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Proteinases

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ProteinasesProtei

    Proteinases

May2008,Volume2,No.5(SerialNo.6)JournalofLifeSciences,ISSN1934-7391,USA

    ;Proteinasesfromthedigestiveorgansofblackcarp(Mylopharyngodon ;piceus):Partialcharacterizationandproteindigestibilityinvitro ;LIUZhong’yLlZhong—hal

    ;(CollegeofFoodScienceandTechnology,CentralSouthUniversityofForestryandTechno

    logy,ChangshaHunan410012,China)

    ;Abstract:Aseriesofexperimentsbasedonelectrophoretical ;andbiochemicalassayswereconductedtopartiallycharacterize ;proteinasespresentinthehepatopancreasandintestineofblack ;carp(Mylopharyngodonpiceus),andinvestigateenzymatic ;activityandproteindigestibilityinvitro.Caseindigestionassays ;revealedthepresenceofacidicproteinaseswithoptimum ;activityintherangeofpH2.02.5andalkalineproteinaseswith

    ;significantlyhigheractivitiesbothintherangeofpH8.18.6and

    ;nearpH9.5.Theinhibitionandsubstratespecificityassays ;showedthattrypsinandchymotrypsinarethemainactive ;componentsofthealkalineproteinases.The

    ;SDSsubstrate.PAGEshowedthatthecrudeextractofblack ;carpintestinehadeighttypesofalkalineproteinaseswiththe ;molecularmassrangeof27.578.5kDawhilethecrudeextract

    ;ofblackcarphepatopancreashadsixtypesofalkaline ;proteinaseswiththemolecularmassrangeof27.5.78.5kI)a. ;Theseenzymeswerecharacterizedastrypsin(27.5kDa.30.1 ;kDa),chymotrypsin(40.5kDa,42.5kDa),serineproteinases ;(32.1kDa,33.2kDa)andnonsefineproteinase(61.5kDa,78.5

    ;kDa1./12vitroproteindigestibilityassaysshowedthatblackcarp ;ca/]beabletoutilizeawiderrangeofproteins.

    ;Keywords:blackcarp;intestine;proteinase;activity;trypsin; ;chymotrypsin;SDS,substratePAGE

    ;1.IntroductioH

    ;Theblackcarp,Mylopharyngodonpiceus,(family

    ;Cyprinidae)isanimportantfarmedfreshwaterspecies ;inChina.Theblackcarpisacarnivorousstomachless

    ;freshwaterspecies,andinnatureresidesonthebottom ;ofrivers,lakesandponds,andmainlycaptures

    ;crustaceanandshellfishsuchassnails,clam,mussel, ;shrimpsandgastropodsforitsfood.Thefarmingof

    ;blackcarphavebeenpracticedformanyyears,butup

    ;LIUZhongyi(1964),male,Ph.D.,associateprofessor;

    ;researchfields:foodbiotechnology,fishphysiologyandfish ;processing.

    ;18

    ;tonow,itsannualyieldreachedonlytoabout180,000 ;toninChinawhiletheannualyieldofgrasscarp ;(CtenopharyngodonidellaVa1.)wasmorethan3 ;millionton(InformationCenterofChineseAcademy ;FisherySciences,http://www.fishinfo.crlO.Themuscle ;ofblackcarpishighlydesiredversusthatofgrasscarp. ;Thefoodandfeedinghabitsofthisspecieswere ;documented,andprotein,unsaturatedfattyacid(C1s:2 ;andCls:3)andmineralsplayveryimportantrolesinits ;diets[-51.Butliteratureonproteinasesfromdigestive ;organsofthisspeciesislimited.onlyWUandZHU【】

    ;reportedthemaindigestiveenzymesactivityinthe ;blackcarpgut.

    ;Proteinplaysanimportantroleinnutritionof ;blackcarp[1-2J.Nowadays.proteinbasedingredients ;fromplantoriginareusedincommercialdryfeedsfor ;blackcarp[1_2.HoweveruseoftheDroteininfish

    ;dependsontheactivitiesofdigestiveproteinases ;presentindigestiveorgansofthisspecies【『1.The

    ;characterizationandquantificationofproteolytic ;enzymesactivitymaycontributetobetter

    ;understandingthephysiologyofdigestion,andleadto ;improveingfeedingregimesandidentifyyingnewfeed ;ingredients.Also,adetailedknowledgeofthe ;proteolyticenzymesactivitythatexistsincaptive ;speciesisuseful,bothtoascertainthemaximum ;periodsfortheirstoragetoavoidautolysisf8J’andto

    ;developpracticalapplicationsforfishproteinasesin ;thefoodindustry19-1~1.

    ;Proteinasesfromfishdigestiveorgansaremainly ;comprisedofacidicproteinasesri.e.aspartic ;proteinases)functioninginanacidicmediumand ;Proteinasesfromthedigestiveorgansofblackcarp(Mylopharyngodonpiceus):

    ;Partialcharacterizationandproteindigestibilityinvitro ;alkalineproteinasesfunctioninginanalkalinemedium. ;Asparticproteinasesincludethedigestiveenzymeslike ;pepsin,gastricsinandchymosine,andthelysosomal ;proteinasescathepsinDandE,aswellasotheraspartic ;proteinaseswithlessknownfunctionsT”.Alkaline

    ;proteinasesaremainlycomprisedofserineproteinases ;liketrypsin,chymotrypsin,elastaseandnonserine

    ;proteinases.Trypsinisamajormemberoftheserine ;proteinasesfoundindifferentisoformsinthedigestive ;systemandplaysanimportantroleindietaryprotein ;digestiveprocessforfishl21.Somestudieshave

    ;characterizeddigestiveenzymesoftheaquatic ;species[13?2o_.

    ;Wereporttheactivityofproteinaseindigestive ;organsofadultblackcarp,theirpartialcharacterization ;andproteindigestibilityinvitroforsomeproteinbased ;ingredients.

    ;2.Materialsandmethods

    ;2.1Experimentalfish

    ;Tenadultblackcarpfedwithcommercialfish ;feedgranules(protein45.2%,oil11.8%)andshellfish, ;werepurchasedinShalingmarket(Xiangtancity). ;Weightandlengthofspecimenswere2.32.5kgand

    ;36.80-38.55cm.respectively.Specimenswere ;maintainedinfreshwaterfor24hours.

    ;2.2Chemicals

    ;MN-methylenbisacrylamid,acylamide,

    ;phenylmethylsulfonylfluoride(PMS,soybean ;trypsininhibitor(SBTI),tosyllysinechloromethyl

    ;kotone(TLCK),tosylphenylalaninechloromethyl

    ;kotone(TPCK),ethylendiaminetetraacetatic(EDTA), ;pepstatinA.N-bezoyl-larginineethylester?HCI

    ;(BAEE),acetyl1tyrosineethylester(ATEE),

    ;bezoylltyrosineethylester(BTEE).

    ;N-toluenesulfonyll-argininemethylester(TAME), ;N-bezoyllarginine-Pnitroanilide(BAPNA)were

    ;obtainedfromSigmaChemicalCo.(St.Louis,USA). ;Bovinehemoglobinandcaseinwerepurchasedfrom ;NativeMedicineCo.(Shanghai,China).Lower ;molecularmassproteinstandardswithrelative ;molecularmassrangeof14,40097,400for

    ;SDSPAGEwereboughtfromShanghaibiochemical ;institute(Shanghai,China).Allotherchemicalsareof ;analyticalgrade.

    ;2.3Preparationofcrudeenzyme

    ;Fishwassacrificed(refertoCARE110.O1). ;Hepatopancreasandintestineswererespectively ;collected.Then,theintestineswerewithscissors. ;Theseoperationswerecompletedontheice. ;Hepatopancreasandintestineswererespectively ;mixedwith0.1Msodiumphosphatebuffer,pH7.0at

;4.Cbyaratioof1:10,homogenized(ModelDS1,

    ;Shanghai,China)for60sandsettledfor2hat4.C. ;Then,theinsolublematerialswererespectively ;removedbycentrifugation(ModelSigma4K15, ;ManufacturedbySigmaCo.,AnderUnterenS6se50, ;D37520Osterode,Germany)at1l,000xgfor30min ;at4.C.Thesupematantcontainingthecrudeenzyme

     ;solutionwasrespectivelystoredat4oC(notmorethan;12h)forfutureproteinasesactivityanalysis. ;Thealkalineproteinaseswerecollectedfromthe ;crudeenzymesolutionbyprecipitationwith1045%

    ;saturationofammoniumsulfate.andtheacidic ;proteinaseswerecollectedfromthecrudeenzyme ;solutionbyprecipitationwith4580%saturationof

    ;ammoniumsulfate.Thecollectionswererespectively ;dialyzedagainst0.02MsodiumphosphatebufferrpH ;7.0)at4.C,andthenlyophilized(ModelABCONCO@ ;6L,ABCONCOLaboratory,USA),andstoredat ;

    ;20.C.

    ;2.4Enzymeassay

    ;2.4.1EffectofpHontotalproteinasesactivity ;TheeffectsofpHontotalproteinasesactivitiesof ;crudeenzymesolutionswerestudiedfollowingthe ;caseindigestionassaywithaslightmodification2l.A

    ;seriesofdifferentbufferswereusedfordifferentpH ;conditions:0.1MKCIHC1(pH1.5),0.2MGlyHC1

    ;(pH2.03.O),0.1MNaAcHAc(pH4.05.63),0.2M

    ;Na2HPO4NaH2PO4(pH6.407.40),0.1MTris.HCl

    ;(pH8.1),0.1MSodiumbarbitalHC1(pH8.62),0.1M

    ;l9

    ;Proteinasesfromthedigestiveorgansofblackcarp(Mylopharyngodonpiceus):

    ;Partialcharacterizationandproteindigestibilityinvitro ;Na2HPO~-NaOH(pH9.019.86),0.2Mmixingbuffer

    ;(pHl0.26l1.40)and0.1MNa2HPO4NaOH(pH

    ;12.04).Theenzymesubstratemixtureconsistedof0.5 ;mlcrudeenzymesolution.1mIselectedbufferand0.5 ;mll%caseinsubstratesolutionin10mMTrisHCl

    ;bufferofpH8.5containing0.02MCaC12was ;incubatedfor30minat37.C.Then2mlof10%(w/v) ;trichloroaceticacid(TCA,wasaddedtostopthe ;reaction.Afterrestingfor10min,thesupernatantwas ;filtered.0.5mloffilteredsolutionwasmixedwith2.5 ;mlOf0.4MNa2CO3and0.5mlFollinreagent.The

    ;mixturewasincubatedfor20minat37.C.andthen ;cooledinicebath.Theabsorbanceofthereaction ;mixtureat680amwasrecordedtomeasuretheamount ;oftyrosineproduced.Theblankusedforthisassaywas ;preparedbyincubatingamixtureofthecrudeenzyme ;solution.bufferandwaterfor30minat37.C.followed ;byadditionofTCAandcasein.OHeunitofenzyme ;activitywasdefinedastheamountofenzymeneeded ;toproduce1tyrosineperminunderaboveassays ;conditions.

    ;2.4.2Acidicproteinase

    ;Theacidicproteinaseactivitywasdetermined ;with2%hemoglobinsolutionin0.04MHC1as ;substrate(pH2and37.C1?.1mlenzymesolution

    ;wasmixedwith1mlsubstratesolution.Themixture ;wasjncubatedfor10minatpH2and37.C.Then2ml ;of1O%(w/v)trichloroaceticacid(TCA)wasaddedto ;stopthereaction.Otherstepswerecompleted ;accordingtothoseofcaseindigestionassaydescribed ;above.Oneunitofenzymeactivitywasdefinedasthe ;amountofenzymecapableofhydrolyzinghemoglobin ;toproduce1tyrosineatpH2and37.Cin1min. ;2.4.3Alkalineproteinase

    ;Thealkalineproteinase(includingtrypsin,E.C. ;3.4.21.4andchymotrypsin,E.C.3.4.21.1)activitywas ;determinedusingl%caseinsolutionassubstrateatpH ;8.5and37DCin50mMTnsHCIbuffercontaining

    ;0,O2mMCaCl,21.Theprocedurewasthesameas

    ;thoseoftheacidicproteinaseactivitydeterminationbut ;incubatedenzymesolutionandsubstratesolutionfor ;10minatpH8.5and37.C.Oneunitofenzyme ;activitywasdefinedastheamountofenzymecapable ;ofhydrolyzingcaseintoproduce1ggtyrosineatpH ;8.5and37.Cin1min.

    ;2.5Proteincontent

    ;Proteincontentwasdeterminedbythemethodof ;Lowry.etall22Jusingbovineserumalbuminas ;standard.

    ;2.6Proteinasesinhibition

    ;PepstatinAinhibitingassaywasconducted ;accordingtoBezerra,etall7J.EDTA,PMSF,SBTI,

    ;TLCKandTPCKinhibitingassayswereconducted ;accordingtoE1.Beltagy,etal231andNatalia,

    ;etalll9.

    ;2.7Substratespecificityofthealkaline ;proteinases

    ;Theactivitiesofthealkalineproteinaseswere ;determinedforhydrolysisofthesyntheticsubstrates ;suchasBAEE,ATEE,BTEE,TAMEandBAPNA

    ;accordingtoNatalia,etal[191andHeu

    ;,

    ;eta1(241.

    ;2.8Classificationofproteinasesbysubstrate ;SDS.PAGE

    ;SubstrateSDSPAGEwasusedc0characterize

    ;theproteinasespresentinthecrudeenzymeas ;describedbyGarcia.Carrefio.etal[Jand ;DfazL6pez,etal26withslightmodification.

    ;Thegel

    ;consistedof4%(w/v)stackinggelanda15%(w/v) ;separatingge1.Theelectrophoresiswasperformedat ;pH8.3,4oCand110VusingtheMiniProteanIII ;electrophoresissystem(BIORADLaboratories, ;California1.Thecrudeenzymeprecipitatedwith ;10.5%saturationofammoniumsulfatewasusedasthe ;alkalineproteinasessample.Thesamplescontaining ;proteinasesweresolvedwithdeionedwaterand ;enzymaticactivityadjustedtomorethan18U/ml, ;thendiluted(I:1)insamplebuffercontainingSDS, ;butnoreducingagents.Thesampleswerenotboiled ;beforeloadingontoge1.

    ;Electrophoresisproteinasescharacterizationwas ;carriedoutusingthespecificinhibitors,using10 ;PepstainAfortheacidicproteinases,and10mM ;PMSF’10mMTPCK,10mMTLCK,10mMEDTA

    ;Proteinasesfromthedigestiveorgansofblackcarp(Mylopharyngodonpiceus):

    ;P——

    ;artialcharacterizationandproteindigestibilityinvitro ;and10pMSBTIf0rthealkalineproteinases.Here.the ;crudeenzymeswereincubatedtogetherwiththe ;variousinhibitorsfor30minat25DC,thendiluted(1:1) ;insamplebufferpriortoelectrophoresis. ;Afterelectrophoresis,gelwasremovedfromthe ;cel1.Thegelwassoakedin3%ofcaseinsolutionin ;0.1MTrisHC1buffercontaining20mMCaC12atpH ;8.5and4.Cfor90mintoallowabsorptionofcasein ;intoge1.Then,gelwiththeabsorbedcaseinwasthen ;removedandplacedinawaterbathat35.Cf0ran

    ;additional120mintoallowthealkalineproteinasesin ;geltodigestthecasein.Gelwaswashedindistilled ;waterandfixedfor20minin15%TCAsolution.then ;stainedwith0.1%rw/v)CoomasieBluefor3hand ;thendestainedindestainingsolutionf95%ethanol: ;aceticacid:water=25:8:67).5u1ofmolecularmass

    PAGEStandards)withrelative ;markers(SDS

    ;molecularmassrangeof14,40097,400wereusedfor

    ;proteinasesrelativemolecularmassdetermination. ;Forcharacterizingtheacidicproteinases, ;substratePAGEatneutralpHcondition(pH7.01was ;employed.Thecrudeenzymeprecipitatedwith ;458O%saturationofammoniumsulfatewasusedas ;thealkalineproteinasessample.Thesamples ;containingacidicproteinaseswerediluted(1:1)in ;samplebuffercontaining3Murea.butnoSDSand ;reducingagents.Thesampleswerenotboiledbefore ;loadingontoge1.Theelectrophoresiswasperformed ;atpH7.0,4.Cand120V.Electrophoresisbuf_ferand ;gel’sbufferdidalsonotcontainSDS.Theporcine

    ;pepsinwasusedascontro1.Afterelectrophoresis,the ;gelwasremovedfromthecel1.andsoakedin0.1M ;HCltoreducethepHto2.0.fortheenzymesto ;becomeactive.After20min.thege1wasremovedand ;soakedfor90mininasolutioncontaining0.18% ;hemoglobinin0.1MGlyHC1buffer,pH2.0and4.C,

    ;thenremovedandincubatedfor120minat35.C.Ge1 ;waswashedindistilledwaterandfixedfor20minin ;l5%TCAsolution.thenstainedwith0.1%(w/v) ;CoomasieBluefor3handthendestained.

    ;2?9Invitrodeterminationofprotein

    ;digestibility

    ;/nvitroproteindigestibilityofthecrudeblack ;carpintestinalorhepatopancreasenzymesolutionwas ;determinedaccordingtoLemos.etal{JandSunI

    ;withslightmodifications.Groundproteinbased ;ingredients(1istedinTable3)powderof1.2gwas ;placedintograduatedcentrifugaltubewithground ;stopper(15m1).Then,crudeenzymesolutionsof10ml, ;adjustingproteinaseactivitytobeabout80U/ml,were ;addedintothetube,andstopperwascovered.Thetube ;wasthenplacedintheconstanttemperatureshakerand ;vibratedat25.Cfor12h.Theshakingfrequencywas ;adjustedatabout125min.ApHcontroller(Shanghai,

    ;China)wasusedtoadjustpHofmixturetobe8.0with ;0.2MNaOHsolution.Thereactionwasthenstopped ;byadding10mlTCAsolutionsof30%.Thetubewas ;centrifugedandthesupernatantwasremoved.And ;then,theremainderwaswashedtwicewithdeionized ;water,anddriedatvacuumadditionat80.C.The ;proteincontentofthedriedremainderandtheground ;proteinbasedingredientpowderwasdetermined ;accordingtoGB/T6432.1994i91.ThecrudeDrotein ;digestiveratioofproteinbasedingredient(X)was ;calculatedasfollowingequation.

    ;X(%)=(W1Wz)/W1xl00(1)

    ;Where:Wlisthecrudeproteincontentofthe ;proteinbasedingredient;W2isthecrudeprotein ;contentofthedriedremainder.

    ;2.10Dataanalysis

    ;Comparisonofactivitiesvalueswascarriedout ;usinganalysisofvariance(ANOVA)andwhere ;applicable,Tukey’sHSDinSPSS.

    ;3.Results

    ;3.1EffectsofpHonactivityofproteinases ;Fig.1showedthepHdependenceofthe

    ;proteolyticactivitiesintheblackcarphepatopancreas ;andintestine.Theproteinasesfromboth

    ;hepatopancreasandintestineallshowedthreepeak ;21

    ;Proteinasesfromthedigestiveorgansofblackcarp(Mylopharyngodonpiceus):

    ;Partialcharacterizationandproteindigestibilityinvitro ;activitiesatrangesofpH2.02.5pH8.18.6andnear

    ;pH9.8,respectively,indicatingthepresenceofthe ;acidicandthealkalineproteinases.

    ;024681012

    ;pH

    ;F.1Activity-pHprofilesofextractsfromtheblackcarp ;digestiveorgans.Resultsweremean?SDoftrlplicates ;Theacidicproteinaseactivityassaysshowedthat ;activityinintestinesf2.121,0.21U/mgprotein)was ;lowerthanthatinhepatopancreasr3.12~0.15U/rag ;protein)(Fig.2,p<O.05).Therewassignificant ;differencebetweentwomeanactivitiesat0.05Ieve1. ;3.2Proteolysisactivitiesofextractsfromthe ;blackcarpdigestiveorgans

    ;Thealkalineproteinasesactivity(Fig.2)also ;showedthatactivityinintestinesf6.28?0.38U/mg

    ;protein)waslowerthanthatinhepatopancreas(15.63? ;0.46U/mgprotein)(Fig.2,p<0.01).Therewas ;significantdifferencebetweentwomeanactivitiesat ;0.01leve1.Thealkalineproteinasesactivitywas ;significantlyhigherthantheacidicproteinasesactivity ;eitherinintestinesorinhepatopancreas(Fig.2, ;p<0.o1).Significantlyhighactivityinbothintestines ;andhepatopancreassuggestsimportanceofthealkaline ;proteinasesduringproteindigestioninthisspecies. ;g

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