Acomparativeperformanceevaluationofjuteandeggshell

ProcessBiochemistry47(2012)749–757ContentslistsavailableatSciVerseScienceDirectProcessBiochemistryjournalhomepage:∗DepartmentofBiotechnology,IndianInstituteofTechnology,Kharagpur,WestBengal721302,IndiaarticleinfoArticlehistory:Received29December2011Receivedinrevisedform3February2012Accepted7February2012Availableonline15February2012Keywords:LipaseimmobilizationEggshellJuteFTIRSEMAdsorptionisothermabstractApancreaticlipasewasimmobilizedonreadilyavailableandinexpensivejuteandeggshellmatrices.ThepurityofextractedenzymewasconfirmedbySDS-PAGE.Themaximumproteinloadforeggshellwas10.23mg/g,andforjute,itwas5.7mg/g.Thefreeenzymeactivityretentionwasgreaterthan80%foreggshelland43%forjute.TheimmobilizedlipasewasstableoverapHrangefrom7to8foreggshelland7.5to8.5forjutewithoveratemperaturerangefrom25to45◦Cforeggshelland37to40◦Cforthejute.FTIRdataindicatednewbondsonthejuteuponimmobilization.Althoughnonewbondwasobserved,immobilizationdataoneggshellfitwellwiththeLangmuiradsorptionisothermmodel.Themodelconstants,maxandKl,were13.92mg/gand0.382mL/mg,respectively.Mixedadsorptionwithbothionicandhydrophobicinteractionswasobserved.LipaseadsorptionwasreducedsignificantlyinpresenceofTween80,whereastheeffectwaslessincaseofionicstrength,pHandtemperature.Forbothmatrices,scanningelectronmicroscopy(SEM)wasusedtodemonstratethechangesinsurfacemorphologyafterimmobilization.Theperformanceofeggshellwasbetterthanthatofjuteasamatrixforimmobilizingpancreaticlipase.©2012ElsevierLtd.Allrightsreserved.1.IntroductionImmobilizationistheprocessofattachingorentrappinganenzymetoaninsolublematerial,thematrix,usingphysicalorchemicalinteractions.Theprimaryobjectiveofimmobilizationistoreducetheprocesscostbyenhancingenzymestabilityandreusability[1].Forfurthercostminimization,findingacheapandreadilyavailablematrixthatcanretainmaximumenzymeactivityisamajorchallengeincommercialenzymeimmobilizationappli-cations.Enzymescanbeimmobilizedbyseveralmethods,whichrangefromcarrier-boundtocarrier-freetechniques[2,3].Galanetal.[4]extensivelyrevieweddifferentenzymeimmobilizationstrategiesandindicatedthatimmobilizationnotonlyallowustoreusetheenzymebutalsotoimproveitsperformancebyenhanc-ingstability,activityspecificity,etc.Variousmethodshavebeenextensivelyusedforenzymeimmobilization,includingentrapmentusingalginate[5],agarose,polyacrylamide,gelatin,sol–gelandk-carrageenan;adsorptionusingalumina,macroporousresin,silica,celiteandcalciumcarbonate;aswellascovalentbindingusingchi-tosan[6]andcertainbiopolymerssuchasjute[7].Glutaraldehydeisusedasacross-linkingagenttostabilizetheenzyme–matrixinteraction[8,9].Theselectionofmatrixandmethodof∗Correspondingauthor.Tel.:[phone];fax:[phone].E-mailaddress:[e-mail](R.Sen).immobilizationarecriticalforretainingmaximumenzymeactiv-ityandimprovingenzymestability.IyerandAnanthanarayanreviewedtheenhancementinenzymestabilityafterimmobiliza-tioninaqueousandnon-aqueousenvironments[10].Multipointcovalentattachmentofanenzymemoleculetoasupportmatrixaidsinincreasingstability.However,duecaremustbetakentoavoiddistortingtheenzymestructure[11].Enzymeactivesiteinac-cessibilityorconformationdistortionuponimmobilizationcausesmasstransferlimitations,whichresultsinreducedenzymeactivity[12,13].Ontheotherhand,itispossiblethatactivitymaybelostduringthereactionbecausetheenzymeisleachedfromthematrix[14].Modifyingtheareanearenzymeactivesiteduringimmobi-lizationisreportedlybeneficialforactivationofcertainenzymessuchaslipases[11,15,16].Changingtheenzymeorientationonthesupportmaterialisanadditionalapproachemployedforeffectiveimmobilization[17,18].Lipases(glycerolesterhydrolase,E.C.3.1.1.3)areimportantenzymesthatbreakdownoilsandfatsintofattyacidsandglycerol.Lipasesactattheoil–waterinterfaceandmaintaingoodactivityonhydrophobicsubstratesforcatalysisofreactionssuchashydroly-sis,interesterificationandtransesterification[19].Lipaseshavea“lid”initsstructurethatcoverstheactivesiteoftheenzyme.Theenzymecontactstheoil–waterinterface,thelidmovesawaytoprovideaccessforthesubstratetoentertheactivesite[20].Thekineticmechanismdoesnotdependonthetypeofreaction.Itwashypothesizedthatthemodeofactionforlipaseissimilartoaserine1359-5113/$–seefrontmatter©2012ElsevierLtd.Allrightsreserved.doi:10.1016/j.procbio.2012.02.003750S.Chattopadhyay,R.Sen/ProcessBiochemistry47(2012)749–757protease[21].Inahydrolyticreaction,onemoleofoilorfatreactswiththreemolesofwatertoproducethreemolesoffattyacidsandonemoleofglycerol.Recently,lipasedemandincreasedduetoitsextensiveuseinatransesterificationreactionforbiodieselproduc-tion[22].Lipaseobtainedfromvarioussources(frommicrobestothehumanandporcinepancreas)wasimmobilizedprimarilyonhydrophobicsupportsusingvariousstrategies.Porcinepancreaticlipase(PPL)hasbeenwidelyusedforthetransesterificationduetoitslowprice,easyavailabilityandhighstability.Theenzymedoesnotrequireanycofactor.Itwasobservedinmostofthereportst