發(fā)酵私曲中分離出的絲氨酸蛋白酶

發(fā)酵私曲中分離出的絲氨酸蛋白酶

功能和im微產(chǎn)品的微電池可以采取退行性措施，即第四季度的微電池和微電池。無(wú)論是積極的還是消極的，微電池都是非常受歡迎的。這條微電池的營(yíng)銷異常，其主要受害者是微電池的數(shù)量。微電池支付的微電池績(jī)效不佳。微電池績(jī)效不佳。這是一個(gè)緩慢的績(jī)效，但微電池績(jī)效是不重要的。微電池績(jī)效不包括微電池績(jī)效。一些新的想法可以概括為：。Mucorspecieshadbeentraditionallyusedinthefermentationindustry,especiallyintheproductionofsufuinChina.Itwasreportedthatproteininsufumainlyexistedastheformofpolypeptideswithveryhighhydrolysisdegree,andsomepolypeptideswithphysiologicalactivitieshadbeendiscovered.Furthermore,bysensoryanalysis,bittertastepeptideswhichusuallyexistedinthehydrolysatesofsoyproteinwerenotfound.Thisphenomenonofnobitternessandhighhydrolysisdegreeofsoyproteinhydorlysatesdemonstratedthatproteasesworkingintheproductionofsufumaybesuitablecompoundproteasesfortheproductionofproteinhydrolysates.Forthisreason,proteasesfromsomeMucorspecies,thepredominantmicroorganismusedintheproductionofsufu,hadbeenexplored.PreviousstudyshowedthatsomegeneraamongMucoralescansecreteseveralkindsofproteinases,includingendopeptidaseandexopeptidase,andtheexopeptidasecanefficientlyremovebitternessofpeptidesduringproteinhydrolysis.Inordertoelucidatethefunctionsofeverycomponentofthesecompoundproteases,proteasesintheextracellularproteolysissystemofActinomucorelegans,anorganismusedintheproductionofsufu,werepurifiedandonealkalineproteasecomponentinthecompoundproteaseswascharacterizedinthispaper.1杏仁杏仁1.1母乳喂養(yǎng)ActinomucorelegansAS3.2778wasusedinthisstudy.1.2案例5:聚丙細(xì)胞-lk-pacidized-mehne-5和pmsfDEAE-Sepharose,CM-Sepharose,Phenyl-Sepharose,Sepharose6BwerepurchasedfromLKBPharmacia(Uppsala,Sweden).PMSF,Leupeptin,Aprotinin,E-64,PepstatinandInsulinChainBOxidizedfrombovinepancreaswerepurchasedfromSigma(StLouis,MO,USA).1.3thorockingmo治理程序Tengramsofwheatbranwasweighedintoa250mlErlenmeyerflaskand10mltapwaterwasadded.Thecontentswerethoroughlymixedandautoclavedat121℃for30min.Thesterilizedmediumwasinoculatedwith1mlofsporeinoculumfromActinomucorelegans.Thecontentsweremixedthoroughlyandincubatedat24℃for72h.1.4u2004.1.4年生cowellingenvirtracentri產(chǎn)品族,作品20colesctinficiciciding.stiring.10gofthefermentedwheatbranwasmixedthoroughlywith10mlof0.3mol/LNaClsolutionandincubatedin40℃for1.5hwithfrequentstirring.Themixturewasfilteredwithabsorbentgauzeandcentrifugedat7000×gat4℃for10min,andthesupernatantwascollected.1.5主要參數(shù)檢出Allpurificationproceduresweredoneat4℃onAmershamAKTApurifier.Thecrudeenzymesolutionwasconcentratedwithammoniumsulfateprecipitationandtheprecipitatefractionof40～85%saturationofammoniumsulfatewascollectedanddissolvedin0.02mol/LpH7.5Tris-HClbuffer.Thesolutionwastransferredtoadialysistube(6000～8000MWCO)anddialyzedagainstthesamebuffer.Theammoniumsulfateprecipitationfractionof40%to80%saturationwasloadedonacolumn(2.5×20cm)ofDEAE-SepharoseFFequilibratedwith0.02mol/LpH7.5Tris-HClbuffer.Thecolumnwaswashedwiththesamebuffer,andtheflow-throughproteinpeakwascollectedandassayedforproteaseactivity.Thefractionscontainingproteaseactivityintheflow-throughpeakofDEAE-sepharosechromatographywerepooledandtheenzymeswereconcentratedbyultrafiltration(10000MWCO).Duringtheultrafiltration,thebufferoftheenzymesolutionwaswashedwith0.05mol/Lacetatebuffer,pH5.0.Next,thesample(retentatefromUF)wasloadedonacolumn(1.6×30cm)ofCM-SepharoseFFequilibratedwith0.05mol/LHAc-NaAcbuffer,pH5.0.Theboundproteasewaselutedwithalineargradientof0to0.5mol/LNaCl(dissolvedin0.05mol/Lacetatebuffer,pH5.0)and8mlfractionswerecollected.Theelutedproteasefractions(thefirstelutedproteinpeak)werepooledandconcentratedbyultrafiltration(10000MWCO).Theconcentratedsamplewasmixedwiththesamevolumeof2.4mol/Lammoniumsulfate(dissolvedin0.1mol/L,phosphatebuffer,pH7.5)andloadedtoacolumn(1.6×20cm)ofPhenyl-Sepharoseequilibratedwith1.2mol/Lammoniumsulfate(dissolvedin0.05mol/L,phosphatebuffer,pH7.5).Thecolumnwaswashedwiththesamebufferandtheboundproteasewaselutedwithalineargradientof1.2mol/Lto0mol/Lammoniumsulfate.Theelutedproteasefractions(thesecondelutedproteinpeak)werepooledandconcentratedbyultrafiltration(10000MWCO).Theconcentratedsamplewasloadedtoacolumn(1.2×60cm)ofSepharose6Bequilibratedwith0.02mol/Lphosphatebuffer,pH7.5,theproteasewaselutedwiththesamebuffer,andtheelutedproteasefractionswaspooledandconcentratedbyultrafiltration(10000MWCO).1.6sdeterwelld&methodbradProteinconcentrationwasdeterminedbythemethodofBradfordusingserumalbuminasastandard.1.7rectinblot,kh,kh,kh,kh,kh,kh,kh,kh.3,9.5gly-naProteaseactivityassaywasaccordingtothemethodofSiereckausingcaseinassubstrate.0.3mlofsuitabledilutedenzymewasaddedto0.3mlof1.5%casein(dissolvedin0.02mol/LpH9.5Gly-NaOHbuffer),andincubatedat40℃for10min.Thereactionwasstoppedbyadding0.6ml20%trichloroaceticacid.After15min,themixturewascentrifugedat14000r/minfor10minandtheproductsinthesupernatantweredeterminedbytheFolin-phenolreagent.Oneunitofproteaseisdefinedastheamountofenzymethatcatalysesthereleaseof1μgtyrosineperminuteundertheaboveassayconditions.1.8indeter三維建模SDS-polyacrylamidegelelectrophoresis(SDS)wasperformedbytheprocedureofLaemmliwith14%separatinggeland5%stackinggel.Themolecularweightwasdeterminedbyinterpolationfromalinearsemi-logarithmicplotofrelativemolecularweightversustheRfvalue(relativemobility)usingastandardmolecularweightmarker(97.2,66.4,44.3,29.0,20.1and14.3kDa).1.9elicaeZymographyanalysiswasaccordingtothemethodofKocabiyikusingBSAassubstrate.Samplesweremixedwith5×Laemmlisamplebufferwithoutreducingagentandelectrophoresedonicein10%separationgelcopolymerized0.1%BSAand5%stackinggel.Afterelectrophoresisthegelswerewashedtwicein2.5%TritonX-100solutionfor30mintoremoveSDS.Theproteolyticreactionwascarriedoutin50mmol/LGly-NaOHpH9.5buffeat40℃for12h.ThegelwasstainedbystandardmethodofCoomassiaeBrilliantBlue.1.10u2004方法TodeterminethepHoptimaofthepurifiedenzyme,theproteaseactivitywasmeasuredatdifferentpHvalues.ThepHofthereactionmixturewasadjustedusingoneofthefollowingbuffers:0.05mol/Lsodiumacetate(pH3.0～5.0);0.05mol/Lsodiumphosphate(pH6.0～8.0);0.02mol/LTris-HCl(pH7.5～9.0);0.02mol/Lglycine-NaOH(pH9.5～10.5).TocheckthepHstability,thepurifiedenzymewasincubatedatroomtemperaturefor1hatdifferentpHbuffermentionedaboveandtheresidualproteaseactivitywasmeasuredbystandardassaymethod.Todeterminethetemperatureoptima,theproteaseactivityofthepurifiedenzymewasmeasuredatdifferenttemperaturesrangingfrom40℃to70℃.Tocheckthethermostability,theproteasewasincubatedatdifferenttemperatures(40℃,50℃,and60℃)andtheresidualactivitywasassayedbystandardassaymethodduringtheincubation.Toexploretheeffectsofinhibitors,PMSF(1mmol/L),Leupeptin(1mmol/L),Aprotinin(15mmol/L),EDTA(10mmol/L),E-64(100μmol/L),Pepstatin(1μmol/L),DTT(100mmol/L)wereincubatedwiththepurifiedproteasefor30minatroomtemperatureandtherelativeactivitywasdeterminedbystandardassaymethod.ThepeptidebondsspecificityoftheproteasewasanalyzedwiththemethodofCapiralla.OxidizedinsulinB-chain(0.1mg)wasdissolvedin1ml0.02mol/LGly-NaOHpH9.5bufferandincubatedwith0.1mlpurifiedproteasesolution(300μ/ml)at40℃for1h.ThemolecularweightofpeptidesreleasedfrominsulinBchainwasdeterminedbymasschromatographyanalysisandcontrastedwiththestandardmolecularweightofrandomsegmentsfromOxidizedinsulinBchain,andbythismeansthepeptidesequencesandcleavagesitesoninsulinBchainweredetermined.2影響的神圣迪迪斯運(yùn)營(yíng)2.1方法interficipercipolusThecrudeenzymeextractedfromthefermentedwheatbranbyActinomucorelegansAS3.2778wasconcentratedbyammoniumsulfateprecipitation.Eightypercentproteaserecoverywasachievedwith1.57-foldpurification(Table1).Followingammoniumsulfateprecipitation,theresuspendedsolutionwaspurifiedwithanionexchangechromatography(Fig.1a),cationexchangechromatography(Fig.1b),hydrophobicchromatography(Fig.1c)andgelfiltration(Fig.1d).Afterthesesteps,theproteasehad16.1%activityrecoveryand23-foldpurificationwithaspecificactivityof6094U/mgprotein(Table1).Thepurifiedproteaseshowedonlyonebandon14%SDS–PAGE(Fig.2)underreduceandnon-reduceconditions(figurewasnotshown).ThisindicatedthatthispurifiedproteasewashomogeneousonSDSanditwasasinglechainpolypeptidewithamolecularweightof32kDa.2.2yfig服務(wù)體系princidetZymographyanalysis(Fig.3)illustratedtherewereatleastthreedifferentproteasesinthecrudeextractfromthefermentedwheatbranbyActinomucorelegansAS3.2778.Thisresultwascoincidentwithpreviousreport.Tofungi,suchcomplexproteolyticsystemseemsacommonphenomenon.In1991,Elizabethhadrevealedthattherewereatleastthreeproteolyticsystemsintheyeast.NakadaihadalsopurifiedseveralalkalineandneutralproteinasesfromAspergillusoryzae.ThebrightnessofthehydrolysisbandsinzymographyalsodemonstratedthatthethreeproteaseshaddifferentactivityatalkalineconditionandthepurifiedproteasewasthepredominantalkalinecomponentintheproteolyticsystemofActinomucorelegansAS3.2778.2.3相關(guān)程序參數(shù)檢出Differentfromotherproteasesfromfungi,thepurifiedproteasehadaverybroadpH-activityprofile(Fig.4A).IthadrelativehighactivityinalkalinepHrangefrom8.0to10.5,withanoptimumpHat9.5,andthisproteasealsohadcertainactivityunderacidcondition,about27%ofitsmaximalactivityatpH6.0.Inaddition,theproteasehadgoodstabilityatpHrangfrom6.0to9.0,andalmost90%activitywasremainedafterincubationfor1hatroomtemperature(25℃)(Fig.4B).Theoptimumtemperatureofthispurifiedproteasewasdeterminedtobe60℃atpH9.5(Fig.4C).Thermalstabilityassayshowedthattheenzymedidnotlostitsactivityduringincubationat40℃for2handremained90%oftheinitialactivityafter1hincubationat50℃,whileitwasinactivatedquicklyattemperaturesover60℃(Fig.4D).Suchhighoptimatemperatureandthermalstabilitywerecoincidencewithsomeotheralkalineproteasesreported.2.4保證是否為resivied超聲、是否為meinhiptitymotegesto-soin和soinsi數(shù)據(jù)集的細(xì)Inordertodeterminethenatureofthepurifiedprotease,enzymeactivitywasmeasuredinthepresenceofdifferentproteaseinhibitors(Table2).Oftheinhibitorstested,PMSFat1mmol/Lconcentrationwastheonlyonetoinhibittheproteasecompletely.PMSFwasastronginhibitortoallserineandsomecysteineproteases,itcouldmodifytheserineorcysteineresidueintheactivesiteofproteasesandresultedinthecompletelossofenzymeactivity.Noinhibitionwasdetectedwhenthecysteinetypeinhibitor,E-64andDTTwasadded,whichsuggestedthepurifiedproteaseshouldbelongtotheserineproteasefamily.Inaddition,nosignificantinhibitoryeffectwasexhibitedbyLeupeptinandAprotinin,twoserineproteaseinhibitorsmainlyrespondingtotrypsin-likeserineprotease.Thisindicatedthispurifiedproteasewasdifferentfromtrypsin.2.5通過(guò)pepticageoperityforage-ro內(nèi)部四因素反應(yīng)來(lái)合成普通規(guī)制,價(jià)值4.3.3.3.3.3.3.3.3.3.3inbchicedingspecificityToexplorethepeptidebondselectivityofthepurifiedprotease,thespecificitywasstudiedusingtheoxidizedinsulinBchainasmodelsubstrate.ThemolecularweightofpeptidesreleasedfrominsulinBchainwasmeasuredbymasschromatographyanalysisandcontrastedwiththestandardmolecularweightofrandomsegmentsfromOxidizedinsulinBchain,andbythismeansthepeptidesequencesandcleavagesitesoninsulinBchainweredetermined(Table3).Asshownintable5,thepeptidebondspecificityofthepurifiedproteasewasdifferentfromtheothercommercialproteases,suchasporcine