产甲烷条件下吲哚类有机物的降解途径

2002,8(5):514519ChinJApplEnvironBiol=ISSN1006-687X2002210225:2001209227:20012112163Correspondingauthor1111,23(1;2),..,3232.,323232.,,23.12,.322,3,.322,.3H,2,3.1425;;CLCX173DEGRADATIONPATHWAYSANDMECHANISMSOFSUBSTITUTEDINDOLESUNDERMETHANOGENICCONDITIONWANGYingying1,QIUWanfei1,FANYanzhen1&GUJidong1,23(1LaboratoryofEnvironmentalToxicology,DepartmentofEcology&Biodiversity;2TheSwireInstituteofMarineScience,theUniversityofHongKong,HongKongSAR)AbstractDegradationofindolebyanindole2degradingmethanogenicconsortiumenrichedfromsewagesludgeproceededthroughatwo2stephydroxylationpathwayyieldingoxindoleandisatin.Theabilityofthisconsortiumtohydroxylateandsubsequentlydegradesubstitutedindoleswasinvestigated.Ofthesub2stitutedindolestested,theconsortiumwasabletotransformordegrade32methylindoleand32indolylac2etate.Oxindole,32methyloxindole,andindoxylwereidentifiedasdegradationmetabolitesofindole,32methylindole,and32indolylacetate,respectively.Isatin(indole22,32dione)wasproducedasaninterme2diatewhentheconsortiumwasamendedwithoxindole,whichprovidedtheevidencethatdegradationofindoleproceededthroughsuccessivehydroxylationofthe22and32positionspriortoringcleavagebetweentheC22andC23atomsonthepyrroleringofindole.Thepresenceofamethylgroup(CH3)ateitherthe12or22positionofindoleinhibitedtheinitialhydroxylationreaction.Thesubstitutedindole,32methylin2dole,washydroxylatedatthe22positionbutnotatthe32positionandcouldnotbefurthermetabolizedthroughtheoxindole2isatinpathway.Indoxyl(indole232one),thedeacetylatedproductof32indolylac2etate,wasnothydroxylatedatthe22positionandthuswasnotfurthermetabolizedbytheconsortium.WhenanHatomorelectron2donatinggroup(i.e.,CH3)waspresentatthe32position,hydroxylationproceededatthe22position,butthepresenceofelectron2withdrawingsubstituentgroups(i.e.,2OHor2COOH)atthe32positioninhibitedhydroxylation.Tab1,Fig4,Ref25Keywordsindoles;pathway;methanogenicconditionCLCX172©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.,[1,2].[3].[4].,32[58].,[9,10].,[11]..,.,[12].,.,,[13,14].,[14].[15][13,16,17],.().11.112223232232.,3232.32,90d.0.5L1mg32.(m/z147),(max=249nm[17]),(1H2NMR):(CDCl3),1.52(d,CH3,J=7.7Hz),3.48(q,H23,J=7.6Hz),6.92(d,H27,J=7.9Hz),7.05(dd,H26,J=7.0Hz,J=7.9Hz),7.23(dd,H25,H24,J=6.4Hz,J=7.9Hz).1.246.,.2L.(0.43mmol0.1mL)(0.43mmol/L).(HPLC)().190d.(=120)15min,N245..:75mL160mL25mL,.,10d,20mL80mL().44,.20mL2143mol80mL.24.3.(1mL),.(CK)()..CK3d(120)20min.0.2m,(500L)CK.1.3,,1;1(),0.2m..CM34003100.25cm(Supelcosil5m).1.0mL/min(60;40)122232.32323223225;10;65()--(60;39.5;0.5),1.0mL/min.271,290,270,260,260,273,247,249243nm,3222122323232.0.05mmol/L0.5mmol/L.5155:©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.50L(HP5890),.1.8m2.1mm80100PorapakN.15050.()20mL/min.1.43..5mL,Na2SO4,N20.2mL..0.25mm.CH2Cl2(6;3;1)(Rf=0.46),32100;1CH2Cl2(Rf=0.62),6;1;6CH2Cl2(Rf=0.69)..,32,N2..32(19;1)..N2,.BeckmanDU27.7070E2HF(VGAnalytical,Manchester,UK)32.Bruker2702MHz..22.150h,325h(1).CK,.[16,17],.(max,247nm),HPLC(3.43min)(m/z,133)(1),.75%,.325h,C8H7N+7H2O4.5CH4+3.5CO2+NH3(78%)..1Tab1ComparisonofthedataforauthenticandauthenticatedindolesstandardsandmetabolitesCompoundsHPLCRetentiontime(tr/min)UVUVpeaks(max/nm)Rf(TLC)Massspectra(m/z)Oxindole(Standard)3.432470.46133Oxindole(Metabolite)3.432470.46133Isatin(Standard)3.232430.69147Isatin(Metabolite)3.232430.691473232methyloxindole(Standard)4.102490.621473232methyloxindole(Metabolite)4.102490.621471Fig1Metabolismofindolewithsubsequentproductionofoxindoleandmethane5(r20.95)Concentrationdatashowingthedisappearanceofindole,theappearanceanddisappearanceofoxindole,andtheappearanceofmethanewerefitintoafifth2orderpolynomialregressionmodel(r20.95)[17],,,.,.,.(m/z,147),(max,243nm)HPLC(3.23min)(1).CK.2.212.1222,100h,32615ChinJApplEnvironBiol8©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved..,,(4.10min)(max,249nm[18])32(1).3232.600h,,32.,32,.3232(2).23232Fig2Transformationof32methylindolewithsubsequentproductionof32methyloxindole43(r20.96)Concentrationdatashowingdisappearanceof32methylindoleandappearanceof32methyloxin2dolewerefittofourth2orderandthird2orderpolynomialregressionmodels,respectively(r20.96)2.332232232323.32,.32,(3).,50h,322(100h)(1,2).1/33248h,480h.32.,.,.,(3.73min)..,.,,,[19,20].(max,284,600nm),Rf(0.61).,,.3232.,3210%(3).CK.32,232.332Fig3Degradationof32indolylacetatewithsubsequentproductionofmethaneandproposedmetabolite,indoxyl33Indoxylconcentrationinculturemediumisrepresentedbyarelativeab2sorbancescale3[17].,[21],[22],[23],23.[19]Alcaligenes,3(),2().,..(Aspergillusniger)3(),,N22[24].,.,.7155:©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.,32.,,.,,.,323.,2.3,,.,32,2.32,.,,[25].,2,.,:22(pKa=0.28)12(pKa=2.3)(pKa=3.6)32(pKa=4.6)232(pKa=6.13).12().3.13(),.32,(0.1mmol/L),.,122,12.,1(N),.2,22[13,14,17].2,2,.43232Fig4Proposedpathwaysfordegradationofindole,32methylindole,and32indolylacetateundermethanogeniccondition32.32.,,3..32815ChinJApplEnvironBiol8©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.32(4),.12,22232.,,.References1BlumDJ,He