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This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information
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Journal of Hazardous Materials 177 (2010) 990–1000
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Journal of Hazardous Materials
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Immobilisation of horseradish peroxidase on Eupergit®C for the enzymaticelimination of phenol
L. Pramparoa, F. Stübera, J. Fonta, A. Fortunyb, A. Fabregata, C. Bengoaa,∗
a Departament d’Enginyeria Quimica, Escola Tècnica Superior d’Enginyeria Química, Universitat Rovira i Virgili, Av. Països Catalans 26, 43007 Tarragona, Spainb EPSEVG, Universitat Politécnica de Catalunya, Av. Víctor Balaguer s/n, 08800 Vilanova i la Geltrú, Spain
a r t i c l e i n f o
Article history:Received 23 July 2009Received in revised form29 December 2009Accepted 5 January 2010Available online 11 January 2010
In this study, three different approaches for the covalent immobilisation of the horseradish peroxidase(HRP) onto epoxy-activated acrylic polymers (Eupergit®C) were explored for the first time, direct HRPbinding to the polymers via their oxirane groups, HRP binding to the polymers via a spacer made fromadipic dihydrazide, and HRP binding to hydrazido polymer surfaces through the enzyme carbohydratemoiety previously modified by periodate oxidation. The periodate-mediated covalent immobilisationof the HRP on hydrazido Eupergit®C was found to be the most effective method for the preparation ofbiocatalysts. In this case, a maximum value of the immobilised enzyme activity of 127 U/gsupport was foundusing an enzyme loading on the support of 35.2 mg/gsupport. The free and the immobilised HRP were usedto study the elimination of phenol in two batch reactors. As expected, the activity of the immobilisedenzyme was lower than the activity of the free enzyme. Around 85% of enzyme activity is lost duringthe immobilisation. However, the reaction using immobilised enzyme showed that it was possible toreach high degrees of phenol removal (around 50%) using about one hundredth of the enzyme used inthe soluble form.
Enzyme immobilisation onto solid supports provides severaladvantages such as the possibility to re-use the biocatalyst due tothe ability to work with a confined bioreactor [1]. This fact allowsprocesses to operate in continuous mode, an easy separation ofenzyme from products, rapid stopping of reactions, and improve-ment of the enzyme’s stability [1,2]. All these advantages allow theeconomical enhancement of enzymatic processes [3].
There are several methods of enzyme immobilisation, amongwhich the carrier-binding method or covalent binding is the oldestand most used technique [4], despite the fact that the conditionsfor immobilisation are much more complicated and aggressive thanphysical adsorption and ionic binding.
In covalent immobilisation, the supporting matrix needs selec-tive activation and coupling procedures. In the last decades,substantial attention has been devoted to the covalent immobil-isation of enzymes to porous and insoluble supports such as glass[5], alumina [6], silica [7], and chitosan [8–11].
Among the large number of supports available for enzymeimmobilisation, the epoxy- activated supports are being widely
used and have received attention for large scale applica-tions. Epoxy-activated beads are bead polymers formed from ahydrophilic acrylamide with allyl glycidyl (epoxide) groups as theactive components responsible for binding. These groups are con-venient for the covalent binding of enzymes. The O–C and N–Cbonds formed by the epoxide groups are extremely stable, so thatthe epoxide-containing polymers can be used for the immobilisa-tion of enzymes and proteins [12]. These epoxy-activated supportsare able to form very stable covalent linkages with different pro-tein groups (amino, thiol, phenolic ones) under mild experimentalconditions (e.g. pH 7.0) mainly if a former mild physical adsorp-tion between the protein and the support has been promoted [13].Several research works has studied the immobilisation of enzymeonto epoxy-activated supports remarking the improvement in thestability of the support towards pH, temperature and storage time[14–16].
In Bayramoglu and Arica [17], they have studied the enzy-matic removal of phenol and p-chlorophenol using horseradishperoxidase immobilised on magnetic beads. In this case, enzymewas immobilised on the magnetic polyglycidylmethacrylate-methylmethacrylate (poly(GMA-MMA)) via covalent bondingusing glutaraldehyde as coupling agent. They have also noted thatthe immobilised enzyme retained a high activity on the magneticbeads and it was more stable during operation and storage com-pared to free counterpart.
L. Pramparo et al. / Journal of Hazardous Materials 177 (2010) 990–1000 991
One of the most interesting epoxy-activated supports isEupergit®C, because it is commercially available worldwide, resis-tant to mechanical and chemical stresses and adaptable to a varietyof configurations and specific processes carried out in reactors [18].Eupergit®C is a copolymer of methacrylamide, N,N′-methylen-bis(acrylamide) and a monomer carrying oxirane groups (resultingin epoxy-activated acrylic beads). This support is very stable, allowseasier immobilisation procedures, has high binding capacity, lowwater uptake, easy filterability, high flow rate in column proceduresand excellent performance in stirred batch reactors [12,19–22].
This polymer has been successfully used for the immobilisationof lipase by several authors and the protocol of preparation wasfound to allow a better stability than the ones used with other sup-ports [3,23,24]. Good results were also obtained in a number ofprevious studies for the immobilisation of other hydrolases such�- and �- galactosidase [25,26], pepsin, trypsin [18], penicillin Gacylase [27].
One of the most used methods to immobilise enzymes onEupergit®C supports involves the direct binding of the enzyme ontothe polymers via the oxirane groups. Knezevic et al. [3] have stud-ied and compared the results of three different methods of covalentimmobilisation of lipase from Candida rugosa on Eupergit®C sup-ports. The procedure yielding the highest activity retention, 43.3%,was based on the coupling of periodate-oxidised lipase via its car-bohydrate moiety. They concluded that this preparation was almost18-fold more stable than the free enzyme and 2-fold more than thelipase conventionally immobilised.
HRP has acquired considerable interest in the field oforganic synthesis in recent years [28] because the enzymecatalyses the synthesis of specialty chemicals, including 3,4-dihydroxyphenylalanine (DOPA) [29] and biphenols [30]. Also, HRPis commercially important for polymerisation reactions in the elim-ination of pollutants such as phenol [31] and aniline in wastewatertreatments [32,33].
In the last ten years, all the work on the HRP immobilisation wasfocused on the preparation of biosensors [34–36].
In order to improve economically the phenol removal processusing enzymes, the enzyme should be used in a continuous regimeover a long time period to exploit it completely. For this reasonshould be necessary to immobilise the enzyme.
Eupergit®C is known to be a good support for enzyme immobil-isation; however, its utilisation as a carrier for the immobilisationof HRP has not been yet explored. Thus, in this study Eupergit®Cwas selected as the support for comparing different immobilisationmethods for HRP.
Finally, the torus reactor is characterised by a toroidal ordoughnut-shaped chamber, and it can be considered as a loop reac-tor. This reactor presents some advantages over other stirred tankreactors including efficient mixing of the reactants, easy scale-up,the absence of dead volume, low power consumption [37], highheat transfer capacity [38], prevention of deposition of the poly-mer or biomaterial on the reactor wall and finally, high efficiency[11,37,39]. In this last work, the hydrolysis of casein by immobilisedenzyme on chitosan in a batch torus reactor was studied. Theyhave shown that the kinetic parameters for free and immobilisedenzyme were of the same order of magnitude, but the activity of theimmobilised protease was only 1/20 that of the free enzyme andthe maximum apparent reaction rate was lower. Nevertheless, ahigh degree of hydrolysis (around 20%) was obtained for the caseinusing immobilised enzyme. This work showed promising results inthe use of the torus reactor in bioprocesses involving immobilisedenzymes.
Therefore, methods for the immobilisation of the HRP onEupergit®C and its utilisation to remove phenol from wastewa-ters are presented in this work. The objectives are to evaluatethe feasibility of covalent HRP immobilisation on Eupergit®C by
Fig. 1. Photograph of the unmodified Eupergit®C beads.
three different methods, to optimise the immobilisation of HRPon Eupergit®C, to evaluate the efficiency of the HRP/Eupergit®Cbiocatalyst in phenol removal, to study the kinetics of the reac-tion using immobilised enzyme and to compare them with thefree enzyme and, finally, to compare the performances of twodifferent types of reactors, the torus reactor and the stirred tankreactor.
2. Methodology
2.1. Materials
Horseradish peroxidase (HRP, EC 1.11.1.7) was purchased fromSigma–Aldrich (ref. P8250; Rz ≥ 1.8). The enzyme, according tothe pyrogallol method performed by the supplier, had a specificactivity of 181 U/mgenzyme (one unit will form 1.0 mg of pur-purogallin from pyrogallol in 20 s at pH 6.0 at 20 ◦C. This unit isequivalent to ∼18 mM units/min at 25 ◦C). Aqueous stock solu-tions of peroxidase were prepared from 85 mg of enzyme and100 mL of distilled water. The stock solution was separated inseveral aliquots of 10 mL and stored at 4 ◦C. Hydrogen peroxide(30% w/v, specific gravity 1.1) was purchased from Panreac (ref.121076.1211). Phenol crystallized was also purchased from Pan-reac (ref. 144852.1211). A stock solution of phenol was preparedwith 500 mg of phenol and distilled water to reach the final vol-ume of 1000 mL (5.3 mM). Acetonitrile was supplied by Fluka (ref.00687).
For enzyme immobilisation, sodium phosphate dibasic (ref.S0876), sodium phosphate monobasic (ref. S9638), adipic dihy-drazide (ref. A0638), 4-aminoantipyrine (ref. 06800), sodium peri-odate (ref. 311448), ethylene glycol (ref. 102466) and Eupergit®C(ref. 46115) were purchased from Sigma–Aldrich. Eupergit®C isa copolymer of methacrylamide, N,N′-methylen-bis(acrylamide)and a monomer carrying oxirane groups (epoxy-activated acrylicbeads). The matrixes are hydrophilic acrylic beads with approx.800 �mol of epoxy groups per g of dry support and three atomsmatrix spacers (when ligands are coupled through the free oxi-rane groups). The beads have a particle size of approx. 150 �m(macroporous particles). Fig. 1 shows a photograph of the unmod-ified Eupergit®C beads taken using an optic microscopy with 30×zoom.
Dialysis membrane, size 18/32 in. with MWCO of12,000–14,000 Da (ref. 9.206.022), was supplied by Medicell.
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Fig. 2. Schemes of the HRP immobilisation methods.
2.2. Immobilisation of HRP
In order to optimise the immobilisation of horseradish peroxi-dase on Eupergit®C, three different procedures of immobilisationwere explored:
(a) direct HRP binding to the beads via their oxirane groups;(b) HRP binding to the beads via a spacer made from adipic dihy-
drazide (hydrazido Eupergit®C);(c) HRP binding to hydrazido Eupergit®C through the enzyme car-
bohydrate moiety previously modified by periodate oxidation.
2.2.1. Direct immobilisation of the HRP by oxirane groupsThe immobilisation was carried out by the direct binding of the
enzyme onto the support through the oxirane groups present onthe solid matrix of the copolymer. A scheme of the procedure ofimmobilisation is presented in Fig. 2a.
This procedure was performed by incubating 50 mg of unmodi-fied Eupergit®C with different amounts, between 0.44 and 5.28 mg(0.18–0.66 g/L), of a (native) HRP solution in a sodium phosphatebuffer 0.1 M, pH 7.4 for 24 h at 4 ◦C. After the incubation, the beadswere washed with deionised water and with sodium phosphatebuffer, filtered and then stored in sodium phosphate buffer at4 ◦C.
The effect of the amount of the enzyme incubated with theEupergit®C support was studied, measuring the activity of theimmobilised enzyme by the 4-aminoantipyrine (4-AAP) methodas detailed in Section 2.3.2. For each initial concentration of theenzyme, several samples were taken consecutively in order todetermine the variations of the enzyme activity in the solution. Theincubation process was ended when the activity of the remainedfree native enzyme, present in the solution, stopped decreasing andkept constant.
2.2.2. Immobilisation of the HRP on adipic dihydrazide treatedEupergit®C
This immobilisation procedure had two main steps: the pre-treatment of the copolymer beads with adipic dihydrazide (ADH),to make “hydrazido beads”, followed by coupling of the enzyme tothe beads surface as shown in Fig. 2b.
The activation of the acrylic particles of the Eupergit®C was car-ried out with 0.1 M of adipic dihydrazide solution in phosphatebuffer 0.1 M, pH 7.4 for 4 h at room temperature and slow shak-ing. In all experiments, 3 mL of the adipic dihydrazide solution wasadded to 50 mg of the solid beads of Eupergit®C. After activation,the beads were washed several times with water and phosphatebuffer.
The hydrazido Eupergit®C was then incubated with differentamounts of native horseradish peroxidase solution, between 0.44and 3.52 mg (0.18–0.59 g/L), in sodium phosphate buffer 0.1 M, pH7.4 for 24 h at 4 ◦C and under slow shaking. Again, several samplesof supernatant enzyme solution were taken in order to determinethe variations of the enzymatic activity in the solution. The enzymeactivity was determined using the 4-aminoantipyrine method.
After the binding, the support was washed with water andsodium phosphate buffer 0.1 M, pH 7.4 and stored in buffer at 4 ◦Cuntil use. The activity of the enzyme immobilised on hydrazidoEupergit®C was determined for each of the initial amount of freeenzyme used in the process.
2.2.3. Immobilisation of the HRP by the periodate methodThe immobilisation procedure consisted of three main steps:
oxidation of the horseradish peroxidase by sodium periodate,pre-treatment of the polymer with adipic dihydrazide to make“hydrazido beads” and, coupling of the oxidised enzyme to thehydrazido Eupergit®C support. A schematic illustration of thismethod is also shown in Fig. 2c.
The oxidation of HRP was initiated by mixing 10 mL of the stocksodium metaperiodate at 8 mM and 10 mL of the stock enzyme [40].
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The reaction mixture was incubated for 2 h at room temperature incomplete darkness. Excess periodate was degraded with the addi-tion of 0.1 mL of ethylene glycol and then incubated for 20 minat room temperature in complete darkness. The resulting oxi-dised HRP was dialysed to remove the small-molecule by-productsafter the enzyme modification. The characteristics of the cellulosedialysis tube were size 18/32 in. with a molecular weight cut-off(MWCO) of 12,000–14,000 Da. The sealed bag was introduced in acontainer and the dialysis was carried out against phosphate buffer0.1 M, at pH 7.4 during 4 h at room temperature. The activity ofthe oxidised enzyme before and after the dialysis process werechecked by spectrophotometric analysis using the 4-AAP methodas described in Section 2.3.1.
In parallel, the Eupergit®C (50 mg) support was conjugated with0.1 M of ADH in phosphate buffer 0.1 M, at pH 7.4 for 4 h at roomtemperature. Then the support was washed several times withwater and sodium phosphate buffer.
Finally, the oxidised enzyme was conjugated with the hydrazidosupport in sodium phosphate buffer 0.1 M, at pH 7.4 at 4 ◦C for24 h and slow shaking [40,41]. Again, the effect of the enzymeloading was studied by varying the amount of the HRP, between0.22 and 2.64 mg (0.09–0.33 g/L), to be fixed to the support(50 mg). The activity of the enzyme was again determined bythe 4-aminoantipyrine method. The immobilisation procedure wasstopped when the activity of the free enzyme in the solution wasconstant.
2.3. Activity assays
2.3.1. Enzyme in solutionThe measurements of pH were performed with a Crison pH
meter, model GL-21 equipped with a Hamilton electrode.Horseradish peroxidase activity was analysed according to the
4-aminoantipyrine (4-AAP) method. In this procedure the reactionbetween phenol and H2O2 was catalysed by the enzyme, the prod-ucts of the reaction reacted with the 4-AAP to form a red colouredsolution which was measured at 510 nm.
The assay mixture consisted of 95 �L of 100 mM phenol in0.5 M sodium phosphate buffer pH 7.4, 0.48 mg of 4-AAP, 1.9 �Lof 100 mM H2O2, 50 �L of the enzyme sample and water to a finalvolume of 1 mL. Immediately after the addition of the enzyme, thecuvette was shaken and the change of absorbance with time wasmonitored at 510 nm.
A DINKO UV–VIS spectrophotometer, model 8500, in combi-nation with a personal computer equipped with UV/vis softwarewas employed for measuring the enzyme activity. The values ofthe absorbance presented a linear relationship with time, and theinitial rate was calculated from the slope of the line, taking intoaccount the dilution of the sample and the extinction coefficient ofthe product. One activity unit was defined as the amount of enzymethat converted 1 �mol of hydrogen peroxide per minute at pH 7.4and 25 ◦C. In this case, the enzymatic activity of the native enzymesolution was 166 ± 3 U/mL (R2 = 0.99) or the equivalent 188 U/mgenzyme.
2.3.2. Immobilised enzymeThe activity test for the immobilised enzyme was carried
out using the 4-AAP method but making some modifications toanalyse solid particles. In a first step, 15 mL of assay mixture(prepared as described in Section 2.3.1) were placed in a beaker,then, around 300 �L of a suspension containing the immobilisedenzyme-Eupergit®C in buffer were also added to the beaker. Themixture was very quickly stirred and a sample of the supernatantsolution was filtered and used to measure the absorbance at 510 nmat 30 s intervals over 5 min. The values of the absorbance presenteda linear relationship within time, and the slope of the line allowed
Fig. 3. Schematic representation of the torus reactor.
calculating the rate, taking into account the dilution of the sample.After the assay, the solid was filtered, washed and then dried in acrucible to obtain the dry weight of the solid.
2.4. Phenol elimination and kinetic study
The enzymatic elimination of phenol was studied in two reac-tors, a stirred tank reactor and a torus reactor, both in batchconditions. The stirred tank reactor consisted of a thermostattedglass vessel of 100 mL where the agitation was done by a magneticbar. The torus reactor was a thermostatted reactor of 100 mL builtwith poly(methylmethacrylate) (PMMA). The torus reactor had anannular square section with a gap width of D = 25 mm. A scheme ofthe torus reactor used in this work can be seen in Fig. 3. The agita-tion in the reactor was done by a three blades marine impeller witha blade pitch angle of 45◦ and an external diameter of 15 mm. A vari-able speed motor controlled by a tachometer (Heidolph model RZR2021) was used to manage the rotation of the impeller. An impellerrotation speed of 1500 rpm was utilised in all experiments.
The phenol solutions were prepared by adding distilled waterto the appropriate quantity of crystallized phenol so as to achievea concentration of 500 ppm (5.3 mM). This stock solution was thenused to prepare the dilute phenol solutions tested in the experi-mental work.
The stock solution of hydrogen peroxide was prepared by dilut-ing 285 �L of the concentrated solution by the addition of deionisedwater so as to achieve the final volume of 25 mL, and a concentra-tion of 100 mM. The different H2O2 concentrations were obtainedby dilution with deionised water in order to achieve the respectiveconcentration inside the reactor. Different initial concentrationsof phenol (0.5–1.6 mM) and H2O2 (0–2.65 mM) were used for theexperimental study.
In a typical experiment, the phenol and HRP preparation, freeor immobilised, were kept into the reactor to reach the operatingtemperature (T = 20 ◦C) before adding the corresponding amountof hydrogen peroxide. The reaction time started when hydrogenperoxide was introduced into the reactor and it was terminatedby the addition of acetonitrile to the sample in 1:1 proportion. Theacetonitrile solution denatured the enzyme [42]. In all experiments,several liquid samples (1 mL) were withdrawn at different times ofreaction, centrifuged and the supernatant analysed by HPLC.
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2.5. Analytical determination of the phenol concentration
The phenol concentration of the liquid samples was analysedand quantified by HPLC (High Performance Liquid Chromatogra-phy, Agilent Technologies 1100 Series) using a C18 reverse phasecolumn (Hypersil ODS, 5 �m, 25 × 0.4 cm). To properly separatephenol from the partial oxidation or polymerisation products, themobile phase was a 40/60 vol. mixture of methanol and deionisedwater, acidified to a pH of 1.41, at a flow rate of 1 mL/min. Thedetection of phenol was performed with an UV with diode arraydetector (DAD) at a wavelength of 270 nm. The phenol concen-tration was determined using a calibration curve obtained withdifferent phenol concentrations.
The conversion of phenol XPh was defined as (Eq. (1)):
Xph (%) = Cph0− Cph(t)
Cph0
× 100 (1)
where CPh0was the initial phenol concentration and CPh(t) the phe-
nol concentration at a given time.
3. Results and discussion
3.1. Variations of the enzyme activity in the solutions of thedifferent immobilisation methods
The incubation process of each immobilisation method wasmonitored by measuring the activity of the remained free enzymepresent in the solution. While this activity was diminishing itwas assumed that HRP was still binding free linkers present inthe support. Once all linkers were occupied, the remained HRPin solution was kept constant and thus, the measured activity ofthis free enzyme was also constant. Fig. 4 shows the evolution ofthe enzyme activity in the solution for the different immobilisa-tion methods. Each immobilisation method presented a differentbehaviour reaching the maximum immobilisation at a differentrate.
Fig. 4a presents the activity of the enzyme in solution as a func-tion of time for the immobilisation method by oxirane groups. As itcan be seen in the figure, the activity of the free enzyme in the solu-tion diminished from 83–996 to 69–976 U depending of the amountof enzyme initially added to the support in solution. The last val-ues of each experiment corresponded to the remaining activity ofthe enzyme in solution after the immobilisation procedure ontothe support. Almost all the linked enzymatic activity was coupledbefore the first 3 h of experimentation and then, the enzyme activ-ity remained constant for most of the tested cases. After this timeit would not be necessary to continue the incubation because theenzyme was not being immobilised onto the support. As seen inthe figure, almost no differences were found for the several studiedamounts of enzyme in solution, denoting the same relative linkingin all cases.
The activity profile obtained for the enzymatic activity in thesolution for the method of immobilisation by adipic dihydrazideis shown in Fig. 4b. As seen in the figure, almost all the linkedenzymatic activity was coupled in the first 16 h of incubation andin some cases before 3 h. After this time, the enzymatic activityof the solution remained constant. Unlike the previous immobili-sation method (3 h of coupling time), the immobilisation methodusing ADH took in some cases 16 h for the same purpose. This dif-ference can be explained as the extra time used by the enzyme toget attached to a more specific binder that will also suppose animprovement in the immobilised activity.
In the case of using the immobilisation procedure by periodatemethod, the activity profile obtained is shown in Fig. 4c. As seen inthe figure, almost the entire free enzyme was coupled before 3 h
Fig. 4. Influence of the extent of the immobilisation on the enzymatic activity of theloading enzyme solution: (a) by oxirane groups, (b) by ADH and, (c) by periodate.50 mg Eupergit®C, Phosphate buffer 0.1 M, pH 7.4 and temperature of 4 ◦C.
when an amount of 0.22 mg of enzyme was in solution with the50 mg of support. As more enzyme was available in the solution,more enzyme activity remained in solution after the immobili-sation and/or more time should be used for the immobilisationprocedure. Also, as seen in the figure, no linear relationship existedbetween the initial amount of enzyme in solution and the enzy-matic activity that remained in solution after the immobilisation,e.g. using 0.22 or 0.44 mg of initial enzyme in solution, the sameactivity remained after 24 h. The use of specific binders increasedthe needed time for the immobilisation of the enzyme on the sup-port, but also provided an improvement in the immobilised activity.
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Table 1Influence of the quantity of enzyme in contact with the support for the different immobilisation procedures. 50 mg Eupergit®C, Phosphate buffer 0.1 M, pH 7.4 and temperatureof 4 ◦C.
Table 1 presents the influence of the quantity of enzyme solu-tion in contact with the support. The table shows in the differentcolumns the amount of enzyme in contact with the support, theactivity of the immobilised enzyme calculated by the 4-AAP methodand the activity coupling yield (�act) after each batch calculated asthe ratio between the specific activity of immobilised enzyme andthe coupled activity of the free enzyme.
The table shows that the activity of the immobilised enzyme wasnot proportional to the amount of the free enzyme exposed to thesupport. Below 17.6 mg/gsupport (or equivalent 3320 U/gsupport) ofHRP loading on the support, the activity of the immobilised enzymeincreased. Additionally, a larger amount of immobilised enzyme onthe support did not represent always a higher activity of the immo-bilised enzyme. This can be due to the packing of the immobilisedenzyme that limits their movement and thus reduces its activity.The maximum activity of the immobilised enzyme (73 U/gsupport)was obtained when 17.6 mg/gsupport of enzyme was in contact withthe support. However, after this value, the activity of the immo-bilised enzyme decreased. The decrease of this activity can beattributed to the formation of multiple layers onto the support thatmasked the active sites of the enzyme. In fact, only very few units ofactivity of the enzyme in solution were really linked to the support,between 14 and 54 U, depending of the initial amount of enzymein contact with the support.
On the other hand, it can be observed that the coupling yieldof the activity was decreasing with an increase in the amount offree enzyme loading on the support. This fact indicated that, eventhough higher amounts of enzyme were available, they were notlinked to the support or multiple layers were formed.
In summary, the maximum activity of the immobilised enzymethat could be attained is 73 U/mgsupport with 17.6 mg/gsupport ofenzyme exposed to the support.
3.3. Immobilisation of HRP on adipic dihydrazide treatedEupergit®C
The objective of this procedure was to increase the enzymaticactivity of the immobilised enzyme by direct binding via oxiranegroups. As seen in Table 1, an increase in the initial amount ofenzyme in solution gave a greater amount of enzymatic activitycoupled to the support. However, not all the enzyme available waslinked to the support. Depending on the initial amount in con-tact with the support, the enzyme activity that was removed from
the solution increased from 29 to 80 U. This fact thus denoted anincrease in the activity of the immobilised enzyme as seen in thetable. The continuous increase in the activity of the immobilisedenzyme with the increase of the initial amount of enzyme available,could suggest that no multiple layers are present in the support.Also in this immobilisation method, a fast diminution was obtainedfor the coupling yield of the activity.
The maximum enzymatic activity of the immobilised enzymewas found to be 129 U/gsupport using the immobilisation methodof HRP on hydrazido Eupergit®C. However, the most efficient ratiobetween initial free enzyme and immobilised enzyme suggest befor 4960 U/gsupport of enzyme. No further experiments were carriedout using greater initial amounts of enzyme because this fact wouldimpose a greater cost on the procedure.
3.4. The immobilisation of HRP by the periodate method
The objective to study this procedure was to increase the enzy-matic activity of the immobilised enzyme obtained by the previousmethods of immobilisation and to get a better use of the enzymeunits present in the solution. ADH was chosen to conjugate theenzyme because the hydrazide groups of ADH offer several advan-tages over simple amino compounds [41,43]. The main advantageof the reaction of a hydrazide and an aldehyde, in neutral and acidicpH, was the production of a hydrazone linkage. This functionallink is stable and does not need to be reduced with cyanoborohy-dride, thereby, circumventing one of the reactions associated withlabelling with amino compounds.
The periodate reaction was chosen because of the absence ofimpact on enzyme activity due to reactions in the glycan chains, itshigh yield of formation of aldehyde groups and the fact that it canbe carried out in aqueous solution at or near neutral pH.
A possible side-effect of the sodium periodate treatment isthe oxidation of some amino acid residues which may alter theconformation of the protein molecules. The oxidation of aminoacid residues with periodate appears to be a problem only underconditions of extensive oxidation (room temperature for severalhours and a very high concentration of periodate). The necessaryconditions for the optimal periodate oxidation of the HRP wereinvestigated by Tijssen and Kurstak [40]. In their investigations,4–8 mM was found to be the optimum concentration range.
At the periodate concentration used, with a 2 h reaction period,the specific activity of the oxidised enzyme was almost the sameas the original (specific activity of oxidised and original enzymeswere 160 and 159–188 U/mg, respectively).
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Table 2Effect of the adipic dihydrazide (ADH) concentration on the enzymatic activity forthe immobilisation method by periodate. 50 mg Eupergit®C, Phosphate buffer 0.1 M,pH 7.4 and temperature of 4 ◦C.
The influence of the amount of HRP in the attachment solutionwas analysed. Table 1 shows the effect of the amount of enzymeloading onto the support. It can be seen that the enzyme activ-ity yield was in the range of 1.3–5.3%. It can be also noticed thatthe activity of the immobilised enzyme was not proportional to theamount of enzyme bound. Almost all the available enzymatic activ-ity was bound when small amounts of enzyme were loading on thesupport. In these cases, for 0.22 and 0.44 mg of enzyme in solu-tion (or 4.4 and 8.8 mg/gsupport), the remaining enzymatic activityin the solution was negligible after the immobilisation procedure.However, when higher amounts of enzyme were available in thesolution, not all the enzyme was linked to the support. Depend-ing on the initial amount in contact with the support, the enzymeactivity linked to the support increased from 50 to 226 U, but,this increase in the enzymatic activity linked did not represent anincrease in the activity of the immobilised enzyme. After a certainvalue of enzyme loading on support (17.6 mg/gsupport), the activityyield did not show an increase possibly due to the close packing ofthe enzyme on the support surface, which could limit the access ofsubstrates needed in the hydrolysis reaction. This idea is supportedby the quantity of enzyme exposed to the support ranged from 1%to 2000% of the maximum theoretical enzyme load on support.
It is known that the catalytic efficiency of immobilisation pro-cesses decreased when enzyme loading exceeded a certain valueand an optimum activity should be selected [44]. Using the perio-date method as enzyme immobilisation technique, the maximumvalue of enzymatic activity was found to be 127 U/gsupport. Thisvalue of enzymatic activity was very close to the one obtained usingthe immobilisation method by adipic dihydrazide (129 U/gsupport).However, the higher efficiency in the activity coupling seemed tobe most appropriate for a loading of 2000 U/gsupport (or 0.44 mg ofenzyme loading in 50 mg of Eupergit®C support).
In order to obtain an industrially feasible biocatalyst with higherenzymatic activity, the immobilisation method by periodate wasoptimised.
3.5. Optimisation of the ADH concentration in the periodatetreatment
The effect of the ADH amount on the activity retention was stud-ied. Different amounts of ADH ranging from 0.01 to 0.09 M wereused for immobilising HRP to Eupergit®C by the periodate method.In all cases, 300 U of enzymatic activity were initially in contact with50 mg of support. The effect of the ADH concentration is illustratedin Table 2. As seen in the table, an increase in the ADH concentra-tion increased the amount of enzyme covalently attached to thematrix, up to a point. At low concentrations of ADH there seemedto be a smaller number of bonds formed between enzyme andthe support. Hence, a lower enzymatic activity of the immobilisedenzyme was obtained. As the concentration of ADH increased, moreenzyme molecules were covalently bound to the support throughthe bonds between the hydrazido groups on Eupergit®C and thealdehyde groups on the enzyme. Moreover, when the concentra-tion was increased further, a decrease in the enzymatic activity
Table 3Effect of the incubation time on activity. 50 mg Eupergit®C, Phosphate buffer 0.1 M,pH 7.4 and temperature of 4 ◦C.
of the immobilised enzyme was observed, which may be due tothe non-specific activation of the groups. Therefore, the optimumvalue of ADH to obtain a high enzymatic activity in the immobilisedenzyme was found to be 0.03 M.
The effect of the incubation period on the activity was studied.In this study, 400 U of enzymatic activity were initially in contactwith 50 mg of support. As seen in Table 3, an increase in the incuba-tion time, up to 24 h, provoked a higher activity of the immobilisedenzyme, thus more enzyme was coupled to the support. Probablymore enzyme was attaching to the support but this fact did notincrease the enzymatic activity. An optimum incubation period of24 h was required to obtain the maximum activity of the immo-bilised enzyme on Eupergit®C.
3.6. Enzymatic elimination of phenol using immobilised HRP
The activity of the immobilised HRP has been determined in bothreactors, stirred and torus. For this, the phenol removal was studiedfor different initial concentrations of HRP, from 0.001 to 0.006 U/mLcorresponding to a mass of support between 3.3 and 25.4 mg, for aphenol initial concentration of 0.5 mM and finally, for a H2O2 ini-tial concentration of 0.662 mM. These initial concentrations werechosen because they were determined as the optimal ratio in a pre-vious preliminary work to obtain higher phenol conversion (datanot shown).
With the stirred reactor it was possible to reach very highdegrees of removal of phenol, around 92% after 4 h of reactionand 0.006 U/mL of the enzyme immobilised by the periodatemethod. This value of phenol conversion was almost the samevalue that those obtained using free enzyme with a concentrationof 1.000 U/mL, 167 times more of enzyme but in 15 min (data notshown).
Fig. 5. Influence of the immobilised HRP initial concentration on the phenol con-version in the stirred tank reactor. [Phenol]0: 0.5 mM, [H2O2]0: 0.662 mM, pH 7 andtemperature of 20 ◦C.
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Fig. 6. Influence of the immobilised HRP initial concentration on the phenol con-version in the torus reactor. [Phenol]0: 0.5 mM, [H2O2]0: 0.662 mM, pH 7 andtemperature of 20 ◦C.
Fig. 5 shows the influence of the immobilised HRP initial activ-ity on the phenol conversion for a phenol initial concentrationof 0.5 mM and for a H2O2 initial concentration of 0.662 mM. Asseen, the conversion of phenol increased with an increase in theimmobilised HRP initial concentration due to a higher formation ofproducts.
The enzymatic activity of the immobilised HRP was determinedcalculating the initial reaction rates. From the value of the slopeit was found an immobilised enzyme activity of 28.3 ± 0.1 U/mg(R2 = 0.999). The immobilisation of the enzyme produced a greatreduction of the specific activity of the enzyme from 204 ± 22 tothe 28.3 ± 0.1 U/mg, for free and immobilized forms, respectively, aloss of 86%. The cause of this diminution could be the formation ofmultiple layers or a bad orientation of the active site of the enzymeduring the procedure of immobilisation.
The same study was carried out using the torus reactor. Fig. 6presents the phenol conversion as a function of time for 0.5 mM ofinitial phenol concentration and 0.662 mM of H2O2 initial concen-tration. Different enzyme concentrations were tested in the range0.001–0.004 U/mL. As seen in the figure, the same trend and almostthe same phenol conversion were obtained using the torus reactor.In the same way, the enzyme activity using the torus reactor wasalso calculated.
In order to determine the enzyme activity in the torus reactor,the initial reaction rate were analysed as a function of the initial HRPconcentration. In this case, the enzyme activity was 28.1 ± 5.0 U/mg(R2 = 0.9). More or less same activity was obtained in both reac-tors, however, the error obtained using the stirred reactor waslower than using the torus reactor. This fact denotes a better sta-bility in the stirred reactor. Therefore, the activity of the enzymeis not affected by the type of reactor used as it had been deducedalready with free enzyme (data not shown). Again, the diminutionof enzyme activity is exactly the same. Therefore, the reaction wasnot affected by the type of reactor used. As both reactors have thesame behaviour, the continuation of the study was realised onlywith the stirred reactor.
3.7. Influence of the hydrogen peroxide initial concentration
To study the influence of the H2O2 initial concentration, differ-ent experiments were carried out in the stirred reactor in batchconditions. In all cases the concentration of immobilised HRP waskept in 0.002 U/mL. The conversion of phenol was studied for differ-ent initial concentrations of H2O2 varying from 0.221 to 2.650 mM
while, three different initial concentrations of phenol of 0.5, 1.1and 1.6 mM were used. The results of the phenol conversion arepresented in Table 4. As seen, for each initial concentration of phe-nol, an increase in the initial H2O2 concentration first provoked anincrease of the conversion of phenol until a maximum conversionwas reached. After this point, an increase of the H2O2 initial con-centration did not provoke any increase of the phenol conversion.The best phenol conversion attained after 150 min of reaction wasaround 45%, 37% and 27% for initial phenol concentrations of 0.5,1.1 and 1.6 mM respectively. These values of conversion are almosta half part of the phenol conversion obtained using free enzyme inall cases as it can be seen also in Table 4. Moreover, the behaviour ofthe curves was similar to those obtained with the free enzyme whenthe initial concentration of H2O2 was lower than the optimum value(data not shown). On the other hand, when higher values of initialH2O2 concentration were used, the inhibition of the immobilisedenzyme did not seem to happen, or the effect was present in asmaller magnitude than using free enzyme. In this sense, the immo-bilisation of the enzyme gave additional protection from inhibitoryeffects.
Finally, it was remarkable that the quantity of immobilisedenzyme necessary to convert the phenol was lower by two ordersof magnitude than with the free enzyme, but it required shiftingfrom 15 to 30 min of reaction time to 150–240 min.
3.8. Kinetics of the immobilised HRP
The determination of the kinetics of the immobilised enzymein the stirred reactor was realised exactly in the same conditionsas for the free enzyme. The experimental initial reaction rates (Vi)were determined from the plot of the consumption of phenol as afunction of time. The experimental data were fitted to a logarithmiccurve and the reaction rate was determined calculating the valueof derivative of the slope of the curve for a time of 0.5 min. Thistime was chosen to have enough conversion of phenol that can bemeasured by the analytical method and to be as near as possible totime 0 where the initial rates are higher.
The kinetic model used in this study, based on theMichaelis–Menten kinetic model with inhibition by substrate [45],not only contemplated the initial H2O2 concentration, but also theinitial phenol concentration. It is important to notice that the initialphenol concentration was included in two terms. The first one mul-tiplies the maximum reaction rate parameter, Vmax, meaning thatthe phenol initial concentration contributed proportionally to theinitial reaction rate. The second term related the initial phenol con-centration as an inhibitor and it was expressed in the denominatorof the kinetic equation, limiting the initial reaction rate for highervalues of initial phenol concentration. This consideration was pos-sible because, in a previous preliminary study (unpublished), thekinetic parameters presented a linear relationship with the initialphenol concentration. The initial rate (Vi) vs. phenol and H2O2 con-centrations was fitted to the kinetic model represented by the Eq.(2).
Vi = Vmax · [H2O2] · [Phenol]
Km + [H2O2] + K · [Phenol] + K ′ · [H2O2]2(2)
where Vi is the apparent reaction rate of the phenol consumption,[Phenol] and [H2O2] are the phenol and the H2O2 initial concen-trations respectively, Vmax is the apparent maximum reaction ratewhile Km, K and K′ are model constants. In this model, the parame-ter Km is a measurement of the dissociation and the affinity of thecomplex enzyme-substrate. The factor K · [Phenol] in Eq. (2) rep-resents the influence of the phenol concentration on the reactionrate, and finally, K′ is the inhibition constant given by the H2O2concentration.
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Table 4Influence of the H2O2 initial concentration on the phenol conversion in the stirred tank reactor using immobilised (periodate method) and free enzyme.
The initial reaction rates of the enzymatic elimination obtainedin the stirred reactor for immobilised and free enzyme are alsoshown in Table 4 for initial phenol concentrations of 0.5, 1.1 and1.6 mM respectively. The calculated values of the fitted parametersfrom Table 4 data are presented in Table 5. As seen in the table, thereaction using immobilised enzyme had lower reaction rates thanusing free enzyme as it was denoted by lower Vmax constants. TheVmax value was decreased significantly upon covalent immobilisa-tion on the support. The affinity of the substrate for the enzymeseemed to be more important in the case of immobilised enzymeas the Km parameter was higher, being 0.35 and 0.01 mM in thecase of immobilised and free enzyme respectively. The Km valueincreased about 35 fold in the case of immobilised enzyme com-pared to free form. This fact showed an alteration in the affinity ofthe enzyme to the substrate upon covalent immobilisation on thebeads.
Using immobilised enzyme almost no inhibition of the reactionby H2O2 was present as shown by an inhibition parameter near 0.These variations in the kinetics parameters values of the enzymeupon covalent immobilisation can be attributed to several factorssuch as the non-covalent interactions of the immobilised enzymemolecule with the modified polymer surface might have inducedan inactive conformation to the enzyme molecules. It should be alsonoticed that the covalent immobilisation process does not controlthe proper orientation of the immobilised enzyme on the support[41,42].
There are different advantages and disadvantages featured bythe HRP immobilisation protocols studied in this work. All proto-cols allowed the retention of high amounts of immobilised enzyme;however, sometimes compromising the final retained activity.The immobilisation using oxirane groups reached the maximumenzyme loading in about 3 h compared with the 16–24 h neededin the other two protocols. Nevertheless, when using the periodateimmobilisation method a higher enzyme stability was achieved and
Table 5Apparent parameters for the reaction of HRP with inhibition by Phenol and H2O2
a larger number of functional aldehyde groups were available. Also,this immobilisation protocol led to a higher immobilised enzymeactivity using less enzyme compared with the other two protocols.The phenol removal carried out with the HRP immobilised by thistechnique was very high, up to 90%, and the quantity of enzymeused was about 100 times less than when using the free enzyme.
Some authors have reported different methods for the immobil-isation of HRP on various supports, most of them based on the useof covalent bonds between the enzyme and the support throughdifferent functional groups. Zhang et al. [46] presented a methodfor the immobilisation of HRP on activated glassy carbon elec-trodes. This method allowed a fast and selective immobilisationof the enzyme in the operating electrode, but also showed a fastloss in the enzyme activity when performing cyclic operation orwhen changing the pH of the solution. Sree-Divya et al. [47] stud-ied the HRP immobilisation on glassy carbon by three differenttechniques: nitration under drastic conditions, bromination andperoxidation. Each of these techniques used a different strategyfor the enzyme immobilisation by activating different functionalgroups. They reported that bromination and peroxidation led to ahigher enzyme activity compared to nitration. However, all tech-niques presented a low enzyme loading.
Fernandes et al. [48] presented the immobilisation of HRP onHCI-doped polyaniline using glutaraldehyde (GA) as crosslinker.This immobilisation method presented high enzyme stabilityagainst pH and temperature changes, but the reactivity to cer-tain substrates significantly varied between the free and theimmobilised enzyme. Zhang et al. [49] immobilised the HRP onpolyacrylamide gel reporting a long life operation of the immo-bilised enzyme but a low immobilisation rate. Cho et al. [50]reported the immobilisation of HRP on Celite beads, by combiningthe action of a silane and GA. The immobilised enzyme was used, incombination with an electrochemical method, for the conversionof phenol presenting a high conversion (77% in 90 min) and a lowadsorption of the products on the support. Nevertheless, the appli-cation of high voltages significantly decreased the enzyme activitydue to the production of strong oxidants.
4. Conclusions
Three different approaches for the covalent immobilisation ofHRP on Eupergit®C were explored. The direct HRP binding to poly-mers via their oxirane groups coupled almost all the enzymatic
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activity before the first 3 h of experimentation and, a maximumvalue of enzymatic activity of 73 U/gsupport was obtained for theimmobilised enzyme.
Using the HRP binding to the polymer via the spacer madefrom adipic dihydrazide, the maximum enzymatic activity of theimmobilised enzyme was found to be 129 U/gsupport. Around 40%of coupling of the enzyme activity was obtained for an enzymeloading on the support of 70.4 mg/gsupport.
Finally, for the HRP binding to hydrazido Eupergit®C surfacesthrough the enzyme carbohydrate moiety previously modified byperiodate oxidation, a maximum value of the immobilised enzymeactivity of 127 U/gsupport was found using an enzyme loading on thesupport of 35.2 mg/gsupport.
Therefore, almost the same activity of the immobilised HRPon Eupergit®C was obtained using the immobilisation techniquesof pre-treatment of the support by adipic dihydrazide or by theperiodate method. The last method allowed the use of a reducedamount of enzyme in order to obtain the same enzymatic activ-ity. In this way, the periodate-mediated covalent immobilisationof HRP on Eupergit®C was found to be an effective method forthe preparation of stable biocatalysts. The reaction of enzymaticelimination of phenol using immobilised horseradish peroxidaseon Eupergit®C showed that it was possible to reach high degrees ofremoval (around 92%), using 0.006 U/mL of immobilised enzyme.This value of removal of phenol was almost the same that thoseobtained using 1.000 U/mL of the free enzyme. This fact seemed toindicate that the immobilisation process improved the enzymaticstability. Moreover, the immobilisation process could protect theenzyme from its inhibition by products or substrates.
Acknowledgements
Laura Pramparo thanks to the Universitat Rovira i Virgili andthe Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR)of Catalan Government for the pre-doctoral scholarships. Finan-cial support was provided by the European Community, projectREMOVALS, FP6-018525. We thank Dr. K.E. Taylor, University ofWindsor, Canada for helpful discussions.
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