyme inside the silica matrixFigure 6 displays the absorption spectra of CPO sol-gelsheets immersed in 05M phosphate buffers at pH values of60 80 and 100 In comparison to solution spectra morealkaline conditions are necessary to achieve the Soret peakcharacteristic for sixfold coordinated heme iron centers It isconceivable that the silanol groups in the sol-gel impose anadditional buffering effect According toDunn and Zink [35]

0005

01015

02025

03035

04045

300 320 340 360 380 400 420 440 460 480 500

Abso

rban

ce

Wavelength (nm)

Figure 6 Absorbance spectra of sol-gel entrapped CPO immersedin 05M potassium phosphate buffer with pH values of 60 (solidline) 80 (dashed line) and 100 (dotted line)The silica sol-gel sheetsof 1mm thickness loaded with 16mgmL CPO were placed on thewall of plastic cuvette with a path length of 10mm

the pH can be up to one pH unit lower inside a sol-gel porethan in the surrounding aqueous buffer

The low apparent 119896cat values for sol-gel entrapped CPO(see Table 1) indicate that a significant number of entrappedCPO molecules were unable to catalyze the peroxidation ofpyrogallol in an effective manner CD and visible absorptionspectra however did not indicate any enzyme damage Itshould be noted that both spectroscopic methods can onlyaddress specific features of the enzymeThese features are theoverall secondary structure of the protein the electronic con-figuration of the active site chromophore and the ability torearrange the coordination sphere of the heme iron center inresponse to an external change in pH value

35 Influence of Methanol on the Spectroscopic Features andActivity of Free CPO Methanol is released from the sol-gelprecursor TMOS If we assume complete hydrolysis of TMOSand no evaporation of methanol the protein is exposed toa methanol concentration of 11 vv as the buffered enzymesolution and the sol are mixed and pipetted onto the parafilmfor gelation and drying The transfer into storage bufferand the subsequent exchange of buffer drastically lower themethanol content during the maturation phase of the sol-gelAfter the first buffer exchange the methanol concentration isonly 003 vv

To study the influence of methanol on the spectroscopicfeatures of CPO the enzyme was incubated with 11 vvmethanol andUVVIS andCD spectrawere recordedWedidnot detect a change in the spectroscopic features of the CDspectra after several hours of incubation (data not shown)but methanol exposure did have a small and immediate effecton UVVIS absorption spectra (see Figure 7)The absorptionmaximum of the Soret band shifted from 396 nm to 400 nmNotably this shift was reversible via dialysis The methanolremoval caused a 13-fold increase in sample volume Weadjusted the corresponding spectroscopic trace in Figure 7for this dilution effect

Journal of Nanotechnology 7

0

02

04

06

08

1

370 390 410 430 450

Abso

rban

ce

Wavelength (nm)

Figure 7 Absorption spectra of 9120583M CPO in 01M citrate-02Mphosphate buffer pH 42 (solid line) in 11 vv methanol and01M citrate-02M phosphate buffer pH 42 (dashed line) dialyzedsample (grey solid line) and dialyzed sample adjusted for 13-foldvolume increase (grey dotted line)

In agreement with previous studies [36] we observed adrastic decline in CPOrsquos catalytic performance after incuba-tion of CPO with organic solvents Compared to an aqueousreference sample without methanol only 57 residual activ-ity was detected after incubation with 11 vv methanol for2 hours After one day the residual activity still remainedat 57 We further discovered that the detrimental effect ofmethanol was reversible Up to 96 of the samplersquos initialactivity was recovered after dialysis Sample dilution alsoresulted in the enzymersquos recovery Decreasing the methanolcontent to 5 or 1 vv via sample dilution resulted in 90and 100 relative activity in comparison to identically dilutedsamples from the same CPO batch that were not exposed tomethanol Our finding that damage caused by methanol wasreversible in solution has implications for other studies on theuse of CPO in organic solvents or in biphasic solvent systems[36 37] Several CPO substrates which can be convertedinto products of industrial interest have high solubility innonpolar organic solvents [8]

Also if any initial damage caused by methanol exposurewas also reversible for sol-gel entrapped CPO immediatereduction in methanol content via evaporation of methanolfrom the sol solution or daily buffer exchanges during thegel maturation phase would not critically alter the finalperformance of the CPO sol-gel beads This might explainwhy the three different but parallel preparations outlinedin Section 33 resulted in virtually identical performance forthe three different CPO sol-gel bead sets On the otherhand manifestation of unrecoverable enzyme damage wouldexplain the low apparent 119896cat values determined for sol-gelentrapped CPO (see Table 1) It is conceivable that recoveryfrom damage caused by methanol exposure is less effectivefor CPO molecules entrapped within the silica sol-gel hostcompared to free CPO in solution

36 HinderedMaterial Transport in CPO Sol-Gel Beads Aftertheir first use all CPO sol-gel beads adopted a persistentyellow coloration indicating the entrapment of the productpurpurogallin Attractive intermolecular forces and physical

constraints both can delay the release of product moleculesfrom the silica nanostructure If product release becomesrate-limiting the apparent 119896cat value decreases as observedin the enzyme kinetic analysis Hindered substrate diffusionhowever was not supported by enzyme kinetic experimentsas the 119870

119872and 119870

119868values for the main substrate pyrogallol

were virtually identical for sol-gel entrapped and free CPO(see Table 1)We cannot explain why pyrogallol and purpuro-gallin would show different material transport propertiesinside the silica nanostructure Both molecules have similarfunctional groups and purpurogallin (MW 220 gmol) isonly somewhat larger than pyrogallol (MW 116 gmol) Analternative explanation would involve side-reactions formingalternate charged products or the trapping of reactive coloredintermediates

The alternative peroxidation substrate 221015840-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is particularlylarge (MW 515 gmol) and the product of the peroxidationreaction carries a positive charge [24] Deprotonated silanolgroups on the sol-gel surface can provide negative coun-tercharges The catalytic performance of CPO sol-gel beadsdropped from 126 plusmn 3 with the substrate pyrogallol to 9 plusmn04 with the substrate ABTS relative to the correspondingassay with free CPO in solution The CPO sol-gel beadsadopted the green color of the ABTS peroxidation productKadnikova and coworkers observed the formation of severalside products for the peroxidation reaction of ABTS byhorseradish peroxidase in a sol-gel matrix [38] The perox-idation reaction with ABTS and possibly other substratesincluding pyrogallol might therefore be more complex thanin solution

We further observed that preequilibration of CPO sol-gelbeadswith pyrogallol considerably improved catalytic perfor-mance For example preequilibration with 72mM pyrogallolfor 3 hours in comparison to using 35mM pyrogallol withoutpreequilibration increased the relative activity from 123 to196The strategy to preequilibrate enzyme sol-gel materialswith an excess of substrate before adding a cosubstrate wasalready successfully applied by Smith and coworkers [23] intheir study on sol-gel encapsulated horseradish peroxidaseBased on the data presented in Figure 1(a) however wewouldnot expect an increase in catalytic performance for sol-gelentrapped CPO as the pyrogallol concentration is raised from35 to 72mM In fact substrate inhibition should slightly lowerthe activity of CPOAdsorption of pyrogallolmolecules to thesol-gel surface or physical entrapment could further enhancethe local effective concentration of pyrogallol around theenzyme One key difference between the experiment leadingto Figure 1(a) and the preequilibration experiment is thetiming of adding the cosubstrate H

2O2 Manoj et al [29]

argue that substrate inhibition of CPO is not simply causedby blocking the active site of the enzymewith excess substratemolecules Instead they propose more complex substrateinhibition mechanisms that involve secondary conversionof an already formed product or competition by transientintermediates leading to alternate products Both substrateinhibition scenarios require the immediate presence of thecosubstrate H

2O2

8 Journal of Nanotechnology

Table 3 Properties of CPO sol-gel beads prepared with different casting buffers

pH of casting buffer 45 55 65BET surface area (m2g) 710 plusmn 40lowast 740 plusmn 60 470 plusmn 40Total pore volume (cm3g) 058 plusmn 002 060 plusmn 006 032 plusmn 004Average pore diameter (nm) 33 plusmn 03 32 plusmn 01 27 plusmn 01Activity (mIU) 251 plusmn 2 242 plusmn 29 161 plusmn 13Specific activity (IUmg) 157 plusmn 1 151 plusmn 18 100 plusmn 8Activity compared to free CPO () 18 17 11Cumulative leakage ()dagger 13 14 9lowastAll data are presented as mean values plusmn one standard deviation of triplicate data sets The relative activity was based on a reference assay with free CPO insolution yielding 887 plusmn 31 IUmg daggerThe cumulative leakage over ten days of maturation corresponds to the summation of the leakage data shown in Figure 8

37 Modification of Sol-Gel Procedure Using MTMS Tomodify the surface of the sol-gel material we incorporatedMTMS at molar ratios of 5 20 and 40 in the sol solutionThe addition of MTMS will introduce nonpolar methylgroups rendering the surface of the silica nanostructuremorehydrophobic [39] The casting buffer had a pH value of 60and the total CPO loading was 4 120583gmL The addition ofMTMS resulted in longer gelation times for example up to240 minutes for a molar ratio of 40 MTMS Unfortunatelythe beads prepared with MTMS were more brittle and fragilethan any of the other CPO sol-gel beads prepared in thisstudy The brittleness of the beads rendered their handlingmore challenging Regardless of the amount of incorporatedMTMS the activity was approximately 14 plusmn 1 comparedto a solution reference assay The cumulative leakage oftenexceeded 20We cannot rule out that the physical instabilityof the beads during and after a buffer exchange might havecontributed to higher apparent leakage and higher apparentactivity values In contrast to other enzymes notably lipasewhich showed interfacial activation and performed betterinsidemore hydrophobic nanostructures [40] the incorpora-tion of MTMS into the CPO sol-gel material did not improvecatalytic performance in a systematic manner

38 Modification of Sol-Gel Procedure Using More AcidicCasting Buffers The enzyme CPO is stable under acidicconditions [24] We exploited this CPO specific property andprepared CPO sol-gel beads using casting buffers with pHvalues of 45 55 and 65 All sample preparations were con-ducted in parallel with the same batches of CPO TMOS andbuffer reagents The gelation time increased with more acidiccasting buffers but the sol-gel beads remained easy to handleand transparentTheCPO loadingwas 4 120583gmL All CPO sol-gel preparations were divided into two portions One portionwas used for porosimetry studies and the other portion wasused for leakage and activity measurements (see Table 3)The properties of CPO sol-gel beads cast at pH 45 and 55are virtually identical but the CPO sol-gel beads cast at pH65 show significantly lower values in all categories Thisindicates a change in thematrix formation of the sol-gel as thecasting pHdrops to or below pH55 Overall the porosimetrydata is positively correlated with catalytic performance andunfortunately leakage All three porosimetric propertiesincluding larger average pore size BET surface area and pore

7

6

5

4

3

2

1

0

Leak

age (

)

1 2 3 4 5 6 7 8 9 10

Time (d)

Figure 8 The storage buffer of CPO sol-gel beads prepared withcasting buffers at pH 45 (dark grey bars) 55 (grey bars) and 65(light grey bars) was exchanged on a daily basis and monitored forCPO activity All samples were prepared in triplicate with eight CPOsol-gel beads per sample tube The bar height represents the meanvalue and the error bar plusmn one standard deviation

volume indicate reduced steric hindrance for material trans-port inside the sol-gel nanostructure As a consequencecatalytic performance increased Smaller average pore sizeson the other hand can aid in the retention of CPO

The dimensions of the protein CPO are 53 nm times 46 nmtimes 60 nm [19] The average pore diameters of approximately3 nm are only slightly smaller than the size of CPO Never-theless CPO remainedwell entrapped after completion of thesol-gel maturation phase Attractive electrostatic forces didnot most likely aid in the retention of CPO as the storagebuffer had a pH value of 42 which is close to the isoelectricpoint of CPO The isoelectric point of CPO from C fumagowas calculated to be approximately 40 [18 21] Isoelectricfocusing experiments on CPO from Pseudomonas pyrrociniayielded an isoelectric point of 41 [41] For all buffer condi-tions employed in our study the net charge on the surfaceof CPO is therefore either close to zero or negative

Our observation that more acidic casting buffers result ingreater porosimetry of sol-gels agrees well with several previ-ous studies [42 43] However not all enzymes will respondwell to the use of more acidic casting conditions Notablyenzymes have different pH profiles and some enzymesare inactive under acidic conditions Sol-gel entrappedcholinesterase for example showed better performance

Journal of Nanotechnology 9

in silica nanostructures prepared at pH values of 70 and 80and then 60 [44]

4 Conclusion

The enzyme CPO was successfully entrapped inside a silicananostructure prepared from the precursor TMOS with orwithout addition of the hydrophobic modifier MTMS SinceCPO is stabile in acidic buffers we further modified the sol-gel procedure by using casting buffers with pH values of 4555 60 and 65The catalytic performance of optimized CPOsol-gel beads approached 18 relative to free CPO in solutionas assessed via the pyrogallol peroxidation assay A combi-nation of factors such as enzyme leakage from the sol-gelhost insufficient recovery from inactivation caused by initialmethanol exposure hindered product release or alternatereaction pathways are most likely responsible for the declinein catalytic performance of CPOafter sol-gel entrapmentTheuse of more acidic casting buffers in the sol-gel procedureprovided themost leverage for optimization by yieldingmoreporous silica nanostructures Overall our findings are ofimportance for the optimization of other sol-gel materialsdevised for applications in biosensing or biocatalysis ordesigned for the controlled release of bioactive compounds

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Funding for this project was obtained from the ResearchCorporation for Science Advancement (Cottrell College Sci-ence Award to Monika Sommerhalter) and California StateUniversity East Bay (Faculty and Student Research Grantsto Monika Sommerhalter Selina Chan and Tuan Le and aSieber-Tombari award to Monika Sommerhalter) ProfessorDaryl Eggers San Jose State University kindly invited theauthors to perform the CD measurements in his laboratoryThe authors are also grateful to Professor AnnMcPartland forproviding detailed feedback on their paper

References

[1] B C Dave B Dunn J S Valentine and J I Zink ldquoNanocon-fined proteins and enzymes sol-gel-based biomolecular mate-rialsrdquoNanotechnology ACS Symposium Series vol 622 pp 351ndash365 1996

[2] I Gill and A Ballesteros ldquoBioencapsulation within syntheticpolymers (part 1) sol-gel encapsulated biologicalsrdquo Trends inBiotechnology vol 18 no 7 pp 282ndash296 2000

[3] D Avnir T Coradin O Lev and J Livage ldquoRecent bio-applications of sol-gel materialsrdquo Journal of Materials Chem-istry vol 16 no 11 pp 1013ndash1030 2006

[4] D R Morris and L P Hager ldquoChloroperoxidase I Isolationand properties of the crystalline glycoproteinrdquo The Journal ofBiological Chemistry vol 241 no 8 pp 1763ndash1768 1966

[5] V Yazbik and M Ansorge-Schumacher ldquoFast and efficientpurification of chloroperoxidase from C fumagordquo Process Bio-chemistry vol 45 no 2 pp 279ndash283 2010

[6] M Hofrichter and R Ullrich ldquoHeme-thiolate haloperoxidasesversatile biocatalysts with biotechnological and environmentalsignificancerdquo Applied Microbiology and Biotechnology vol 71no 3 pp 276ndash288 2006

[7] V M Dembitsky ldquoOxidation epoxidation and sulfoxidationreactions catalysed by haloperoxidasesrdquoTetrahedron vol 59 no26 pp 4701ndash4720 2003

[8] L Santhanam and J S Dordick ldquoChloroperoxidase catalyzedepoxidation of styrene in aqueous and non-aqueous mediardquoBiocatalysis and Biotransformation vol 20 no 4 pp 265ndash2742002

[9] M Ayala N R Robledo A Lopez-Munguia and R Vazquez-Duhalt ldquoSubstrate specificity and ionization potential inchloroperoxidase-catalyzed oxidation of diesel fuelrdquo Environ-mental Science and Technology vol 34 no 13 pp 2804ndash28092000

[10] R Vazquez-Duhalt M Ayala and F J Marquez-Rocha ldquoBio-catalytic chlorination of aromatic hydrocarbons by chloroper-oxidase of Caldariomyces fumagordquo Phytochemistry vol 58 no6 pp 929ndash933 2001

[11] E Terres M Montiel S Le Borgne and E Torres ldquoImmo-bilization of chloroperoxidase on mesoporous materials forthe oxidation of 46-dimethyldibenzothiophene a recalcitrantorganic sulfur compound present in petroleum fractionsrdquoBiotechnology Letters vol 30 no 1 pp 173ndash179 2008

[12] V Trevisan M Signoretto S Colonna V Pironti and GStrukul ldquoMicroencapsulated chloroperoxidase as a recyclablecatalyst for the enantioselective oxidation of sulfides withhydrogen peroxiderdquo Angewandte Chemie International Editionvol 43 no 31 pp 4097ndash4099 2004

[13] N Spreti R Germani A Incani and G Savelli ldquoStabiliza-tion of chloroperoxidase by polyethylene glycols in aqueousmedia kinetic studies and synthetic applicationsrdquoBiotechnologyProgress vol 20 no 1 pp 96ndash101 2004

[14] J-B Park and D S Clark ldquoNew reaction system for hydrocar-bon oxidation by chloroperoxidaserdquo Biotechnology and Bioengi-neering vol 94 no 1 pp 189ndash192 2006

[15] J-Z Liu and M Wang ldquoImprovement of activity and stabilityof chloroperoxidase by chemical modificationrdquo BMC Biotech-nology vol 7 no 1 article 23 2007

[16] L Zhi Y Jiang Y Wang M Hu S Li and Y Ma ldquoEffects ofadditives on the thermostability of chloroperoxidaserdquo Biotech-nology Progress vol 23 no 3 pp 729ndash733 2007

[17] T A Kadima andM A Pickard ldquoImmobilization of chloroper-oxidase on aminopropyl-glassrdquo Applied and EnvironmentalMicrobiology vol 56 no 11 pp 3473ndash3477 1990

[18] Y-J Han J T Watson G D Stucky and A Butler ldquoCatalyticactivity of mesoporous silicate-immobilized chloroperoxidaserdquoJournal ofMolecular Catalysis B Enzymatic vol 17 no 1 pp 1ndash82002

[19] J Aburto M Ayala I Bustos-Jaimes et al ldquoStability andcatalytic properties of chloroperoxidase immobilized on SBA-16mesoporousmaterialsrdquoMicroporous andMesoporousMaterialsvol 83 no 1ndash3 pp 193ndash200 2005

[20] M Hartmann and C Streb ldquoSelective oxidation of indole bychloroperoxidase immobilized on the mesoporous molecularsieve SBA-15rdquo Journal of Porous Materials vol 13 no 3-4 pp347ndash352 2006

10 Journal of Nanotechnology

[21] S Hudson J Cooney B K Hodnett and E Magner ldquoChlorop-eroxidase on periodic mesoporous organosilanes immobiliza-tion and reuserdquo Chemistry of Materials vol 19 no 8 pp 2049ndash2055 2007

[22] R A Sheldon ldquoEnzyme immobilization the quest for optimumperformancerdquo Advanced Synthesis amp Catalysis vol 349 no 8-9pp 1289ndash1307 2007

[23] K Smith N J Silvernail K R Rodgers T E Elgren M Castroand RM Parker ldquoSol-gel encapsulated horseradish peroxidasea catalytic material for peroxidationrdquo Journal of the AmericanChemical Society vol 124 no 16 pp 4247ndash4252 2002

[24] K M Manoj and L P Hager ldquoChloroperoxidase a janusenzymerdquo Biochemistry vol 47 no 9 pp 2997ndash3003 2008

[25] V M Samokyszyn and P R Ortiz de Montellano ldquoTopology ofthe chloroperoxidase active site regiospecificity of heme mod-ification by phenylhydrazine and sodium aziderdquo Biochemistryvol 30 no 50 pp 11646ndash11653 1991

[26] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[27] D K Eggers and J S Valentine ldquoMolecular confinementinfluences protein structure and enhances thermal proteinstabilityrdquo Protein Science vol 10 no 2 pp 250ndash261 2001

[28] J H Harreld T Ebina N Tsubo and G Stucky ldquoManipulationof pore size distributions in silica and ormosil gels dried underambient pressure conditionsrdquo Journal of Non-Crystalline Solidsvol 298 no 2-3 pp 241ndash251 2002

[29] K M Manoj A Baburaj B Ephraim et al ldquoExplaining theatypical reaction profiles of heme enzymes with a novel mech-anistic hypothesis and kinetic treatmentrdquo PLoS ONE vol 5 no5 Article ID e10601 2010

[30] D Jung and M Hartmann ldquoOxidation of indole with CPO andGOx immobilized on mesoporous molecular sievesrdquo CatalysisToday vol 157 no 1ndash4 pp 378ndash383 2010

[31] M L Ferrer F del Monte and D Levy ldquoA novel and simplealcohol-free sol-gel route for encapsulation of labile proteinsrdquoChemistry of Materials vol 14 no 9 pp 3619ndash3621 2002

[32] M Sundaramoorthy J Terner and T L Poulos ldquoThe crystalstructure of chloroperoxidase a heme peroxidase-cytochromeP450 functional hybridrdquo Structure vol 3 no 12 pp 1367ndash13771995

[33] X W Yi A Conesa P J Punt and L P Hager ldquoExamining therole of glutamic acid 183 in chloroperoxidase catalysisrdquo Journalof Biological Chemistry vol 278 no 16 pp 13855ndash13859 2003

[34] S R Blanke S AMartinis S G Sligar L P Hager J J Rux andJ H Dawson ldquoProbing the heme iron coordination structureof alkaline chloroperoxidaserdquo Biochemistry vol 35 no 46 pp14537ndash14543 1996

[35] B Dunn and J I Zink ldquoProbes of pore environment andmolecule-matrix interactions in sol-gel materialsrdquo Chemistry ofMaterials vol 9 no 11 pp 2280ndash2291 1997

[36] W A Loughlin and D B Hawkes ldquoEffect of organic solvents ona chloroperoxidase biotransformationrdquo Bioresource Technologyvol 71 no 2 pp 167ndash172 2000

[37] E Kiljunen and L T Kanerva ldquoChloroperoxidase-catalysedoxidation of alcohols to aldehydesrdquo Journal of Molecular Catal-ysis B Enzymatic vol 9 no 4ndash6 pp 163ndash172 2000

[38] E N Kadnikova and N M Kostic ldquoOxidation of ABTS byhydrogen peroxide catalyzed by horseradish peroxidase encap-sulated into sol-gel glass Effects of glass matrix on reactivityrdquo

Journal of Molecular Catalysis B Enzymatic vol 18 no 1ndash3 pp39ndash48 2002

[39] A Venkateswara Rao and D Haranath ldquoEffect of methyltri-methoxysilane as a synthesis component on the hydrophobicityand some physical properties of silica aerogelsrdquo Microporousand Mesoporous Materials vol 30 no 2-3 pp 267ndash273 1999

[40] M T Reetz A Zonta and J Simpelkamp ldquoEfficient immo-bilization of lipases by entrapment in hydrophobic sol-gelmaterialsrdquo Biotechnology and Bioengineering vol 49 no 5 pp527ndash534 1996

[41] W Wiesner K-H van Pee and F Lingens ldquoPurification andcharacterization of a novel bacterial non-heme chloroperox-idase from Pseudomonas pyrrociniardquo Journal of BiologicalChemistry vol 263 no 27 pp 13725ndash13732 1988

[42] Y Xi Z Liangying and W Sasa ldquoPore size and pore-sizedistribution control of porous silicardquo Sensors and Actuators BChemical vol 25 no 1-3 pp 347ndash352 1995

[43] C Lin and J A Ritter ldquoEffect of synthesis pH on the structureof carbon xerogelsrdquo Carbon vol 35 no 9 pp 1271ndash1278 1997

[44] M Altstein G Segev N Aharonson O Ben-Aziz A Tur-niansky and D Avnir ldquoSol-gel entrapped cholinesterases amicrotiter plate method for monitoring anti-cholinesterasecompoundsrdquo Journal of Agricultural and Food Chemistry vol46 no 8 pp 3318ndash3324 1998

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials

Page 7: Silica Sol-Gel Entrapment of the Enzyme Chloroperoxidase

Journal of Nanotechnology 7

0

02

04

06

08

1

370 390 410 430 450

Abso

rban

ce

Wavelength (nm)

Figure 7 Absorption spectra of 9120583M CPO in 01M citrate-02Mphosphate buffer pH 42 (solid line) in 11 vv methanol and01M citrate-02M phosphate buffer pH 42 (dashed line) dialyzedsample (grey solid line) and dialyzed sample adjusted for 13-foldvolume increase (grey dotted line)

In agreement with previous studies [36] we observed adrastic decline in CPOrsquos catalytic performance after incuba-tion of CPO with organic solvents Compared to an aqueousreference sample without methanol only 57 residual activ-ity was detected after incubation with 11 vv methanol for2 hours After one day the residual activity still remainedat 57 We further discovered that the detrimental effect ofmethanol was reversible Up to 96 of the samplersquos initialactivity was recovered after dialysis Sample dilution alsoresulted in the enzymersquos recovery Decreasing the methanolcontent to 5 or 1 vv via sample dilution resulted in 90and 100 relative activity in comparison to identically dilutedsamples from the same CPO batch that were not exposed tomethanol Our finding that damage caused by methanol wasreversible in solution has implications for other studies on theuse of CPO in organic solvents or in biphasic solvent systems[36 37] Several CPO substrates which can be convertedinto products of industrial interest have high solubility innonpolar organic solvents [8]

Also if any initial damage caused by methanol exposurewas also reversible for sol-gel entrapped CPO immediatereduction in methanol content via evaporation of methanolfrom the sol solution or daily buffer exchanges during thegel maturation phase would not critically alter the finalperformance of the CPO sol-gel beads This might explainwhy the three different but parallel preparations outlinedin Section 33 resulted in virtually identical performance forthe three different CPO sol-gel bead sets On the otherhand manifestation of unrecoverable enzyme damage wouldexplain the low apparent 119896cat values determined for sol-gelentrapped CPO (see Table 1) It is conceivable that recoveryfrom damage caused by methanol exposure is less effectivefor CPO molecules entrapped within the silica sol-gel hostcompared to free CPO in solution

36 HinderedMaterial Transport in CPO Sol-Gel Beads Aftertheir first use all CPO sol-gel beads adopted a persistentyellow coloration indicating the entrapment of the productpurpurogallin Attractive intermolecular forces and physical

constraints both can delay the release of product moleculesfrom the silica nanostructure If product release becomesrate-limiting the apparent 119896cat value decreases as observedin the enzyme kinetic analysis Hindered substrate diffusionhowever was not supported by enzyme kinetic experimentsas the 119870

119872and 119870

119868values for the main substrate pyrogallol

were virtually identical for sol-gel entrapped and free CPO(see Table 1)We cannot explain why pyrogallol and purpuro-gallin would show different material transport propertiesinside the silica nanostructure Both molecules have similarfunctional groups and purpurogallin (MW 220 gmol) isonly somewhat larger than pyrogallol (MW 116 gmol) Analternative explanation would involve side-reactions formingalternate charged products or the trapping of reactive coloredintermediates

The alternative peroxidation substrate 221015840-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is particularlylarge (MW 515 gmol) and the product of the peroxidationreaction carries a positive charge [24] Deprotonated silanolgroups on the sol-gel surface can provide negative coun-tercharges The catalytic performance of CPO sol-gel beadsdropped from 126 plusmn 3 with the substrate pyrogallol to 9 plusmn04 with the substrate ABTS relative to the correspondingassay with free CPO in solution The CPO sol-gel beadsadopted the green color of the ABTS peroxidation productKadnikova and coworkers observed the formation of severalside products for the peroxidation reaction of ABTS byhorseradish peroxidase in a sol-gel matrix [38] The perox-idation reaction with ABTS and possibly other substratesincluding pyrogallol might therefore be more complex thanin solution

We further observed that preequilibration of CPO sol-gelbeadswith pyrogallol considerably improved catalytic perfor-mance For example preequilibration with 72mM pyrogallolfor 3 hours in comparison to using 35mM pyrogallol withoutpreequilibration increased the relative activity from 123 to196The strategy to preequilibrate enzyme sol-gel materialswith an excess of substrate before adding a cosubstrate wasalready successfully applied by Smith and coworkers [23] intheir study on sol-gel encapsulated horseradish peroxidaseBased on the data presented in Figure 1(a) however wewouldnot expect an increase in catalytic performance for sol-gelentrapped CPO as the pyrogallol concentration is raised from35 to 72mM In fact substrate inhibition should slightly lowerthe activity of CPOAdsorption of pyrogallolmolecules to thesol-gel surface or physical entrapment could further enhancethe local effective concentration of pyrogallol around theenzyme One key difference between the experiment leadingto Figure 1(a) and the preequilibration experiment is thetiming of adding the cosubstrate H

2O2 Manoj et al [29]

argue that substrate inhibition of CPO is not simply causedby blocking the active site of the enzymewith excess substratemolecules Instead they propose more complex substrateinhibition mechanisms that involve secondary conversionof an already formed product or competition by transientintermediates leading to alternate products Both substrateinhibition scenarios require the immediate presence of thecosubstrate H

2O2

8 Journal of Nanotechnology

Table 3 Properties of CPO sol-gel beads prepared with different casting buffers

pH of casting buffer 45 55 65BET surface area (m2g) 710 plusmn 40lowast 740 plusmn 60 470 plusmn 40Total pore volume (cm3g) 058 plusmn 002 060 plusmn 006 032 plusmn 004Average pore diameter (nm) 33 plusmn 03 32 plusmn 01 27 plusmn 01Activity (mIU) 251 plusmn 2 242 plusmn 29 161 plusmn 13Specific activity (IUmg) 157 plusmn 1 151 plusmn 18 100 plusmn 8Activity compared to free CPO () 18 17 11Cumulative leakage ()dagger 13 14 9lowastAll data are presented as mean values plusmn one standard deviation of triplicate data sets The relative activity was based on a reference assay with free CPO insolution yielding 887 plusmn 31 IUmg daggerThe cumulative leakage over ten days of maturation corresponds to the summation of the leakage data shown in Figure 8

37 Modification of Sol-Gel Procedure Using MTMS Tomodify the surface of the sol-gel material we incorporatedMTMS at molar ratios of 5 20 and 40 in the sol solutionThe addition of MTMS will introduce nonpolar methylgroups rendering the surface of the silica nanostructuremorehydrophobic [39] The casting buffer had a pH value of 60and the total CPO loading was 4 120583gmL The addition ofMTMS resulted in longer gelation times for example up to240 minutes for a molar ratio of 40 MTMS Unfortunatelythe beads prepared with MTMS were more brittle and fragilethan any of the other CPO sol-gel beads prepared in thisstudy The brittleness of the beads rendered their handlingmore challenging Regardless of the amount of incorporatedMTMS the activity was approximately 14 plusmn 1 comparedto a solution reference assay The cumulative leakage oftenexceeded 20We cannot rule out that the physical instabilityof the beads during and after a buffer exchange might havecontributed to higher apparent leakage and higher apparentactivity values In contrast to other enzymes notably lipasewhich showed interfacial activation and performed betterinsidemore hydrophobic nanostructures [40] the incorpora-tion of MTMS into the CPO sol-gel material did not improvecatalytic performance in a systematic manner

38 Modification of Sol-Gel Procedure Using More AcidicCasting Buffers The enzyme CPO is stable under acidicconditions [24] We exploited this CPO specific property andprepared CPO sol-gel beads using casting buffers with pHvalues of 45 55 and 65 All sample preparations were con-ducted in parallel with the same batches of CPO TMOS andbuffer reagents The gelation time increased with more acidiccasting buffers but the sol-gel beads remained easy to handleand transparentTheCPO loadingwas 4 120583gmL All CPO sol-gel preparations were divided into two portions One portionwas used for porosimetry studies and the other portion wasused for leakage and activity measurements (see Table 3)The properties of CPO sol-gel beads cast at pH 45 and 55are virtually identical but the CPO sol-gel beads cast at pH65 show significantly lower values in all categories Thisindicates a change in thematrix formation of the sol-gel as thecasting pHdrops to or below pH55 Overall the porosimetrydata is positively correlated with catalytic performance andunfortunately leakage All three porosimetric propertiesincluding larger average pore size BET surface area and pore

7

6

5

4

3

2

1

0

Leak

age (

)

1 2 3 4 5 6 7 8 9 10

Time (d)

Figure 8 The storage buffer of CPO sol-gel beads prepared withcasting buffers at pH 45 (dark grey bars) 55 (grey bars) and 65(light grey bars) was exchanged on a daily basis and monitored forCPO activity All samples were prepared in triplicate with eight CPOsol-gel beads per sample tube The bar height represents the meanvalue and the error bar plusmn one standard deviation

volume indicate reduced steric hindrance for material trans-port inside the sol-gel nanostructure As a consequencecatalytic performance increased Smaller average pore sizeson the other hand can aid in the retention of CPO

The dimensions of the protein CPO are 53 nm times 46 nmtimes 60 nm [19] The average pore diameters of approximately3 nm are only slightly smaller than the size of CPO Never-theless CPO remainedwell entrapped after completion of thesol-gel maturation phase Attractive electrostatic forces didnot most likely aid in the retention of CPO as the storagebuffer had a pH value of 42 which is close to the isoelectricpoint of CPO The isoelectric point of CPO from C fumagowas calculated to be approximately 40 [18 21] Isoelectricfocusing experiments on CPO from Pseudomonas pyrrociniayielded an isoelectric point of 41 [41] For all buffer condi-tions employed in our study the net charge on the surfaceof CPO is therefore either close to zero or negative

Our observation that more acidic casting buffers result ingreater porosimetry of sol-gels agrees well with several previ-ous studies [42 43] However not all enzymes will respondwell to the use of more acidic casting conditions Notablyenzymes have different pH profiles and some enzymesare inactive under acidic conditions Sol-gel entrappedcholinesterase for example showed better performance

Journal of Nanotechnology 9

in silica nanostructures prepared at pH values of 70 and 80and then 60 [44]

4 Conclusion

The enzyme CPO was successfully entrapped inside a silicananostructure prepared from the precursor TMOS with orwithout addition of the hydrophobic modifier MTMS SinceCPO is stabile in acidic buffers we further modified the sol-gel procedure by using casting buffers with pH values of 4555 60 and 65The catalytic performance of optimized CPOsol-gel beads approached 18 relative to free CPO in solutionas assessed via the pyrogallol peroxidation assay A combi-nation of factors such as enzyme leakage from the sol-gelhost insufficient recovery from inactivation caused by initialmethanol exposure hindered product release or alternatereaction pathways are most likely responsible for the declinein catalytic performance of CPOafter sol-gel entrapmentTheuse of more acidic casting buffers in the sol-gel procedureprovided themost leverage for optimization by yieldingmoreporous silica nanostructures Overall our findings are ofimportance for the optimization of other sol-gel materialsdevised for applications in biosensing or biocatalysis ordesigned for the controlled release of bioactive compounds

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Funding for this project was obtained from the ResearchCorporation for Science Advancement (Cottrell College Sci-ence Award to Monika Sommerhalter) and California StateUniversity East Bay (Faculty and Student Research Grantsto Monika Sommerhalter Selina Chan and Tuan Le and aSieber-Tombari award to Monika Sommerhalter) ProfessorDaryl Eggers San Jose State University kindly invited theauthors to perform the CD measurements in his laboratoryThe authors are also grateful to Professor AnnMcPartland forproviding detailed feedback on their paper

References

[1] B C Dave B Dunn J S Valentine and J I Zink ldquoNanocon-fined proteins and enzymes sol-gel-based biomolecular mate-rialsrdquoNanotechnology ACS Symposium Series vol 622 pp 351ndash365 1996

[2] I Gill and A Ballesteros ldquoBioencapsulation within syntheticpolymers (part 1) sol-gel encapsulated biologicalsrdquo Trends inBiotechnology vol 18 no 7 pp 282ndash296 2000

[3] D Avnir T Coradin O Lev and J Livage ldquoRecent bio-applications of sol-gel materialsrdquo Journal of Materials Chem-istry vol 16 no 11 pp 1013ndash1030 2006

[4] D R Morris and L P Hager ldquoChloroperoxidase I Isolationand properties of the crystalline glycoproteinrdquo The Journal ofBiological Chemistry vol 241 no 8 pp 1763ndash1768 1966

[5] V Yazbik and M Ansorge-Schumacher ldquoFast and efficientpurification of chloroperoxidase from C fumagordquo Process Bio-chemistry vol 45 no 2 pp 279ndash283 2010

[6] M Hofrichter and R Ullrich ldquoHeme-thiolate haloperoxidasesversatile biocatalysts with biotechnological and environmentalsignificancerdquo Applied Microbiology and Biotechnology vol 71no 3 pp 276ndash288 2006

[7] V M Dembitsky ldquoOxidation epoxidation and sulfoxidationreactions catalysed by haloperoxidasesrdquoTetrahedron vol 59 no26 pp 4701ndash4720 2003

[8] L Santhanam and J S Dordick ldquoChloroperoxidase catalyzedepoxidation of styrene in aqueous and non-aqueous mediardquoBiocatalysis and Biotransformation vol 20 no 4 pp 265ndash2742002

[9] M Ayala N R Robledo A Lopez-Munguia and R Vazquez-Duhalt ldquoSubstrate specificity and ionization potential inchloroperoxidase-catalyzed oxidation of diesel fuelrdquo Environ-mental Science and Technology vol 34 no 13 pp 2804ndash28092000

[10] R Vazquez-Duhalt M Ayala and F J Marquez-Rocha ldquoBio-catalytic chlorination of aromatic hydrocarbons by chloroper-oxidase of Caldariomyces fumagordquo Phytochemistry vol 58 no6 pp 929ndash933 2001

[11] E Terres M Montiel S Le Borgne and E Torres ldquoImmo-bilization of chloroperoxidase on mesoporous materials forthe oxidation of 46-dimethyldibenzothiophene a recalcitrantorganic sulfur compound present in petroleum fractionsrdquoBiotechnology Letters vol 30 no 1 pp 173ndash179 2008

[12] V Trevisan M Signoretto S Colonna V Pironti and GStrukul ldquoMicroencapsulated chloroperoxidase as a recyclablecatalyst for the enantioselective oxidation of sulfides withhydrogen peroxiderdquo Angewandte Chemie International Editionvol 43 no 31 pp 4097ndash4099 2004

[13] N Spreti R Germani A Incani and G Savelli ldquoStabiliza-tion of chloroperoxidase by polyethylene glycols in aqueousmedia kinetic studies and synthetic applicationsrdquoBiotechnologyProgress vol 20 no 1 pp 96ndash101 2004

[14] J-B Park and D S Clark ldquoNew reaction system for hydrocar-bon oxidation by chloroperoxidaserdquo Biotechnology and Bioengi-neering vol 94 no 1 pp 189ndash192 2006

[15] J-Z Liu and M Wang ldquoImprovement of activity and stabilityof chloroperoxidase by chemical modificationrdquo BMC Biotech-nology vol 7 no 1 article 23 2007

[16] L Zhi Y Jiang Y Wang M Hu S Li and Y Ma ldquoEffects ofadditives on the thermostability of chloroperoxidaserdquo Biotech-nology Progress vol 23 no 3 pp 729ndash733 2007

[17] T A Kadima andM A Pickard ldquoImmobilization of chloroper-oxidase on aminopropyl-glassrdquo Applied and EnvironmentalMicrobiology vol 56 no 11 pp 3473ndash3477 1990

[18] Y-J Han J T Watson G D Stucky and A Butler ldquoCatalyticactivity of mesoporous silicate-immobilized chloroperoxidaserdquoJournal ofMolecular Catalysis B Enzymatic vol 17 no 1 pp 1ndash82002

[19] J Aburto M Ayala I Bustos-Jaimes et al ldquoStability andcatalytic properties of chloroperoxidase immobilized on SBA-16mesoporousmaterialsrdquoMicroporous andMesoporousMaterialsvol 83 no 1ndash3 pp 193ndash200 2005

[20] M Hartmann and C Streb ldquoSelective oxidation of indole bychloroperoxidase immobilized on the mesoporous molecularsieve SBA-15rdquo Journal of Porous Materials vol 13 no 3-4 pp347ndash352 2006

10 Journal of Nanotechnology

[21] S Hudson J Cooney B K Hodnett and E Magner ldquoChlorop-eroxidase on periodic mesoporous organosilanes immobiliza-tion and reuserdquo Chemistry of Materials vol 19 no 8 pp 2049ndash2055 2007

[22] R A Sheldon ldquoEnzyme immobilization the quest for optimumperformancerdquo Advanced Synthesis amp Catalysis vol 349 no 8-9pp 1289ndash1307 2007

[23] K Smith N J Silvernail K R Rodgers T E Elgren M Castroand RM Parker ldquoSol-gel encapsulated horseradish peroxidasea catalytic material for peroxidationrdquo Journal of the AmericanChemical Society vol 124 no 16 pp 4247ndash4252 2002

[24] K M Manoj and L P Hager ldquoChloroperoxidase a janusenzymerdquo Biochemistry vol 47 no 9 pp 2997ndash3003 2008

[25] V M Samokyszyn and P R Ortiz de Montellano ldquoTopology ofthe chloroperoxidase active site regiospecificity of heme mod-ification by phenylhydrazine and sodium aziderdquo Biochemistryvol 30 no 50 pp 11646ndash11653 1991

[26] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[27] D K Eggers and J S Valentine ldquoMolecular confinementinfluences protein structure and enhances thermal proteinstabilityrdquo Protein Science vol 10 no 2 pp 250ndash261 2001

[28] J H Harreld T Ebina N Tsubo and G Stucky ldquoManipulationof pore size distributions in silica and ormosil gels dried underambient pressure conditionsrdquo Journal of Non-Crystalline Solidsvol 298 no 2-3 pp 241ndash251 2002

[29] K M Manoj A Baburaj B Ephraim et al ldquoExplaining theatypical reaction profiles of heme enzymes with a novel mech-anistic hypothesis and kinetic treatmentrdquo PLoS ONE vol 5 no5 Article ID e10601 2010

[30] D Jung and M Hartmann ldquoOxidation of indole with CPO andGOx immobilized on mesoporous molecular sievesrdquo CatalysisToday vol 157 no 1ndash4 pp 378ndash383 2010

[31] M L Ferrer F del Monte and D Levy ldquoA novel and simplealcohol-free sol-gel route for encapsulation of labile proteinsrdquoChemistry of Materials vol 14 no 9 pp 3619ndash3621 2002

[32] M Sundaramoorthy J Terner and T L Poulos ldquoThe crystalstructure of chloroperoxidase a heme peroxidase-cytochromeP450 functional hybridrdquo Structure vol 3 no 12 pp 1367ndash13771995

[33] X W Yi A Conesa P J Punt and L P Hager ldquoExamining therole of glutamic acid 183 in chloroperoxidase catalysisrdquo Journalof Biological Chemistry vol 278 no 16 pp 13855ndash13859 2003

[34] S R Blanke S AMartinis S G Sligar L P Hager J J Rux andJ H Dawson ldquoProbing the heme iron coordination structureof alkaline chloroperoxidaserdquo Biochemistry vol 35 no 46 pp14537ndash14543 1996

[35] B Dunn and J I Zink ldquoProbes of pore environment andmolecule-matrix interactions in sol-gel materialsrdquo Chemistry ofMaterials vol 9 no 11 pp 2280ndash2291 1997

[36] W A Loughlin and D B Hawkes ldquoEffect of organic solvents ona chloroperoxidase biotransformationrdquo Bioresource Technologyvol 71 no 2 pp 167ndash172 2000

[37] E Kiljunen and L T Kanerva ldquoChloroperoxidase-catalysedoxidation of alcohols to aldehydesrdquo Journal of Molecular Catal-ysis B Enzymatic vol 9 no 4ndash6 pp 163ndash172 2000

[38] E N Kadnikova and N M Kostic ldquoOxidation of ABTS byhydrogen peroxide catalyzed by horseradish peroxidase encap-sulated into sol-gel glass Effects of glass matrix on reactivityrdquo

Journal of Molecular Catalysis B Enzymatic vol 18 no 1ndash3 pp39ndash48 2002

[39] A Venkateswara Rao and D Haranath ldquoEffect of methyltri-methoxysilane as a synthesis component on the hydrophobicityand some physical properties of silica aerogelsrdquo Microporousand Mesoporous Materials vol 30 no 2-3 pp 267ndash273 1999

[40] M T Reetz A Zonta and J Simpelkamp ldquoEfficient immo-bilization of lipases by entrapment in hydrophobic sol-gelmaterialsrdquo Biotechnology and Bioengineering vol 49 no 5 pp527ndash534 1996

[41] W Wiesner K-H van Pee and F Lingens ldquoPurification andcharacterization of a novel bacterial non-heme chloroperox-idase from Pseudomonas pyrrociniardquo Journal of BiologicalChemistry vol 263 no 27 pp 13725ndash13732 1988

[42] Y Xi Z Liangying and W Sasa ldquoPore size and pore-sizedistribution control of porous silicardquo Sensors and Actuators BChemical vol 25 no 1-3 pp 347ndash352 1995

[43] C Lin and J A Ritter ldquoEffect of synthesis pH on the structureof carbon xerogelsrdquo Carbon vol 35 no 9 pp 1271ndash1278 1997

[44] M Altstein G Segev N Aharonson O Ben-Aziz A Tur-niansky and D Avnir ldquoSol-gel entrapped cholinesterases amicrotiter plate method for monitoring anti-cholinesterasecompoundsrdquo Journal of Agricultural and Food Chemistry vol46 no 8 pp 3318ndash3324 1998

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials

Page 8: Silica Sol-Gel Entrapment of the Enzyme Chloroperoxidase

8 Journal of Nanotechnology

Table 3 Properties of CPO sol-gel beads prepared with different casting buffers

pH of casting buffer 45 55 65BET surface area (m2g) 710 plusmn 40lowast 740 plusmn 60 470 plusmn 40Total pore volume (cm3g) 058 plusmn 002 060 plusmn 006 032 plusmn 004Average pore diameter (nm) 33 plusmn 03 32 plusmn 01 27 plusmn 01Activity (mIU) 251 plusmn 2 242 plusmn 29 161 plusmn 13Specific activity (IUmg) 157 plusmn 1 151 plusmn 18 100 plusmn 8Activity compared to free CPO () 18 17 11Cumulative leakage ()dagger 13 14 9lowastAll data are presented as mean values plusmn one standard deviation of triplicate data sets The relative activity was based on a reference assay with free CPO insolution yielding 887 plusmn 31 IUmg daggerThe cumulative leakage over ten days of maturation corresponds to the summation of the leakage data shown in Figure 8

37 Modification of Sol-Gel Procedure Using MTMS Tomodify the surface of the sol-gel material we incorporatedMTMS at molar ratios of 5 20 and 40 in the sol solutionThe addition of MTMS will introduce nonpolar methylgroups rendering the surface of the silica nanostructuremorehydrophobic [39] The casting buffer had a pH value of 60and the total CPO loading was 4 120583gmL The addition ofMTMS resulted in longer gelation times for example up to240 minutes for a molar ratio of 40 MTMS Unfortunatelythe beads prepared with MTMS were more brittle and fragilethan any of the other CPO sol-gel beads prepared in thisstudy The brittleness of the beads rendered their handlingmore challenging Regardless of the amount of incorporatedMTMS the activity was approximately 14 plusmn 1 comparedto a solution reference assay The cumulative leakage oftenexceeded 20We cannot rule out that the physical instabilityof the beads during and after a buffer exchange might havecontributed to higher apparent leakage and higher apparentactivity values In contrast to other enzymes notably lipasewhich showed interfacial activation and performed betterinsidemore hydrophobic nanostructures [40] the incorpora-tion of MTMS into the CPO sol-gel material did not improvecatalytic performance in a systematic manner

38 Modification of Sol-Gel Procedure Using More AcidicCasting Buffers The enzyme CPO is stable under acidicconditions [24] We exploited this CPO specific property andprepared CPO sol-gel beads using casting buffers with pHvalues of 45 55 and 65 All sample preparations were con-ducted in parallel with the same batches of CPO TMOS andbuffer reagents The gelation time increased with more acidiccasting buffers but the sol-gel beads remained easy to handleand transparentTheCPO loadingwas 4 120583gmL All CPO sol-gel preparations were divided into two portions One portionwas used for porosimetry studies and the other portion wasused for leakage and activity measurements (see Table 3)The properties of CPO sol-gel beads cast at pH 45 and 55are virtually identical but the CPO sol-gel beads cast at pH65 show significantly lower values in all categories Thisindicates a change in thematrix formation of the sol-gel as thecasting pHdrops to or below pH55 Overall the porosimetrydata is positively correlated with catalytic performance andunfortunately leakage All three porosimetric propertiesincluding larger average pore size BET surface area and pore

7

6

5

4

3

2

1

0

Leak

age (

)

1 2 3 4 5 6 7 8 9 10

Time (d)

Figure 8 The storage buffer of CPO sol-gel beads prepared withcasting buffers at pH 45 (dark grey bars) 55 (grey bars) and 65(light grey bars) was exchanged on a daily basis and monitored forCPO activity All samples were prepared in triplicate with eight CPOsol-gel beads per sample tube The bar height represents the meanvalue and the error bar plusmn one standard deviation

volume indicate reduced steric hindrance for material trans-port inside the sol-gel nanostructure As a consequencecatalytic performance increased Smaller average pore sizeson the other hand can aid in the retention of CPO

The dimensions of the protein CPO are 53 nm times 46 nmtimes 60 nm [19] The average pore diameters of approximately3 nm are only slightly smaller than the size of CPO Never-theless CPO remainedwell entrapped after completion of thesol-gel maturation phase Attractive electrostatic forces didnot most likely aid in the retention of CPO as the storagebuffer had a pH value of 42 which is close to the isoelectricpoint of CPO The isoelectric point of CPO from C fumagowas calculated to be approximately 40 [18 21] Isoelectricfocusing experiments on CPO from Pseudomonas pyrrociniayielded an isoelectric point of 41 [41] For all buffer condi-tions employed in our study the net charge on the surfaceof CPO is therefore either close to zero or negative

Our observation that more acidic casting buffers result ingreater porosimetry of sol-gels agrees well with several previ-ous studies [42 43] However not all enzymes will respondwell to the use of more acidic casting conditions Notablyenzymes have different pH profiles and some enzymesare inactive under acidic conditions Sol-gel entrappedcholinesterase for example showed better performance

Journal of Nanotechnology 9

in silica nanostructures prepared at pH values of 70 and 80and then 60 [44]

4 Conclusion

The enzyme CPO was successfully entrapped inside a silicananostructure prepared from the precursor TMOS with orwithout addition of the hydrophobic modifier MTMS SinceCPO is stabile in acidic buffers we further modified the sol-gel procedure by using casting buffers with pH values of 4555 60 and 65The catalytic performance of optimized CPOsol-gel beads approached 18 relative to free CPO in solutionas assessed via the pyrogallol peroxidation assay A combi-nation of factors such as enzyme leakage from the sol-gelhost insufficient recovery from inactivation caused by initialmethanol exposure hindered product release or alternatereaction pathways are most likely responsible for the declinein catalytic performance of CPOafter sol-gel entrapmentTheuse of more acidic casting buffers in the sol-gel procedureprovided themost leverage for optimization by yieldingmoreporous silica nanostructures Overall our findings are ofimportance for the optimization of other sol-gel materialsdevised for applications in biosensing or biocatalysis ordesigned for the controlled release of bioactive compounds

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Funding for this project was obtained from the ResearchCorporation for Science Advancement (Cottrell College Sci-ence Award to Monika Sommerhalter) and California StateUniversity East Bay (Faculty and Student Research Grantsto Monika Sommerhalter Selina Chan and Tuan Le and aSieber-Tombari award to Monika Sommerhalter) ProfessorDaryl Eggers San Jose State University kindly invited theauthors to perform the CD measurements in his laboratoryThe authors are also grateful to Professor AnnMcPartland forproviding detailed feedback on their paper

References

[1] B C Dave B Dunn J S Valentine and J I Zink ldquoNanocon-fined proteins and enzymes sol-gel-based biomolecular mate-rialsrdquoNanotechnology ACS Symposium Series vol 622 pp 351ndash365 1996

[2] I Gill and A Ballesteros ldquoBioencapsulation within syntheticpolymers (part 1) sol-gel encapsulated biologicalsrdquo Trends inBiotechnology vol 18 no 7 pp 282ndash296 2000

[3] D Avnir T Coradin O Lev and J Livage ldquoRecent bio-applications of sol-gel materialsrdquo Journal of Materials Chem-istry vol 16 no 11 pp 1013ndash1030 2006

[4] D R Morris and L P Hager ldquoChloroperoxidase I Isolationand properties of the crystalline glycoproteinrdquo The Journal ofBiological Chemistry vol 241 no 8 pp 1763ndash1768 1966

[5] V Yazbik and M Ansorge-Schumacher ldquoFast and efficientpurification of chloroperoxidase from C fumagordquo Process Bio-chemistry vol 45 no 2 pp 279ndash283 2010

[6] M Hofrichter and R Ullrich ldquoHeme-thiolate haloperoxidasesversatile biocatalysts with biotechnological and environmentalsignificancerdquo Applied Microbiology and Biotechnology vol 71no 3 pp 276ndash288 2006

[7] V M Dembitsky ldquoOxidation epoxidation and sulfoxidationreactions catalysed by haloperoxidasesrdquoTetrahedron vol 59 no26 pp 4701ndash4720 2003

[8] L Santhanam and J S Dordick ldquoChloroperoxidase catalyzedepoxidation of styrene in aqueous and non-aqueous mediardquoBiocatalysis and Biotransformation vol 20 no 4 pp 265ndash2742002

[9] M Ayala N R Robledo A Lopez-Munguia and R Vazquez-Duhalt ldquoSubstrate specificity and ionization potential inchloroperoxidase-catalyzed oxidation of diesel fuelrdquo Environ-mental Science and Technology vol 34 no 13 pp 2804ndash28092000

[10] R Vazquez-Duhalt M Ayala and F J Marquez-Rocha ldquoBio-catalytic chlorination of aromatic hydrocarbons by chloroper-oxidase of Caldariomyces fumagordquo Phytochemistry vol 58 no6 pp 929ndash933 2001

[11] E Terres M Montiel S Le Borgne and E Torres ldquoImmo-bilization of chloroperoxidase on mesoporous materials forthe oxidation of 46-dimethyldibenzothiophene a recalcitrantorganic sulfur compound present in petroleum fractionsrdquoBiotechnology Letters vol 30 no 1 pp 173ndash179 2008

[12] V Trevisan M Signoretto S Colonna V Pironti and GStrukul ldquoMicroencapsulated chloroperoxidase as a recyclablecatalyst for the enantioselective oxidation of sulfides withhydrogen peroxiderdquo Angewandte Chemie International Editionvol 43 no 31 pp 4097ndash4099 2004

[13] N Spreti R Germani A Incani and G Savelli ldquoStabiliza-tion of chloroperoxidase by polyethylene glycols in aqueousmedia kinetic studies and synthetic applicationsrdquoBiotechnologyProgress vol 20 no 1 pp 96ndash101 2004

[14] J-B Park and D S Clark ldquoNew reaction system for hydrocar-bon oxidation by chloroperoxidaserdquo Biotechnology and Bioengi-neering vol 94 no 1 pp 189ndash192 2006

[15] J-Z Liu and M Wang ldquoImprovement of activity and stabilityof chloroperoxidase by chemical modificationrdquo BMC Biotech-nology vol 7 no 1 article 23 2007

[16] L Zhi Y Jiang Y Wang M Hu S Li and Y Ma ldquoEffects ofadditives on the thermostability of chloroperoxidaserdquo Biotech-nology Progress vol 23 no 3 pp 729ndash733 2007

[17] T A Kadima andM A Pickard ldquoImmobilization of chloroper-oxidase on aminopropyl-glassrdquo Applied and EnvironmentalMicrobiology vol 56 no 11 pp 3473ndash3477 1990

[18] Y-J Han J T Watson G D Stucky and A Butler ldquoCatalyticactivity of mesoporous silicate-immobilized chloroperoxidaserdquoJournal ofMolecular Catalysis B Enzymatic vol 17 no 1 pp 1ndash82002

[19] J Aburto M Ayala I Bustos-Jaimes et al ldquoStability andcatalytic properties of chloroperoxidase immobilized on SBA-16mesoporousmaterialsrdquoMicroporous andMesoporousMaterialsvol 83 no 1ndash3 pp 193ndash200 2005

[20] M Hartmann and C Streb ldquoSelective oxidation of indole bychloroperoxidase immobilized on the mesoporous molecularsieve SBA-15rdquo Journal of Porous Materials vol 13 no 3-4 pp347ndash352 2006

10 Journal of Nanotechnology

[21] S Hudson J Cooney B K Hodnett and E Magner ldquoChlorop-eroxidase on periodic mesoporous organosilanes immobiliza-tion and reuserdquo Chemistry of Materials vol 19 no 8 pp 2049ndash2055 2007

[22] R A Sheldon ldquoEnzyme immobilization the quest for optimumperformancerdquo Advanced Synthesis amp Catalysis vol 349 no 8-9pp 1289ndash1307 2007

[23] K Smith N J Silvernail K R Rodgers T E Elgren M Castroand RM Parker ldquoSol-gel encapsulated horseradish peroxidasea catalytic material for peroxidationrdquo Journal of the AmericanChemical Society vol 124 no 16 pp 4247ndash4252 2002

[24] K M Manoj and L P Hager ldquoChloroperoxidase a janusenzymerdquo Biochemistry vol 47 no 9 pp 2997ndash3003 2008

[25] V M Samokyszyn and P R Ortiz de Montellano ldquoTopology ofthe chloroperoxidase active site regiospecificity of heme mod-ification by phenylhydrazine and sodium aziderdquo Biochemistryvol 30 no 50 pp 11646ndash11653 1991

[26] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[27] D K Eggers and J S Valentine ldquoMolecular confinementinfluences protein structure and enhances thermal proteinstabilityrdquo Protein Science vol 10 no 2 pp 250ndash261 2001

[28] J H Harreld T Ebina N Tsubo and G Stucky ldquoManipulationof pore size distributions in silica and ormosil gels dried underambient pressure conditionsrdquo Journal of Non-Crystalline Solidsvol 298 no 2-3 pp 241ndash251 2002

[29] K M Manoj A Baburaj B Ephraim et al ldquoExplaining theatypical reaction profiles of heme enzymes with a novel mech-anistic hypothesis and kinetic treatmentrdquo PLoS ONE vol 5 no5 Article ID e10601 2010

[30] D Jung and M Hartmann ldquoOxidation of indole with CPO andGOx immobilized on mesoporous molecular sievesrdquo CatalysisToday vol 157 no 1ndash4 pp 378ndash383 2010

[31] M L Ferrer F del Monte and D Levy ldquoA novel and simplealcohol-free sol-gel route for encapsulation of labile proteinsrdquoChemistry of Materials vol 14 no 9 pp 3619ndash3621 2002

[32] M Sundaramoorthy J Terner and T L Poulos ldquoThe crystalstructure of chloroperoxidase a heme peroxidase-cytochromeP450 functional hybridrdquo Structure vol 3 no 12 pp 1367ndash13771995

[33] X W Yi A Conesa P J Punt and L P Hager ldquoExamining therole of glutamic acid 183 in chloroperoxidase catalysisrdquo Journalof Biological Chemistry vol 278 no 16 pp 13855ndash13859 2003

[34] S R Blanke S AMartinis S G Sligar L P Hager J J Rux andJ H Dawson ldquoProbing the heme iron coordination structureof alkaline chloroperoxidaserdquo Biochemistry vol 35 no 46 pp14537ndash14543 1996

[35] B Dunn and J I Zink ldquoProbes of pore environment andmolecule-matrix interactions in sol-gel materialsrdquo Chemistry ofMaterials vol 9 no 11 pp 2280ndash2291 1997

[36] W A Loughlin and D B Hawkes ldquoEffect of organic solvents ona chloroperoxidase biotransformationrdquo Bioresource Technologyvol 71 no 2 pp 167ndash172 2000

[37] E Kiljunen and L T Kanerva ldquoChloroperoxidase-catalysedoxidation of alcohols to aldehydesrdquo Journal of Molecular Catal-ysis B Enzymatic vol 9 no 4ndash6 pp 163ndash172 2000

[38] E N Kadnikova and N M Kostic ldquoOxidation of ABTS byhydrogen peroxide catalyzed by horseradish peroxidase encap-sulated into sol-gel glass Effects of glass matrix on reactivityrdquo

Journal of Molecular Catalysis B Enzymatic vol 18 no 1ndash3 pp39ndash48 2002

[39] A Venkateswara Rao and D Haranath ldquoEffect of methyltri-methoxysilane as a synthesis component on the hydrophobicityand some physical properties of silica aerogelsrdquo Microporousand Mesoporous Materials vol 30 no 2-3 pp 267ndash273 1999

[40] M T Reetz A Zonta and J Simpelkamp ldquoEfficient immo-bilization of lipases by entrapment in hydrophobic sol-gelmaterialsrdquo Biotechnology and Bioengineering vol 49 no 5 pp527ndash534 1996

[41] W Wiesner K-H van Pee and F Lingens ldquoPurification andcharacterization of a novel bacterial non-heme chloroperox-idase from Pseudomonas pyrrociniardquo Journal of BiologicalChemistry vol 263 no 27 pp 13725ndash13732 1988

[42] Y Xi Z Liangying and W Sasa ldquoPore size and pore-sizedistribution control of porous silicardquo Sensors and Actuators BChemical vol 25 no 1-3 pp 347ndash352 1995

[43] C Lin and J A Ritter ldquoEffect of synthesis pH on the structureof carbon xerogelsrdquo Carbon vol 35 no 9 pp 1271ndash1278 1997

[44] M Altstein G Segev N Aharonson O Ben-Aziz A Tur-niansky and D Avnir ldquoSol-gel entrapped cholinesterases amicrotiter plate method for monitoring anti-cholinesterasecompoundsrdquo Journal of Agricultural and Food Chemistry vol46 no 8 pp 3318ndash3324 1998

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials

Page 9: Silica Sol-Gel Entrapment of the Enzyme Chloroperoxidase

Journal of Nanotechnology 9

in silica nanostructures prepared at pH values of 70 and 80and then 60 [44]

4 Conclusion

The enzyme CPO was successfully entrapped inside a silicananostructure prepared from the precursor TMOS with orwithout addition of the hydrophobic modifier MTMS SinceCPO is stabile in acidic buffers we further modified the sol-gel procedure by using casting buffers with pH values of 4555 60 and 65The catalytic performance of optimized CPOsol-gel beads approached 18 relative to free CPO in solutionas assessed via the pyrogallol peroxidation assay A combi-nation of factors such as enzyme leakage from the sol-gelhost insufficient recovery from inactivation caused by initialmethanol exposure hindered product release or alternatereaction pathways are most likely responsible for the declinein catalytic performance of CPOafter sol-gel entrapmentTheuse of more acidic casting buffers in the sol-gel procedureprovided themost leverage for optimization by yieldingmoreporous silica nanostructures Overall our findings are ofimportance for the optimization of other sol-gel materialsdevised for applications in biosensing or biocatalysis ordesigned for the controlled release of bioactive compounds

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Funding for this project was obtained from the ResearchCorporation for Science Advancement (Cottrell College Sci-ence Award to Monika Sommerhalter) and California StateUniversity East Bay (Faculty and Student Research Grantsto Monika Sommerhalter Selina Chan and Tuan Le and aSieber-Tombari award to Monika Sommerhalter) ProfessorDaryl Eggers San Jose State University kindly invited theauthors to perform the CD measurements in his laboratoryThe authors are also grateful to Professor AnnMcPartland forproviding detailed feedback on their paper

References

[1] B C Dave B Dunn J S Valentine and J I Zink ldquoNanocon-fined proteins and enzymes sol-gel-based biomolecular mate-rialsrdquoNanotechnology ACS Symposium Series vol 622 pp 351ndash365 1996

[2] I Gill and A Ballesteros ldquoBioencapsulation within syntheticpolymers (part 1) sol-gel encapsulated biologicalsrdquo Trends inBiotechnology vol 18 no 7 pp 282ndash296 2000

[3] D Avnir T Coradin O Lev and J Livage ldquoRecent bio-applications of sol-gel materialsrdquo Journal of Materials Chem-istry vol 16 no 11 pp 1013ndash1030 2006

[4] D R Morris and L P Hager ldquoChloroperoxidase I Isolationand properties of the crystalline glycoproteinrdquo The Journal ofBiological Chemistry vol 241 no 8 pp 1763ndash1768 1966

[5] V Yazbik and M Ansorge-Schumacher ldquoFast and efficientpurification of chloroperoxidase from C fumagordquo Process Bio-chemistry vol 45 no 2 pp 279ndash283 2010

[6] M Hofrichter and R Ullrich ldquoHeme-thiolate haloperoxidasesversatile biocatalysts with biotechnological and environmentalsignificancerdquo Applied Microbiology and Biotechnology vol 71no 3 pp 276ndash288 2006

[7] V M Dembitsky ldquoOxidation epoxidation and sulfoxidationreactions catalysed by haloperoxidasesrdquoTetrahedron vol 59 no26 pp 4701ndash4720 2003

[8] L Santhanam and J S Dordick ldquoChloroperoxidase catalyzedepoxidation of styrene in aqueous and non-aqueous mediardquoBiocatalysis and Biotransformation vol 20 no 4 pp 265ndash2742002

[9] M Ayala N R Robledo A Lopez-Munguia and R Vazquez-Duhalt ldquoSubstrate specificity and ionization potential inchloroperoxidase-catalyzed oxidation of diesel fuelrdquo Environ-mental Science and Technology vol 34 no 13 pp 2804ndash28092000

[10] R Vazquez-Duhalt M Ayala and F J Marquez-Rocha ldquoBio-catalytic chlorination of aromatic hydrocarbons by chloroper-oxidase of Caldariomyces fumagordquo Phytochemistry vol 58 no6 pp 929ndash933 2001

[11] E Terres M Montiel S Le Borgne and E Torres ldquoImmo-bilization of chloroperoxidase on mesoporous materials forthe oxidation of 46-dimethyldibenzothiophene a recalcitrantorganic sulfur compound present in petroleum fractionsrdquoBiotechnology Letters vol 30 no 1 pp 173ndash179 2008

[12] V Trevisan M Signoretto S Colonna V Pironti and GStrukul ldquoMicroencapsulated chloroperoxidase as a recyclablecatalyst for the enantioselective oxidation of sulfides withhydrogen peroxiderdquo Angewandte Chemie International Editionvol 43 no 31 pp 4097ndash4099 2004

[13] N Spreti R Germani A Incani and G Savelli ldquoStabiliza-tion of chloroperoxidase by polyethylene glycols in aqueousmedia kinetic studies and synthetic applicationsrdquoBiotechnologyProgress vol 20 no 1 pp 96ndash101 2004

[14] J-B Park and D S Clark ldquoNew reaction system for hydrocar-bon oxidation by chloroperoxidaserdquo Biotechnology and Bioengi-neering vol 94 no 1 pp 189ndash192 2006

[15] J-Z Liu and M Wang ldquoImprovement of activity and stabilityof chloroperoxidase by chemical modificationrdquo BMC Biotech-nology vol 7 no 1 article 23 2007

[16] L Zhi Y Jiang Y Wang M Hu S Li and Y Ma ldquoEffects ofadditives on the thermostability of chloroperoxidaserdquo Biotech-nology Progress vol 23 no 3 pp 729ndash733 2007

[17] T A Kadima andM A Pickard ldquoImmobilization of chloroper-oxidase on aminopropyl-glassrdquo Applied and EnvironmentalMicrobiology vol 56 no 11 pp 3473ndash3477 1990

[18] Y-J Han J T Watson G D Stucky and A Butler ldquoCatalyticactivity of mesoporous silicate-immobilized chloroperoxidaserdquoJournal ofMolecular Catalysis B Enzymatic vol 17 no 1 pp 1ndash82002

[19] J Aburto M Ayala I Bustos-Jaimes et al ldquoStability andcatalytic properties of chloroperoxidase immobilized on SBA-16mesoporousmaterialsrdquoMicroporous andMesoporousMaterialsvol 83 no 1ndash3 pp 193ndash200 2005

[20] M Hartmann and C Streb ldquoSelective oxidation of indole bychloroperoxidase immobilized on the mesoporous molecularsieve SBA-15rdquo Journal of Porous Materials vol 13 no 3-4 pp347ndash352 2006

10 Journal of Nanotechnology

[21] S Hudson J Cooney B K Hodnett and E Magner ldquoChlorop-eroxidase on periodic mesoporous organosilanes immobiliza-tion and reuserdquo Chemistry of Materials vol 19 no 8 pp 2049ndash2055 2007

[22] R A Sheldon ldquoEnzyme immobilization the quest for optimumperformancerdquo Advanced Synthesis amp Catalysis vol 349 no 8-9pp 1289ndash1307 2007

[23] K Smith N J Silvernail K R Rodgers T E Elgren M Castroand RM Parker ldquoSol-gel encapsulated horseradish peroxidasea catalytic material for peroxidationrdquo Journal of the AmericanChemical Society vol 124 no 16 pp 4247ndash4252 2002

[24] K M Manoj and L P Hager ldquoChloroperoxidase a janusenzymerdquo Biochemistry vol 47 no 9 pp 2997ndash3003 2008

[25] V M Samokyszyn and P R Ortiz de Montellano ldquoTopology ofthe chloroperoxidase active site regiospecificity of heme mod-ification by phenylhydrazine and sodium aziderdquo Biochemistryvol 30 no 50 pp 11646ndash11653 1991

[26] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[27] D K Eggers and J S Valentine ldquoMolecular confinementinfluences protein structure and enhances thermal proteinstabilityrdquo Protein Science vol 10 no 2 pp 250ndash261 2001

[28] J H Harreld T Ebina N Tsubo and G Stucky ldquoManipulationof pore size distributions in silica and ormosil gels dried underambient pressure conditionsrdquo Journal of Non-Crystalline Solidsvol 298 no 2-3 pp 241ndash251 2002

[29] K M Manoj A Baburaj B Ephraim et al ldquoExplaining theatypical reaction profiles of heme enzymes with a novel mech-anistic hypothesis and kinetic treatmentrdquo PLoS ONE vol 5 no5 Article ID e10601 2010

[30] D Jung and M Hartmann ldquoOxidation of indole with CPO andGOx immobilized on mesoporous molecular sievesrdquo CatalysisToday vol 157 no 1ndash4 pp 378ndash383 2010

[31] M L Ferrer F del Monte and D Levy ldquoA novel and simplealcohol-free sol-gel route for encapsulation of labile proteinsrdquoChemistry of Materials vol 14 no 9 pp 3619ndash3621 2002

[32] M Sundaramoorthy J Terner and T L Poulos ldquoThe crystalstructure of chloroperoxidase a heme peroxidase-cytochromeP450 functional hybridrdquo Structure vol 3 no 12 pp 1367ndash13771995

[33] X W Yi A Conesa P J Punt and L P Hager ldquoExamining therole of glutamic acid 183 in chloroperoxidase catalysisrdquo Journalof Biological Chemistry vol 278 no 16 pp 13855ndash13859 2003

[34] S R Blanke S AMartinis S G Sligar L P Hager J J Rux andJ H Dawson ldquoProbing the heme iron coordination structureof alkaline chloroperoxidaserdquo Biochemistry vol 35 no 46 pp14537ndash14543 1996

[35] B Dunn and J I Zink ldquoProbes of pore environment andmolecule-matrix interactions in sol-gel materialsrdquo Chemistry ofMaterials vol 9 no 11 pp 2280ndash2291 1997

[36] W A Loughlin and D B Hawkes ldquoEffect of organic solvents ona chloroperoxidase biotransformationrdquo Bioresource Technologyvol 71 no 2 pp 167ndash172 2000

[37] E Kiljunen and L T Kanerva ldquoChloroperoxidase-catalysedoxidation of alcohols to aldehydesrdquo Journal of Molecular Catal-ysis B Enzymatic vol 9 no 4ndash6 pp 163ndash172 2000

[38] E N Kadnikova and N M Kostic ldquoOxidation of ABTS byhydrogen peroxide catalyzed by horseradish peroxidase encap-sulated into sol-gel glass Effects of glass matrix on reactivityrdquo

Journal of Molecular Catalysis B Enzymatic vol 18 no 1ndash3 pp39ndash48 2002

[39] A Venkateswara Rao and D Haranath ldquoEffect of methyltri-methoxysilane as a synthesis component on the hydrophobicityand some physical properties of silica aerogelsrdquo Microporousand Mesoporous Materials vol 30 no 2-3 pp 267ndash273 1999

[40] M T Reetz A Zonta and J Simpelkamp ldquoEfficient immo-bilization of lipases by entrapment in hydrophobic sol-gelmaterialsrdquo Biotechnology and Bioengineering vol 49 no 5 pp527ndash534 1996

[41] W Wiesner K-H van Pee and F Lingens ldquoPurification andcharacterization of a novel bacterial non-heme chloroperox-idase from Pseudomonas pyrrociniardquo Journal of BiologicalChemistry vol 263 no 27 pp 13725ndash13732 1988

[42] Y Xi Z Liangying and W Sasa ldquoPore size and pore-sizedistribution control of porous silicardquo Sensors and Actuators BChemical vol 25 no 1-3 pp 347ndash352 1995

[43] C Lin and J A Ritter ldquoEffect of synthesis pH on the structureof carbon xerogelsrdquo Carbon vol 35 no 9 pp 1271ndash1278 1997

[44] M Altstein G Segev N Aharonson O Ben-Aziz A Tur-niansky and D Avnir ldquoSol-gel entrapped cholinesterases amicrotiter plate method for monitoring anti-cholinesterasecompoundsrdquo Journal of Agricultural and Food Chemistry vol46 no 8 pp 3318ndash3324 1998

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials

Page 10: Silica Sol-Gel Entrapment of the Enzyme Chloroperoxidase

10 Journal of Nanotechnology

[21] S Hudson J Cooney B K Hodnett and E Magner ldquoChlorop-eroxidase on periodic mesoporous organosilanes immobiliza-tion and reuserdquo Chemistry of Materials vol 19 no 8 pp 2049ndash2055 2007

[22] R A Sheldon ldquoEnzyme immobilization the quest for optimumperformancerdquo Advanced Synthesis amp Catalysis vol 349 no 8-9pp 1289ndash1307 2007

[23] K Smith N J Silvernail K R Rodgers T E Elgren M Castroand RM Parker ldquoSol-gel encapsulated horseradish peroxidasea catalytic material for peroxidationrdquo Journal of the AmericanChemical Society vol 124 no 16 pp 4247ndash4252 2002

[24] K M Manoj and L P Hager ldquoChloroperoxidase a janusenzymerdquo Biochemistry vol 47 no 9 pp 2997ndash3003 2008

[25] V M Samokyszyn and P R Ortiz de Montellano ldquoTopology ofthe chloroperoxidase active site regiospecificity of heme mod-ification by phenylhydrazine and sodium aziderdquo Biochemistryvol 30 no 50 pp 11646ndash11653 1991

[26] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[27] D K Eggers and J S Valentine ldquoMolecular confinementinfluences protein structure and enhances thermal proteinstabilityrdquo Protein Science vol 10 no 2 pp 250ndash261 2001

[28] J H Harreld T Ebina N Tsubo and G Stucky ldquoManipulationof pore size distributions in silica and ormosil gels dried underambient pressure conditionsrdquo Journal of Non-Crystalline Solidsvol 298 no 2-3 pp 241ndash251 2002

[29] K M Manoj A Baburaj B Ephraim et al ldquoExplaining theatypical reaction profiles of heme enzymes with a novel mech-anistic hypothesis and kinetic treatmentrdquo PLoS ONE vol 5 no5 Article ID e10601 2010

[30] D Jung and M Hartmann ldquoOxidation of indole with CPO andGOx immobilized on mesoporous molecular sievesrdquo CatalysisToday vol 157 no 1ndash4 pp 378ndash383 2010

[31] M L Ferrer F del Monte and D Levy ldquoA novel and simplealcohol-free sol-gel route for encapsulation of labile proteinsrdquoChemistry of Materials vol 14 no 9 pp 3619ndash3621 2002

[32] M Sundaramoorthy J Terner and T L Poulos ldquoThe crystalstructure of chloroperoxidase a heme peroxidase-cytochromeP450 functional hybridrdquo Structure vol 3 no 12 pp 1367ndash13771995

[33] X W Yi A Conesa P J Punt and L P Hager ldquoExamining therole of glutamic acid 183 in chloroperoxidase catalysisrdquo Journalof Biological Chemistry vol 278 no 16 pp 13855ndash13859 2003

[34] S R Blanke S AMartinis S G Sligar L P Hager J J Rux andJ H Dawson ldquoProbing the heme iron coordination structureof alkaline chloroperoxidaserdquo Biochemistry vol 35 no 46 pp14537ndash14543 1996

[35] B Dunn and J I Zink ldquoProbes of pore environment andmolecule-matrix interactions in sol-gel materialsrdquo Chemistry ofMaterials vol 9 no 11 pp 2280ndash2291 1997

[36] W A Loughlin and D B Hawkes ldquoEffect of organic solvents ona chloroperoxidase biotransformationrdquo Bioresource Technologyvol 71 no 2 pp 167ndash172 2000

[37] E Kiljunen and L T Kanerva ldquoChloroperoxidase-catalysedoxidation of alcohols to aldehydesrdquo Journal of Molecular Catal-ysis B Enzymatic vol 9 no 4ndash6 pp 163ndash172 2000

[38] E N Kadnikova and N M Kostic ldquoOxidation of ABTS byhydrogen peroxide catalyzed by horseradish peroxidase encap-sulated into sol-gel glass Effects of glass matrix on reactivityrdquo

Journal of Molecular Catalysis B Enzymatic vol 18 no 1ndash3 pp39ndash48 2002

[39] A Venkateswara Rao and D Haranath ldquoEffect of methyltri-methoxysilane as a synthesis component on the hydrophobicityand some physical properties of silica aerogelsrdquo Microporousand Mesoporous Materials vol 30 no 2-3 pp 267ndash273 1999

[40] M T Reetz A Zonta and J Simpelkamp ldquoEfficient immo-bilization of lipases by entrapment in hydrophobic sol-gelmaterialsrdquo Biotechnology and Bioengineering vol 49 no 5 pp527ndash534 1996

[41] W Wiesner K-H van Pee and F Lingens ldquoPurification andcharacterization of a novel bacterial non-heme chloroperox-idase from Pseudomonas pyrrociniardquo Journal of BiologicalChemistry vol 263 no 27 pp 13725ndash13732 1988

[42] Y Xi Z Liangying and W Sasa ldquoPore size and pore-sizedistribution control of porous silicardquo Sensors and Actuators BChemical vol 25 no 1-3 pp 347ndash352 1995

[43] C Lin and J A Ritter ldquoEffect of synthesis pH on the structureof carbon xerogelsrdquo Carbon vol 35 no 9 pp 1271ndash1278 1997

[44] M Altstein G Segev N Aharonson O Ben-Aziz A Tur-niansky and D Avnir ldquoSol-gel entrapped cholinesterases amicrotiter plate method for monitoring anti-cholinesterasecompoundsrdquo Journal of Agricultural and Food Chemistry vol46 no 8 pp 3318ndash3324 1998

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials

Page 11: Silica Sol-Gel Entrapment of the Enzyme Chloroperoxidase

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials


Recommended