tate Key Laboratory of Materials-Oriented Chemical Engineering, College of Life Science and Pharmaceutical Engineering, Nanjing University ofechnology, Nanjing 210009, PR China
r t i c l e i n f o
rticle history:eceived 28 August 2013eceived in revised form4 November 2013ccepted 28 December 2013vailable online 7 January 2014
dsorptionuclease P1
a b s t r a c t
The reversible immobilization of crude nuclease P1 fermented by Penicllium citrinum and used withoutfurther purification was performed via adsorption on a weak base anion resin activated by polyethylen-imine (PEI). The immobilization conditions, including PEI concentration, the amount of support, theimmobilization time, and the reusability of the PEI-activated resin were investigated. The results haveshown that the PEI-activated resin has the capability of selectively adsorbing nuclease P1. Subsequently,the functional properties of the immobilized nuclease P1 were studied and compared to those of the freeenzyme. The apparent Km value for immobilized nuclease P1 on the activated resin (15.31 g L−1) was about4.41-fold higher than that of the free enzyme (3.47 g L−1), and the apparent Vmax value of the immobilized
enzyme (530 U g ) was about 3.9-fold less than that of the free enzyme (2082 U mL ). The optimumtemperature was observed to be 70 ◦C, 15 ◦C higher than that of the free enzyme. The optimum pH wasthe same for both free and immobilized nuclease P1 (pH 5.0). The apparent activation energies (Ea) of thefree and immobilized nuclease P1 were 163.09 kJ mol−1 and 156.32 kJ mol−1, respectively, implying thatthe catalytic efficiency of the immobilized enzyme was restricted by mass-transfer rather than kineticlimitations.
. Introduction
Taking into account the numerous problems associated withractical applications of free enzymes (e.g., stability, reusability,eparation from substrates and products, and operational costs),he development of cost-effective and efficient strategies fornzyme immobilization has attracted significant attention inhe scientific and engineering communities [1–3]. During theast decade, immobilized enzymes have been mostly used inhe production of food, pharmaceuticals, and other biologicallymportant fine products [4]. Among the immobilization techniquessed, adsorption may have a higher commercial potential thanther methods because the adsorption process is simple and lessxpensive, causes minimal inactivation of enzymes, and, most
mportantly, the support can easily be repeatedly reused after inac-ivation of the immobilized enzyme [5–8]. But the main drawbackf the simple reversible adsorption protocols (e.g., on conventional
anionic exchange resins) is that the interaction between theenzyme and the support is generally not very strong so that someof the adsorbed enzyme may be released from the support duringwashing and other procedures [6,9,10]. To overcome this draw-back, reversible enzyme immobilization via strong hydrophobic orhydrophilic interactions, especially ionic interactions between theenzyme and support as described in previous reports [3,6,11], canpreclude enzyme leakage under mild conditions.
Reversible immobilization techniques are based on thereversible interaction between different amino acid residues on theenzyme and groups present on the surface of the solid support [12].In the reversible enzyme immobilization, Ionic adsorption, as thefirst method used on an industrial scale [13–15], seems to be asuitable methodology for immobilizing enzymes because it is verysimple and requires very little work or time. Moreover, the supportmaterials can be regenerated after desorption of the inactivatedenzyme using the suitable condition and suitable desorption agentwith subsequent reduction of industrial waste and economic sav-
ings [11,16–19]. On the other hand, enzyme adsorption on thesupport could induce minimal distortion because the flexible sup-port adapts itself to the protein structure as a result of the intenseelectrostatic interaction [1,16,18]. The reversible immobilization of
nzymes onto composites coated with flexible polymers containingigh-density ion-exchange groups [3,6,20–22] has been proposeds a very suitable method for reversible but very strong proteinmmobilization, and desorption of protein from the functionalizedupport was found to require a change in pH or an increase in ionictrength after inactivation of the enzyme upon use [6].
PEI is a polymer having a high density of ionizable tertiary, sec-ndary and primary amino groups. Most multilayer enzymes mayecome strongly adsorbed via ionic exchange on supports coatedith PEI at neutral pH values [23]. We also tried to immobilize
′-adenylic deaminase, �-galactosidase on PEI-activated resin andhowed good results. Garcia-Galan et al. [24] have researched thattabilization of glutamate dehydrogenase (GDH) which is a hexam-ric protein was increased and the activity was maintained in theresence of polyethyleneimine (PEI) because subunit dissociationas restricted.
Nuclease P1 (EC 3.1.30.1) is a multi-zinc hydrolase with a molec-lar mass of about 36 kDa, and it is a key enzyme used in theydrolysis of ribonucleic acid (RNA) to nucleotides which areidely used in the field of medicine and in the food and phar-aceutical industries [25–27]. It has been shown to be a single
trand-specific nuclease by Fujimoto et al. [28] and a glycol-enzymeonsisting of 270 amino acid residues with two disulfide bands25]. Nuclease P1 has been immobilized on natural (organic andnorganic) and synthetic supports [29–32]. In particular, Roku-awa et al. [33,34] have researched the reversible immobilization ofighly purified nuclease P1 on titanium-activated supports. How-ver, such supports need to be made reusable by modificationsing titanium tetrachloride solution. To overcome this problemnd decrease the cost of the commercial enzyme, a weak base anionesin activated by polyethylenimine (PEI) was used for reversiblemmobilization of crude nuclease P1 solution that was fermentednd isolated in our laboratory, and the properties of the immobi-ized enzyme were investigated in this study.
. Materials and methods
.1. Materials
The weak base anion resin, AD-5, which has polyamine groupsn a methyl acrylate-divinylbenzene copolymer with macroporoustructure, was a gift from National Engineering Technique Researchenter for Biotechnology (Nanjing, China). According to the instruc-ions, the resin was successively soaked for 8 h each in ethanol,
M sodium hydroxide, and 3 M hydrochloric acid to remove impu-ities which were residual in the synthesis process. After eachtep, the resin should be washed to neutral pH with deionizedater. The RNA for analyzing enzyme activity was purchased from
hen-Ao Company (purity 90%). The RNA for circular catalysis wasindly provided by Nanjing Biotogether Co. Ltd. (purity about 80%).olyethylenimine (PEI, MW 70 kDa) was purchased from Aladdinhemistry Co. Ltd. All other chemicals were of analytical grade andsed without further purification.
.2. Microorganism and nuclease P1 preparation
The nuclease P1-producing strain Penicllium citrinum was iso-ated and stored in our laboratory. The fermentation conditionsave been previously described by He et al. [35]. The fermentationolution containing nuclease P1 was harvested by centrifugation
GL-21M, Shanghai, China) at 11,392 × g for 15 min at 4 ◦C. Then,he supernatant was enriched approximately 5-fold using a hollowber ultrafiltration machine (Tianjin, China). The specific activityf the crude enzyme was approximately 3734 U mg−1. The enzymeas stored at 4 ◦C and analyzed in subsequent studies.
B: Enzymatic 101 (2014) 92– 100 93
2.3. Preparation of PEI-activated supports
The activation of the weak base anion resin (AD-5) was based onthe method of Arica et al. [6]. The AD-5 resin was suspended in twicethe volume of different concentrations of PEI (ranging from 0% to12.5% (w/v)) at 65 ◦C for 5 h with stirring. After this period, the acti-vated beads were removed from the PEI solution and washed withdeionized water, and then collected by vacuum suction filtration.
The surface morphologies of the resin, PEI-activated resin andimmobilized enzyme were examined by using scanning electronmicroscope (SEM, PHILIPS, XL-30ESEM, Holland). The nitrogencontent on the surface of resins was determined using an energy-dispersive X-ray spectroscopy (EDS, PHILIPS, XL-30ESEM, Holland).
2.4. Enzyme immobilization
During this procedure, unless stated, immobilization was per-formed by suspending 2 g PEI-activated support in 5 mL of aqueousnuclease P1 solution at natural pH (approximately pH 4.0). The mix-ture was incubated at room temperature and 150 rpm in a rotaryshaker (HYG-A, Taicang, China) for different time (from 5 min to180 min). The schematic representation of reversible immobiliza-tion of nuclease P1 on PEI-activated resin is presented in Fig. 1.After immobilization, the immobilized enzyme was collected andwashed intensively with deionized water to remove the unboundenzyme and stored at 4 ◦C in aqueous solution.
2.5. Activity assays of free and immobilized enzyme
The activities of both free and immobilized nuclease P1 werebased on the method described previously [27,32]. 0.1 mL freeenzyme or 0.1 g immobilized nuclease P1 was incubated with theheated substrate solution (1 mL of 5% RNA; 0.9 mL of 0.2 M acetatebuffer at pH 5.0; 3 mM Zn2+) at 70 ◦C for 15 min. Then, 2.0 mL ofice-cold nucleic acid precipitator (0.25% ammonium molybdate dis-solved in 2.5% perchloric acid, w/v) was added and the mixturewas settled in an ice-bath for 10 min. The mixture was centrifuged(Centrifuge 5415D, Eppendorf, Hamburg, Germany) at 14,645 × gfor 5 min. The supernatant was diluted 250-fold and measured at260 nm with a UV–vis spectrophotometer (UV-2000, UNICO, USA)against a blank incubation without enzyme. One unit of enzymeactivity was defined as the amount of enzyme that produced anincrease in optical density of 1.0 in 1 min at 260 nm.
The effects of pH and temperature on the free and immobilizedenzyme were studied in the pH range 3.5–7.0 and the temperaturerange 30–80 ◦C, respectively. Relative activities were calculated asthe ratio of the activity of the enzyme measured at different pHand temperature values to the maximal activity of the enzyme.In addition, below the inactivation temperature, the temperaturedependence of the rate constant can be described by the Arrheniusequation [36]
k = A × e−Ea/RT
where k is the rate constant, A is the pre-exponential factor, Ea is theapparent activation energy, R is the gas constant (8.31 J mol−1 K−1),and T is the absolute temperature. The apparent activation energy(Ea) of catalysis was determined by the slope of the Arrhenius plotusing the following equation:
Slope = −Ea
2.303R
2.6. Determination of protein concentration
Protein concentration was determined as described by Bradford[37] using bovine serum albumin as a reference. The amount of
94 B. Li et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 92– 100
F on PEr
ai
Q
wre(i
2
i(dalM
2
rrm1os
ig. 1. The schematic representation of reversible immobilization of nuclease P1
eusability of the PEI-activated resin.
dsorbed protein (i.e., adsorption capacity) was calculated accord-ng to the following equation:
= (C0 − C) × V
m
here Q is the amount of enzyme adsorbed on 1 g of PEI-activatedesin (mg g−1), C0 and C are the concentrations of protein in thenzyme solution before and after immobilization, respectivelymg mL−1), V is the volume of the enzyme solution (mL), and ms the mass of the support (g).
.7. Determination of kinetic parameters
The activity profiles of free or immobilized enzyme werenvestigated by reaction with various concentrations of RNA2–20 mg mL−1) in acetate buffer (0.2 M, pH 5.0) at 70 ◦C forifferent time periods. The Michaelis–Menten constant (Km)nd maximum initial reaction rate (Vmax) of free and immobi-ized enzyme were determined by non-linear analysis using the
ichaelis–Menten equation.
.8. Reusability of the PEI-activated resin
In order to determine the reusability of the PEI-activatedesin, the enzyme adsorption–catalysis–desorption cycle wasepeated six times using the same support. For these experi-
ents, 10 g immobilized enzyme were packed in a glass column (˚
2 mm × 200 mm), equipped with a water jacket at 70 ◦C. 250 mLf 2% RNA solution, at pH 5.0 containing 0.5 M zinc sulfate andtored in a flask, were continuously recirculated for 2 h through
I-activated resin and device for continuously hydrolysis of RNA to evaluate the
the immobilized enzyme column from top to bottom at a flow rateof 5 mL min−1 using a peristaltic pump (seen from Fig. 1). At the endof each batch, the matrices containing the inactivated nuclease P1were suspended in 2 M NaCl solution with a stirring rate of 150 rpmin a rotary shaker (HYG-A, Taicang, China) at room temperature for30 min (longer incubation times (up to 2 h) did not result in signifi-cant increments in desorbed protein). The support was thoroughlywashed with deionized water and then suspended in fresh enzymesolution for immobilization studies as described previously.
3. Results and discussion
3.1. Properties of AD-5 resins
The representative processes of polycationic polymer (i.e., PEI)-activated AD-5 resin and reversible immobilization are presentedin Fig. 1. Morphology of support matrices is an important factorin terms of kinetic behavior of the immobilized enzyme. Scanningelectron microscope images of the AD-5 resins in the unmodified,modified and immobilized enzyme states are shown in Fig. 2. Asseen in Fig. 2, the AD-5 resins have a smooth and corrugated sur-face with some macroporous structures. The surface properties ofthe AD-5 resins would increase the surface area for binding PEI andthe enzyme. Although the surfaces of the unmodified and modifiedresins have no significant changes, the nitrogen content on the sur-
face of resins has increased significantly (shown in Table 1). Whenenzyme was immobilized on the modified polymers, the contentof nitrogen decreased. These phenomena were presented becauseof the different nitrogen content of the resin (18.06%), PEI (33.33%)
B. Li et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 92– 100 95
Table 1The nitrogen content on the surface of resins in the non-immobilized and immobilized enzyme states.
PEI concentration (%) 0 2.5 5 12.5
20.719.1
ae
sdsbr
3
aibiZ
i5
Ft
Non-immobilized enzyme 18.06 ± 0.816
Immobilized enzyme 18.21 ± 2.393
nd enzyme (16%). These results demonstrated that the PEI andnzyme were bound on the polymers.
The water content of the polymers was determined as 48%. Thisupport was highly stable both chemically and mechanically, andid not swell or shrink in aqueous solution. Thus this support isuitable to use as column packing material for application of immo-ilized enzyme in a continuous flow system, even in a stirred tankeactor.
.2. Immobilization of nuclease P1 on PEI-activated resin
In order to optimize the immobilization of nuclease P1 on PEI-ctivated resin, the PEI concentration, the amount of resin, and themmobilization time were investigated for each individual set ofatch immobilization experiments. The activities of the free and
mmobilized nuclease P1 have been determined using RNA from
hen-Ao Company as substrate.
The activity of the immobilized enzyme is one of the mostmportant aspects in the immobilization process. The effect of AD-
resin activated degree by different concentration of PEI on the
ig. 2. SEM micrographs: (A) unmodified resin; (B) modified resin with 5% PEI solu-ion; and (C) immobilized enzyme.
immobilization of the crude nuclease P1 fermented by P. citrinumwas studied. In order to saturate the support with proteins, 1 g sup-port was suspended in 5 mL enzyme solution. As seen in Fig. 3, anincrease in the PEI concentration led to an increase in activity ofthe immobilized enzyme, but this leveled off at a PEI concentra-tion of 5–10%. Although the weak base anion resin has polyaminegroups with a macroporous structure, very little activity of immo-bilized enzyme was detected on unactivated resin, implying thatthe amine groups are inactive so that it hardly binds the enzyme.If the resin soaked order in NaOH and HCl solution is reversed, theimmobilized enzyme will be rarely active. Moreover, it is interest-ing that the activities of the derivatives decreased when the PEIconcentration was higher than 10%.
Optimum pH of polyethyleneimine and resin has been studiedand the results presented in Table 2. In acidic and concentratedalkaline conditions, the immobilized enzyme activities were com-parable to that immobilized on unactivated resin. However, theimmobilized enzyme showed the higher activity at natural pH(approximately pH 11.0) than that at below and above pH 11.0. Sothe natural pH was considered optimum pH of polyethyleneimineand resin. In the present study, the crude enzyme solution pH wasobserved at around 4.0, and was shifted about 0.5 unit to less alka-line pH than the isoelectric point (PI) of nuclease P1 (around 4.5)[38]. Thus, nuclease P1 had a net positive charge. On the otherhand, the pKa value of the amino groups of PEI is about 7.1 [6],so the surface of activated resins had a negative charge at natu-ral pH. Theoretically, there is mutual repulsion between enzymeand support when the medium pH is 4.0. However, research hasshown that the maximum activities of the immobilized nucleaseP1 were obtained and the immobilized enzyme properties, such asstability of the immobilized enzyme, had minor differences in themedium pH range 3.5 to 5.0 (data not shown). This result showsthat hydrogen bonding, hydrophobic interactions, and multipoint
ionic interactions between the support and the protein are the pref-erential forces, the latter between the amino groups of the PEI andthe groups of carboxylic acid side-chains of the enzyme molecules.
Fig. 3. Effect of AD-5 resin activated degree by different concentration of PEI on thenuclease P1 immobilization. Immobilization was carried out at room temperaturefor 2 h with 1 g support suspended in 5 mL enzyme solution.
96 B. Li et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 92– 100
Table 2Effect of PEI solution pH on the activated extent of resins; the activated extent was shown through the activities of the immobilized enzyme.
pH Natural (about 11.0) 4 7 9 12 2 mol L−1 NaOH
P 5% sol
car[pnistaeabiac
laWeiatbtsitpry
FP
Enzyme activities (U g−1) 530 ± 90 63.6 ± 4.6
EI solution pH was adjusted by 3 M HCl and 5 M NaOH, and then was diluted into
Protein adsorption does not necessitate an overall oppositeharge between the protein and the ionic exchangers becausedsorption to the support may occur through oppositely chargedegions on the surface of the protein [22]. Fuentes and co-worker39] reported that a support may be able to ionically exchangeroteins under identical experimental conditions even if it has noet charge but has a mixture of cationic and anionic groups, or if
t is coated with anionic or cationic exchangers, principally if theupport is grafted with a polymer (e.g., polyethylenimine or dex-ran sulfate). Thus, to some extent, nuclease P1 is able to becomedsorbed on PEI-activated resin at a pH under and below its iso-lectric point, implying that the enzyme involved in cationic andnionic rich regions. However, when the electrostatic repulsionetween the enzyme and the support achieves a certain degree,
t would become the preferential force. This may explain why thectivity of the immobilized enzyme decreased when the PEI con-entration was higher than 10%.
Fig. 4 shows that the quantity of support affected the immobi-ized enzyme activity, residual enzyme activity in the supernatant,nd the amount of protein adsorbed on the PEI-activated resin.hen the amount of support was less than 2.0 g, the immobilized
nzyme displayed maximal enzyme activity, whereas the activ-ty in the supernatant decreased linearly. It is interesting that,lthough the protein adsorbed per gram support decreased whenhe amount of resin was from 1.5 g to 2.0 g, the activity of the immo-ilized enzyme remained constant. In addition, only 56% of theotal offered protein was immobilized when 2 g of PEI-activatedupport were added to 5 mL of enzyme solution, while 71% of thenitial activity was immobilized. These phenomena may imply that
he PEI-activated support tends to selectively adsorb the targetrotein, i.e., nuclease P1, because the affinity between PEI-AD-5esin and nuclease P1 was stronger than others. However, anal-sis of the activity of the immobilized enzyme has shown that
ig. 4. Effect of the quantity of support on the immobilized enzyme activity, residual
EI-activated resin. The immobilization experiments were performed for 2 h as described
90.1 ± 5.6 260 ± 13 480 ± 63 57.3 ± 2.4
ution.
the retained activity, expressed as a percentage of the theoreti-cal enzyme activity immobilized on the support, was only 14.6%.This result indicated that nuclease P1 immobilization brings abouta drop in the activity which may be due to: (1) a change in spatialconformation of the enzyme induced by the immobilization; (2)poorer accessibility of the enzyme active sites caused by randomimmobilization, which restricts diffusion of the macromolecularsubstrate into the active sites [12,40,41]; (3) the formation of mul-tiple enzyme layers on the surface of the support [42]; or (4)adsorption of the product on the resin.
Fig. 5 shows the time courses of immobilization of nuclease P1from P. citrinum on the PEI-activated support. These data showthat the amount of adsorbed protein was positively correlatedwith the activity of the immobilized enzyme up to 60 min. Thisresult indicated that more than 55% of the activity and 56.1% ofthe protein was immobilized after 60 min. Although the amountof adsorbed protein increased only slightly thereafter, the activityof the immobilized enzyme increased by nearly 18%. Furthermore,when immobilized enzyme was suspended in 0.4 M NaCl solutionfor 30 min with agitation, the enzyme activity in the supernatantwas almost equal to the loss of the raw enzyme and there is onlyone main band by SDS-PAGE analysis (shown in Fig. 6). This resultfurther demonstrated that the PEI-activated support had the abilityto selectively adsorb nuclease P1. As shown in Fig. 5, an immobiliza-tion time of 120 min was considered ideal to reach the maximumimmobilization of enzyme on the support.
Several reports have described nuclease P1 immobilization[30–34]. The activities of the enzyme immobilized on oxiraneacrylic resins [30] and porous ceramics [32] were 15 U g−1
and 20 U g−1 support, respectively. The appropriate amount ofimmobilized protein was considered to be 8–10 mg g−1 titaniumchloride-activated pumice and the maximum activity of the deriva-tive was 448 U g−1 support [34]. The specific activity of the
enzyme activity in the supernatant, and the amount of protein adsorbed on the in Section 2.4.
B. Li et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 92– 100 97
e PEI-
iptesh
Fasbt
Fig. 5. Immobilization courses of nuclease P1 from Penicllium citrinum on th
mmobilized enzyme on avicel was 16.6 U mg−1 proteins [32]. In theresent study, the PEI-activated resin resulted in a lower adsorp-
ion capacity (about 0.87 mg g−1 resin) and a higher immobilizednzyme activity (approximately 530 U g−1 support). In addition, theupport also has the ability of selectively adsorbed nuclease P1 andas the advantage of reusability.
ig. 6. SDS-PAGE gels of protein which was desorbed from the immobilized enzymet growing ionic strength. Line 1 to line 4 were 0.8 M, 0.6 M, 0.4 M and 0.2 M NaClolution as desorption reagents, respectively. Line 5: molecular weight markers (theand were 116 kDa, 66.2 kDa, 45.0 kDa, 35.0 kDa, 25.0 kDa, 18.4 kDa, 14.4 kDa fromop to bottom). Nuclease P1 is a protein with a molecular mass of about 36 kDa.
activated support; experiments were carried out as described in Section 2.
3.3. Kinetic parameters
The kinetic parameters, Km and Vmax, for nuclease P1 were cal-culated from the Michaelis–Menten equation at 70 ◦C and pH 5.0,while varying the substrate concentration. Vmax reflects the intrin-sic characteristics of the enzyme, and may be affected by diffusionlimitations. Km, or apparent Km, reflects the effective characteris-tics of the enzyme and depends upon both partition and diffusioneffects [41]. The values of Km and Vmax for the free and immobilizedenzyme are shown in Table 3. As expected, Km and Vmax were sig-nificantly affected by immobilization onto the PEI-activated resin.The apparent Km value of the immobilized nuclease P1 on the PEI-activated resin was higher than that of the free form, and theapparent Vmax value of the immobilized enzyme was 530 ± 34 U g−1
support, while Vmax of the free enzyme was 2082 ± 33 U mL−1. Theapparent Km values were 3.47 ± 0.21 g L−1 and 15.31 ± 1.82 g L−1 forthe free and immobilized enzyme, respectively. An increase in Km
value indicates that the immobilized enzyme has an apparentlylower affinity for its substrate than the free enzyme. This may beattributed to substrate diffusional limitations, steric hindrance ofthe active site by the support, or the loss of enzyme flexibility nec-essary for substrate binding [36,42]. The decrease in Vmax value asa result of immobilization is considered to be associated with theKm value since the lower the value of Km, the greater the affinitybetween the enzyme and substrate [20].
3.4. Effect of pH and temperature on nuclease P1 activity
The effect of pH on immobilized enzyme activity depends on thefree enzyme, the immobilization method, and the carrier used [43].
Table 3Enzymatic properties of free and immobilized nuclease P1.
98 B. Li et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 92– 100
Fig. 7. Effect of pH (A) and temperature (B) on the activity of free and immobilizedna
Tdpinbpccb
nartiFwTomm
e
uclease P1; the relative activities at the optimum pH and temperature were takens 100% for free and immobilized nuclease P1, respectively.
he effect of pH on the activity of free and immobilized enzyme wasetermined in the pH range of 3.5–7.0 at 70 ◦C, and the results areresented in Fig. 7(A). As can be seen from these curves, enzyme
mmobilization did not cause a shift in the optimal pH. However,uclease P1 immobilized on the PEI-activated resin exhibited aroader pH tolerance than the free enzyme, implying that the sup-ort microenvironment affects the reaction conditions. Filho ando-workers [22] reported that �-galactosidase immobilized on PEI-oated supports presented a greater activity at alkaline pH valuesecause of the increased stability of the enzyme.
The effect of temperature on the activity of free and immobilizeduclease P1 was determined in the temperature range 30 ◦C to 80 ◦Ct pH 5.0. Although the effects of changes in temperature on theates of enzyme-catalyzed reactions do not provide much informa-ion on the mechanism of catalysis, these effects could be importantn indicating structural changes in the enzymes [20,44] As shown inig. 7(B), the apparent temperature optimum for the free enzymeas about 55 ◦C, while that for the immobilized enzyme was 70 ◦C.
he increase in optimum temperature may be due to the tendencyf the PEI-activated support to absorb nuclease P1 because the opti-um temperature of the purified nuclease P is 70 ◦C [27,28], or it
1ay be because of the improvement in the enzyme rigidity [8,36].The apparent activation energies (Ea) of free and immobilized
nzyme were also evaluated using an Arrhenius plot. The regression
Fig. 8. Thermal stability of the free and immobilized nuclease P1; the relative activitywas expressed as a percentage of the original activity assayed without heating.
equations for the Arrhenius plots of free and immobilized nucleaseP1 were y = −8.522x + 33.796 (R2 = 0.99) and y = −8.168x + 30.349(R2 = 0.97), respectively. As shown in Table 3, the catalytic effi-ciency of the immobilized enzyme was slightly higher than thatof the free enzyme because immobilization lowered the activationenergy (from 163.09 ± 7.50 kJ mol−1 to 156.32 ± 13.91 kJ mol−1).This result also demonstrated that the decrease in the retainedactivity (i.e., immobilized yield) was mainly caused by mass-transfer effects rather than kinetic limitations because of nosignification difference in the apparent activation energies of bothfree and immobilized enzyme.
The thermal stability of the immobilized and free enzyme wasmeasured at 70 ◦C for different time periods (the immobilizedenzyme should be stayed moist) and the results of residual activityare showed in Fig. 8. It is obvious that the stability of the immo-bilized enzyme at 70 ◦C shows remarkably higher durability thanits free form. Approximately 54% of the free enzyme activity waslost after 10 h of heat treatment, while the immobilized enzymeretained about 80% of initial activity after 18 h of heat treatment.These results were very promising for industrial use.
3.5. Reusability of the PEI-activated resin
Since the yeast RNA from Nanjing Biotogether Co. Ltd. wasof a technical grade (purity about 80%), pigments and sedimentsderived from the RNA preparation were adsorbed (or adhered)to the resin with an accompanying loss in enzymatic activity inthe course of the continuously recirculated reaction. The immo-bilized enzyme must be washed by heavily brine (1 mol L−1) withthe enzyme leakage from the support. So the reusability of theimmobilized enzyme was not unsatisfactory. Regeneration of thesupport is a crucial step for reversible enzyme immobilizationtechniques. Thus, it is necessary to evaluate the regenerationefficiency of the support after inactivation of the adsorbed enzyme.In order to demonstrate the reusability of the PEI-activated resin,the adsorption–hydrolysis–desorption cycle of nuclease P1 wasrepeated six times using the same resin. Although the adsorptioncapacity of the PEI-activated resin did not significantly changeduring the adsorption–hydrolysis–desorption cycles, the finalabsorbance of the RNA hydrolysate decreased slightly after eachcycle (seen from Fig. 9). According to the residual enzyme activity
in the supernatant (data not shown) and the amount of adsorbedprotein after each cycle, we can surmise that the selective adsorp-tion ability of PEI-activated resin to nuclease P1 was weakened.
B. Li et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 92– 100 99
F ilizatic
Tt
stdmIseascelscnohis2iwKibR
4
iurta
ig. 9. Reusability of the PEI-activated weak base anion resin in nuclease P1 immobycles.
his may be attributed to the change of the surface properties ofhe resin caused by the pigments and sediments.
In the previous papers [38,45,46], nuclease P1 did not onlyhow the phosphodiesterase activity toward RNA and DNA andhe phosphomonesterase activity toward 2′- and 3′-AMP, but beiscovered to catalyze asymmetric aldol reactions between aro-atic aldehydes and cyclic ketones under solvent-free condition.
n the process of RNA preparation, DNA and other by-products areeparated from the RNA with great difficulty. Immobilization ofnzymes has been discovered to enhance the enzyme observedctivity, specificity or selectivity [47–49]. In order to evaluate theelectivity of the immobilized nuclease P1, the RNA hydrolysateatalyzed by free enzyme, immobilized enzyme and a immobilizednzyme packed-bed system was analyzed by high performanceiquid chromatography (HPLC, Agilent Technologies 1200 series)upplied with a BF-C18(B) column (GALAK, Wuxi, China). Theolumn was eluted at room temperature with 20 mmol L−1 Ammo-ium dihydrogen phosphate and 3.5% (v/v) methanol at a flow ratef 1.0 mL min−1. The results showed that the purity of the RNAydrolysate catalyzed by free enzyme, immobilized enzyme and a
mmobilized enzyme packed-bed system, defined as the ratio of theum peak area of four ribonucleotides to the total peak area, was3.2%, 43.1% and 78.6%, respectively. These results indicated that
mmobilized enzyme could enhance the purity of RNA hydrolysatehich was very favorable for separation of the four nucleotides.eller et al. [47] investigated the selectivity of the nuclease P1
mmobilized on the copolymers, which demonstrated the immo-ilized enzyme had a surprisingly high selectivity with respect toNA of 100%.
. Conclusion
In this work, a weak base anion resin activated by polyethylen-mine has been used to immobilize nuclease P1. This process opened
p a simple route of enzyme immobilization on the PEI-activatedesin without costly enzyme purification, and it was demonstratedhat the support had the capability of selectively adsorbing nucle-se P1. In addition, this support was highly stable both chemically
on; the relative absorbance at the first cycle was taken as 100% for the subsequent
and mechanically, and did not swell or shrink in aqueous solu-tion. Thus, the immobilized enzyme could be applied in packed-bedreactors for continuous hydrolysis of RNA. The optimum pH wasnot affected by immobilization, whereas the optimum temperaturewas shifted from 55 ◦C to 70 ◦C upon immobilization. Additionally,immobilization of nuclease P1 on the PEI-activated resin enhancedthe tolerance of the enzyme to changes in pH and temperature.Moreover, the regenerated resin could be reused for the reversibleimmobilization of the same or a different enzyme, which couldsignificantly lower the cost in possible large-scale applications.
Acknowledgement
This work was supported by the National Outstanding YouthFoundation of China (Grant no.: 21025625), the National High-Tech Research and Development Program of China (863) (Grantno.: 2012AA021200), the National Basic Research Program of China(973) (Grant no.: 2011CBA00806), the National Key TechnologyR&D Program (2012BAI44G01), the Program Changjiang Scholarsand Innovative in University (Grant no. IRT1066), the NationalNatural Science Foundation of China, Youth Program (Grant no.:21106070), the Jiangsu Provincial Natural Science Foundation ofChina (Grant no.: BK2011031), the Priority Academic ProgramDevelopment of Jiangsu Higher Education Institutions (PAPD)and the Jiangsu Provincial Graduate Student innovation Project(CXZZ13 0457).
References
[1] J. Wang, G. Zhao, Y. Li, X. Liu, P. Hou, Appl. Microbiol. Biotechnol. 2 (2013)681–692.
[2] J.K. Lai, T.H. Chuang, J.S. Jan, S.S.S. Wang, Colloids Surf., B 80 (2010) 51–58.[3] B.C.C. Pessela, R. Fernandez-Lafuente, M. Fuentes, A. Vian, J.L. Garcia, A.V. Gar-
[7] G. Bayramoglu, B. Karagoz, B. Altintas, M.Y. Arica, N. Bicak, Bioprocess Biosyst.Eng. 34 (2011) 735–746.
[8] G. Bayramoglu, A.U. Metin, B. Altintas, M.Y. Arica, Bioresour. Technol. 101(2010) 6881–6887.
[9] A.C. Olson, W.L. Stanley, J. Agric. Food Chem. 21 (1973) 440–445.10] R.A. Sheldon, Adv. Synth. Catal. 349 (2007) 1289–1307.11] M. Fuentes, B.C.C. Pessela, J.V. Maquiese, C. Ortiz, R.L. Segura, J.M. Palomo, O.
Abian, R. Torres, C. Mateo, R. Fernandez-Lafuente, J.M. Guisan, Biotechnol. Progr.20 (2004) 1134–1139.
12] I. Ardao, G. Alvaro, M.D. Benaiges, Biochem. Eng. J. 56 (2011) 190–197.13] G.P. Royer, Catal. Rev. 22 (1980) 29–73.14] A.M. Klibanov, Science 219 (1983) 722–727.15] E. Katchalski-Katzir, Trends Biotechnol. 11 (1993) 471–478.16] R. Torres, B.C.C. Pessela, C. Mateo, C. Ortiz, M. Fuentes, J.M. Guisan, R. Fernandez-
Lafuente, Biotechnol. Progr. 20 (2004) 1297–1300.17] M. Fuentes, J.V. Maquiese, B.C.C. Pessela, O. Abian, R. Fernandez-Lafuente, C.
Mateo, J.M. Guisan, Biotechnol. Progr. 20 (2004) 284–288.18] G. Bayramoglu, B. Karagoz, M. Yilmaz, N. Bicak, M.Y. Arica, Bioresour. Technol.
102 (2011) 3653–3661.19] M. Bartolini, V. Cavrini, V. Andrisano, J. Chromatogr. A 1144 (2007) 102–110.20] G. Bayramoglu, M.Y. Arica, J. Mol. Catal. B: Enzym. 62 (2010) 297–304.21] M. Fuentes, J.V. Maquiese, B.C.C. Pessela, R. Torres, V. Grazu, R. Fernandez-
Lafuente, J.M. Guisan, C. Mateo, Enzyme Microb. Technol. 39 (2006) 332–336.22] M. Filho, B.C. Pessela, C. Mateo, A.V. Carrascosa, R. Fernandez-Lafuente, J.M.
Guisan, Process Biochem. 43 (2008) 1142–1146.23] C. Mateo, O. Abian, R. Fernandez-Lafuente, J.M. Guisan, Biotechnol. Bioeng. 68
(2000) 98–105.24] C. Garcia-Galan, Q. Barbosa, R. Fernandez-Lafuente, Enzyme Microb. Technol.
52 (2013) 211–217.25] K. Maekawa, S. Tsunasawa, G. Dibo, F. Sakiyama, Eur. J. Biochem. 200 (1991)
652–661.26] B. Li, X. Chen, H. Ren, L. Li, J. Xiong, J. Bai, Y. Chen, J. Wu, H. Ying, Bioprocess
Biosyst. Eng. 35 (2012) 415–422.27] B. Li, Y. Chen, X. Chen, D. Liu, H. Niu, J. Xiong, J. Wu, J. Xie, J. Bai, H. Ying, Process
Biochem. 47 (2012) 665–670.
[[[
[
B: Enzymatic 101 (2014) 92– 100
28] M. Fujimoto, A. Kuninaka, H. Yoshino, Agric. Biol. Chem. 38 (1974) 777–783.29] Q.Q. Ying, L.E. Shi, X.Y. Zhang, W. Chen, Y. Yi, Appl. Biochem. Biotechnol. 136
(2007) 119–126.30] F. Olmedo, F. Iturbe, J. Gomez-Hernandez, A. Lopez-Munguia, World J. Micro-
biol. Biotechnol. 10 (1994) 36–40.31] W.C. Suh, B.S. Lim, M. Chun, M. Sernetz, Korean Biochem. J. 20 (1987) 17–23.32] S. Noguchi, G. Shimura, K. Kimura, H. Samejima, J. Solid-Phase Biochem. 1
(1976) 105–118.33] K. Rokugawa, T. Fujishima, A. Kuninaka, H. Yoshino, J. Ferment. Technol. 58
(1980) 423–429.34] K. Rokugawa, T. Fujishima, A. Kuninaka, H. Yoshino, J. Ferment. Technol. 58
(1980) 509–515.35] Q. He, N. Li, X. Chen, Q. Ye, J. Bai, J. Xiong, H. Ying, Korean J. Chem. Eng. 28 (2011)
544–549.36] N. Ortega, M. Perez-Mateos, M.C. Pilar, M.D. Busto, J. Agric. Food Chem. 57 (2009)
109–115.37] A. Bradford, Anal. Biochem. 72 (1976) 248–254.38] M. Fujimoto, A. Kuninaka, H. Yoshino, Agric. Biol. Chem. 39 (1975) 1991–1997.39] M. Fuentes, P. Batalla, V. Grazu, B.C.C. Pessela, C. Mateo, T. Montes, J.A.
Hermoso, J.M. Guisan, R. Fernandez-Lafuente, Biomacromolecules 8 (2007)703–707.
40] M. Karra-Châabouni, I. Bouaziz, S. Boufi, A.M.B. Rego, Y. Gargouri, Colloids Surf.,B 66 (2008) 168–177.
41] J.F. Liu, H. Liu, B. Tan, Y.H. Chen, R.J. Yang, J. Mol. Catal. B: Enzym. 82 (2012)64–70.
42] A. Pal, F. Khanum, Process Biochem. 46 (2011) 1315–1322.43] N. Kharrat, Y.B. Ali, S. Marzouk, Y.T. Gargouri, M. Karra-Châabouni, Process
Biochem. 46 (2011) 1083–1089.44] M.Y. Arica, Polym. Int. 49 (2000) 775–781.45] M. Fujimoto, A. Kuninaka, H. Yoshino, Agric. Biol. Chem. 38 (1974) 1555–1561.
46] H.H. Li, Y.H. He, Y. Yuan, Z. Guan, Green Chem. 13 (2011) 185–189.47] R. Keller, M. Schlingmann, U.S. 4649111, 1987.48] R.C. Rodrigues, C. Ortiz, A. Berenguer-Murcia, R. Torres, R. Fernandez-Lafuente,
