红平菇木质素降解酶系统漆酶-锰过氧化物酶及木质素过氧化物酶的检测-池玉杰

4512200912SCIENTIASILVAESINICAEVol.45,No.12Dec.,2009、池玉杰　闫洪波(　150040):　;;;;:S782.23　　　:A　　　:1001-7488(2009)12-0154-05:2009-05-29。:(30671700)。DetectiononLaccase,ManganesePeroxidaseandLigninPeroxidaseinLigninolyticEnzymesofPleurotusdjamorChiYujie　YanHongbo(CollegeofForestry,NortheastForestryUniversity　Harbin150040)Abstract:　WhiterotfungusPleurotusdjamorH1wasstillyculturedat28℃under4differentkindsofLNASculturesolution,theextracellularenzymesolutionsweresampledatdifferentinterval,O.D.valuesofthesolutions,representingmanganeseperoxidase(MnP),laccaseandligninperoxidase(LiP)activities,weremeasuredspectrophotometricallybymonitoringtheoxidationof2,6-DMPat470nm,ABTSat420nmandveratrylalcohol(VA)at310nm,thelignin-degradingenzymesofthefungusandtherelationshipsbetweenenzymeactivitiesandmediumcompositionandsubstratesweregained.ResultsindicatedthatPleurotusdjamorcouldproduceMnPandlaccasesimultaneously,butnoLip.Substrateswoodsawdustand2,6-DMPcouldsignificantlyenhancedMnPandlaccaseactivities,thebiggestMnpandlaccaseactivitywere5U·L-1.and38U·L-1whennosubstrateswereadded,and69U·L-1and205U·L-1whensubstratesadded.ThepaperofferedabasicligninolyticenzymologyofPleurotusdjamorforfutureutilizationoftheenzymesandfurtherstudyonMnPandlaccasemechanismofthefungus.Keywords:　Pleurotusdjamor;ligninolyticenzymes;manganeseperoxidase;laccase;enzymeactivitydetection　　,[(manganeseperoxidase,MnP)、(ligninperoxidase,LiP)][(laccase)]。,,、、DDT、、、。3,21,3。MnPlaccase,LiP。,MnPlaccase(Hofrichter,2002;Hatakka,1994)。MnP(EC1.11.1.13)H2O2,,、、MnP。laccase(EC1.10.3.2),、、,。LiP(EC1.11.1.14)MnP。(Phanerochaetechrysosporium)、(Ceriporiopsissubvermispora)、(Pleurotusostreatus)、(Trametesversicolor),、、(Alicetal.,1997;Giardinaetal.,2000;Irieetal.,2000;Telloetal.,2000;Johanssonetal.,2002;Yaveretal.,2003);MnPlaccase(,1993;,1998;,2002;,2005),MnPlaccase(,2003;,2008;,2008)。laccase、,(Neurosporacrassa)、(Flammulinavelutipes)(,2004;,2006),MnP。,;,、。(Pleurotusdjamor),、、,。H1-(LNAS,lownitrogenasparaginesuccinicacid)4,MnP,laccaseLiP,,,,。1　1.1　菌种来源　H1,(PDA,potatodextroseagar)4℃。1.2　培养基　:PDA。:-(Kirketal.,1978;Hatakkaetal.,1983)。1.3　培养方式　250mL70mLLNAS,4:A.LNASMn2+,MnSO4·H2O;B.LNASMn2+(2.67μmol·L-1),MnSO4·H2O;C.LNASMn2+(2.67μmol·L-1),2g(Populusussuriensis);D.LNASMn2+(2.67μmol·L-1),10mmol·L-12,6-(2,6-DMP)0.1mL。15min,5mL15%。PDA(9cm),28℃10,8mm,,5,28℃,3。3,5,7,9,11,13,15,1721,1.3mL,(13200r·min-1,)5min,-20℃。1.4　酶活测定方法　MnP(Ultrospec4300pro,Amersham)470nm2,6-DMP(Wariishietal.,1992)。1mL,(50mmol·L-1)840μL,MnSO4(10mmol·L-1)50μL,2,6DMP(10mmol·L-1)50μL,50μL,H2O2(10mmol·L-1)10μL,1mL,470nm1min,10mmol·L-1H2O2。laccase420nm2,2′--(3--6-)(ABTS)(Eggertetal.,1996)。1mL,(50mmol·L-1)850μL,100μL,ABTS(20mmol·L-1)50μL,1mL,420nm3min。Lip(VA)(Tienetal.,1988)。1mL,(100mmol·L-1,pH3.0)340μL,(20mmol·L-1)100μL,550μL,H2O2(54mmol·L-1)10μL,1mL,310nm3min。(U):,1μmol2,6-DMP,ABTSVA。ε470=49600(mol·L-1cm)-1,ε420=36000(mol·L-1cm)-1,ε310=9300000(mol·L-1cm)-1。2　2.1　红平菇4种培养液中LiP的酶活测定　4LiP,310nm,LiP。2.2　红平菇培养液A酶活测定结果　Mn2+LNAS21,MnP,laccase,laccase9,38U·L-1(1)。2.3　红平菇培养液B酶活测定结果　Mn2+LNAS21,MnP,,15,5U·L-1;laccase9,36U·L-1(2)。A,B2:155　12:、1　AMnPlaccaseFig.1　MnPandlaccaseactivityofculturemethodAMn2+MnP,MnP,。A,B2。2　BMnPlaccaseFig.2　MnPandlaccaseactivityofculturemethodB2.3　红平菇培养液C酶活测定结果　Mn2+LNAS21,MnP,MnP13,69U·L-1;laccase9,205U·L-1(3)。MnPlaccase,MnP,。3　CMnPlaccaseFig.3　MnPandlaccaseactivityofculturemethodC2.4　红平菇培养液D酶活测定结果　Mn2+LNAS2,6-DMP21,MnP,MnP11,46U·L-1;laccase9,199U·L-1(4)。2,6-DMPMnPlaccase,MnP。C,D2,MnPlaccase2,6-DMPMnPlaccase。2.5　红平菇4种培养方式下MnP和laccase活性的比较　4,MnPlaccase。laccase,3,,9,,11,13～15;MnP3,,,11～13～15,,1721,(Chietal.,2007)。MnPMn2+,laccaseMn2+,。2,6-DMPMnP15645　4　DMnPlaccaseFig.4　MnPandlaccaseactivityofculturemethodDlaccase,laccase4,MnP10;,2,6-DMPMnPlaccase,MnP(5)。5　4MnPlaccaseFig.5　ComparisionofMnPandlaccaseactivityamongfourculturemethod3　LNAS4MnP,laccaseLiP。,MnPlaccase,LiP,2,laccase9。2,。4,Mn2+MnP,Mn2+MnP,,。Mn2+,MnPMn2+,,Mn3+,Mn3+,,,,,(,2007)。2,6-DMP,MnPlaccase,。,2,6-DMPMnPlaccase。2,6-DMP,,2,6-DMP,。MnPlaccase,-,MnP,,。,,、,。　,,　.2006..,25(4):43-54.,.2007.-、.,26(1):153-160.,,,.1998..,17(3):251-255.,.2002..:,17(2):76-78.,,,.2008.TP21.:,29(5):54-57.,.2008.G1502.,29(5):287-291.,,.2003.PhanerochaetesordidaYK-157　12:、624.,23(4):22-26.,,,.2005..,32(5):98-102.,　,,.2004..,44(6):775-779.,,.1993.Ⅰ..,33(5):387-391.AlicM,AkileswaranL,GoldMH.1997.Characterizationofthegeneencodingmanganeseperoxidaseisozyme3fromPhanerochaetechrysosporium.BiochimicaetBiophysicaActa,1338:1-7.ChiYJ,HatakkaA,MaijalaP.2007.Canco-culturingoftwowhite-rotfungiincreaselignindegradationandtheproductionoflignin-degradingenzymes?InternationalBiodeteriorationandBiodegradation,59(1):32-39.EggertC,TempU,ErikssonKE.1996.Theligninolyticsystemofthewhite-rotfungusPycnoporuscinnabarinus:purificationandcharacterizationofthelaccase.AppliedandEnvironmentalMicrobiology,62:1151-1158.GiardinaP,PalmieriG,FontanellaB,etal.2000.ManganeseperoxidaseisoenzymesproducedbyPleurotusostreatusgrownonwoodsawdust.ArchivesofBiochemistryandBiophysics,376(1):171-179.HatakkaA.1994.Lignin-modifyingenzymesfromselecte