Immobilisation of horseradish peroxidase on Eupergit ®C for the enzymatic elimination of phenol

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Immobilisation of horseradish peroxidase on Eupergit ®C for the enzymatic elimination of phenol
  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 institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites 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 informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:  Author's personal copy  Journal of Hazardous Materials 177 (2010) 990–1000 Contents lists available at ScienceDirect  JournalofHazardousMaterials  journal homepage: Immobilisation of horseradish peroxidase on Eupergit ® C for the enzymaticelimination of phenol L. Pramparo a , F. Stüber a , J. Font a , A. Fortuny b , A. Fabregat a , C. Bengoa a , ∗ a Departament d’Enginyeria Quimica, Escola Tècnica Superior d’Enginyeria Química, Universitat Rovira i Virgili, Av. Països Catalans 26, 43007 Tarragona, Spain b 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 2010 Available online 11 January 2010 Keywords: Horseradish peroxidaseEupergit ® CImmobilisationPhenol removalTorus reactor a b s t r a c t 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 of biocatalysts.Inthiscase,amaximumvalueoftheimmobilisedenzymeactivityof127U/g support  wasfoundusinganenzymeloadingonthesupportof35.2mg/g support .ThefreeandtheimmobilisedHRPwereusedto 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. © 2010 Elsevier B.V. All rights reserved. 1. Introduction 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 of enzyme from products, rapid stopping of reactions, and improve-mentoftheenzyme’sstability[1,2].Alltheseadvantagesallowtheeconomical enhancement of enzymatic processes [3].There are several methods of enzyme immobilisation, among whichthecarrier-bindingmethodorcovalentbindingistheoldestand most used technique [4], despite the fact that the conditionsforimmobilisationaremuchmorecomplicatedandaggressivethanphysical 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 ∗ Corresponding author. Tel.: +34 977 55 8619; fax: +34 977 55 9621. E-mail address: (C. Bengoa). 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-tionofenzymesandproteins[12].Theseepoxy-activatedsupportsare 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. 0304-3894/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2010.01.017  Author's personal copy 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-tanttomechanicalandchemicalstressesandadaptabletoavarietyofconfigurationsandspecificprocessescarriedoutinreactors[18].Eupergit ® C is a copolymer of methacrylamide, N,N  -methylen-bis(acrylamide)andamonomercarryingoxiranegroups(resultinginepoxy-activatedacrylicbeads).Thissupportisverystable,allowseasier immobilisation procedures, has high binding capacity, lowwateruptake,easyfilterability,highflowrateincolumnproceduresand excellent performance in stirred batch reactors [12,19–22].Thispolymerhasbeensuccessfullyusedfortheimmobilisationof lipase by several authors and the protocol of preparation was foundtoallowabetterstabilitythantheonesusedwithothersup-ports [3,23,24]. Good results were also obtained in a number of previous studies for the immobilisation of other hydrolases such  - and   - galactosidase [25,26], pepsin, trypsin [18], penicillin G acylase [27].One of the most used methods to immobilise enzymes on Eupergit ® Csupportsinvolvesthedirectbindingoftheenzymeontothe polymers via the oxirane groups. Knezevic et al. [3] have stud-iedandcomparedtheresultsofthreedifferentmethodsofcovalentimmobilisation of lipase from  Candida ru gosa  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-bohydratemoiety.Theyconcludedthatthispreparationwasalmost18-foldmorestablethanthefreeenzymeand2-foldmorethanthelipase conventionally immobilised.HRP has acquired considerable interest in the field of organic synthesis in recent years [28] because the enzymecatalyses the synthesis of specialty chemicals, including 3,4-dihydroxyphenylalanine(DOPA)[29]andbiphenols[30].Also,HRP iscommerciallyimportantforpolymerisationreactionsintheelim-inationofpollutantssuchasphenol[31]andanilineinwastewater treatments [32,33].Inthelasttenyears,alltheworkontheHRPimmobilisationwasfocused on the preparation of biosensors [34–36]. In order to improve economically the phenol removal processusingenzymes,theenzymeshouldbeusedinacontinuousregime over a long time period to exploit it completely. For this reasonshould be necessary to immobilise the enzyme.Eupergit ® Cisknowntobeagoodsupportforenzymeimmobil-isation; however, its utilisation as a carrier for the immobilisationof HRP has not been yet explored. Thus, in this study Eupergit ® Cwasselectedasthesupportforcomparingdifferentimmobilisationmethods for HRP.Finally, the torus reactor is characterised by a toroidal ordoughnut-shapedchamber,anditcanbeconsideredasaloopreac-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].Inthislastwork,thehydrolysisofcaseinbyimmobilised enzyme on chitosan in a batch torus reactor was studied. Theyhave shown that the kinetic parameters for free and immobilisedenzymewereofthesameorderofmagnitude,buttheactivityoftheimmobilised protease was only 1/20 that of the free enzyme andthe maximum apparent reaction rate was lower. Nevertheless, ahighdegreeofhydrolysis(around20%)wasobtainedforthecaseinusingimmobilisedenzyme.Thisworkshowedpromisingresultsinthe 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 was purchased fromSigma–Aldrich (ref. P8250; Rz ≥ 1.8). The enzyme, according tothe pyrogallol method performed by the supplier, had a specificactivity of 181U/mg enzyme  (one unit will form 1.0mg of pur-purogallin from pyrogallol in 20s at pH 6.0 at 20 ◦ C. This unit isequivalent to  ∼ 18mM units/min at 25 ◦ C). Aqueous stock solu-tions of peroxidase were prepared from 85mg of enzyme and100mL of distilled water. The stock solution was separated inseveral aliquots of 10mL 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 500mg of phenol and distilled water to reach the final vol-ume of 1000mL (5.3mM). 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 18/32in. with MWCO of 12,000–14,000Da (ref. 9.206.022), was supplied by Medicell.  Author's personal copy 992  L. Pramparo et al. / Journal of Hazardous Materials 177 (2010) 990–1000 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 groups The immobilisation was carried out by the direct binding of theenzyme onto the support through the oxirane groups present onthe solid matrix of the copolymer. A scheme of the procedure of immobilisation is presented in Fig. 2a.This procedure was performed by incubating 50mg of unmodi-fiedEupergit ® Cwithdifferentamounts,between0.44and5.28mg(0.18–0.66g/L), of a (native) HRP solution in a sodium phosphatebuffer 0.1M, pH 7.4 for 24h 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 todeterminethevariationsoftheenzymeactivityinthesolution.Theincubation process was ended when the activity of the remainedfreenativeenzyme,presentinthesolution,stoppeddecreasingandkept 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.TheactivationoftheacrylicparticlesoftheEupergit ® Cwascar-ried out with 0.1M of adipic dihydrazide solution in phosphatebuffer 0.1M, pH 7.4 for 4h at room temperature and slow shak-ing.Inallexperiments,3mLoftheadipicdihydrazidesolutionwasadded to 50mg 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 3.52mg (0.18–0.59g/L), in sodium phosphate buffer 0.1M, pH7.4 for 24h at 4 ◦ C and under slow shaking. Again, several samplesof supernatant enzyme solution were taken in order to determinethevariationsoftheenzymaticactivityinthesolution.Theenzymeactivity was determined using the 4-aminoantipyrine method.After the binding, the support was washed with water andsodium phosphate buffer 0.1M, 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 method The 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.TheoxidationofHRPwasinitiatedbymixing10mLofthestocksodiummetaperiodateat8mMand10mLofthestockenzyme[40].  Author's personal copy L. Pramparo et al. / Journal of Hazardous Materials 177 (2010) 990–1000  993 Thereactionmixturewasincubatedfor2hatroomtemperatureincomplete darkness. Excess periodate was degraded with the addi-tion of 0.1mL of ethylene glycol and then incubated for 20minat room temperature in complete darkness. The resulting oxi-disedHRPwasdialysedtoremovethesmall-moleculeby-productsafter the enzyme modification. The characteristics of the cellulosedialysis tube were size 18/32in. with a molecular weight cut-off (MWCO) of 12,000–14,000Da. The sealed bag was introduced in acontainerandthedialysiswascarriedoutagainstphosphatebuffer0.1M, at pH 7.4 during 4h at room temperature. The activity of the oxidised enzyme before and after the dialysis process werechecked by spectrophotometric analysis using the 4-AAP methodas described in Section 2.3.1.Inparallel,theEupergit ® C(50mg)supportwasconjugatedwith0.1M of ADH in phosphate buffer 0.1M, at pH 7.4 for 4h at roomtemperature. Then the support was washed several times withwater and sodium phosphate buffer.Finally,theoxidisedenzymewasconjugatedwiththehydrazidosupport in sodium phosphate buffer 0.1M, at pH 7.4 at 4 ◦ C for24h and slow shaking [40,41]. Again, the effect of the enzymeloading was studied by varying the amount of the HRP, between0.22 and 2.64mg (0.09–0.33g/L), to be fixed to the support (50mg). The activity of the enzyme was again determined bythe4-aminoantipyrinemethod.Theimmobilisationprocedurewasstopped when the activity of the free enzyme in the solution wasconstant.  2.3. Activity assays 2.3.1. Enzyme in solution The measurements of pH were performed with a Crison pHmeter, model GL-21 equipped with a Hamilton electrode.Horseradish peroxidase activity was analysed according to the4-aminoantipyrine(4-AAP)method.Inthisprocedurethereactionbetween phenol and H 2 O 2  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 510nm.The assay mixture consisted of 95  L of 100mM phenol in0.5M sodium phosphate buffer pH 7.4, 0.48mg of 4-AAP, 1.9  L of 100mM H 2 O 2 , 50  L of the enzyme sample and water to a finalvolume of 1mL. Immediately after the addition of the enzyme, thecuvette was shaken and the change of absorbance with time wasmonitored at 510nm.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 of the absorbance presented a linear relationship with time, and theinitial rate was calculated from the slope of the line, taking intoaccountthedilutionofthesampleandtheextinctioncoefficientof theproduct.Oneactivityunitwasdefinedastheamountofenzymethat 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 ± 3U/mL ( R 2 =0.99) or the equivalent 188U/mg enzyme .  2.3.2. Immobilised enzyme The activity test for the immobilised enzyme was carriedout using the 4-AAP method but making some modifications toanalyse solid particles. In a first step, 15mL 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 supernatantsolutionwasfilteredandusedtomeasuretheabsorbanceat510nmat30sintervalsover5min.Thevaluesoftheabsorbancepresenteda 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 100mL where the agitation was done by a magneticbar. The torus reactor was a thermostatted reactor of 100mL builtwith poly(methylmethacrylate) (PMMA). The torus reactor had anannularsquaresectionwithagapwidthof  D =25mm.Aschemeof the torus reactor used in this work can be seen in Fig. 3. The agita-tioninthereactorwasdonebyathreebladesmarineimpellerwithabladepitchangleof45 ◦ andanexternaldiameterof15mm.Avari-ablespeedmotorcontrolledbyatachometer(HeidolphmodelRZR 2021)wasusedtomanagetherotationoftheimpeller.Animpellerrotation speed of 1500rpm 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 500ppm (5.3mM). This stock solution was thenused to prepare the dilute phenol solutions tested in the experi-mental work.Thestocksolutionofhydrogenperoxidewaspreparedbydilut-ing285  Loftheconcentratedsolutionbytheadditionofdeionisedwater so as to achieve the final volume of 25mL, and a concentra-tion of 100mM. The different H 2 O 2  concentrations were obtainedbydilutionwithdeionisedwaterinordertoachievetherespectiveconcentration inside the reactor. Different initial concentrationsof phenol (0.5–1.6mM) and H 2 O 2  (0–2.65mM) 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. Theacetonitrilesolutiondenaturedtheenzyme[42].Inallexperiments,severalliquidsamples(1mL)werewithdrawnatdifferenttimesof reaction, centrifuged and the supernatant analysed by HPLC.
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