分子酶工程学研究进展

18420027ChineseJournalofBiotechnologyVol.18No.4July2002:2002202210,:2002204222:(No.39870204)23Tel:[phone];Fax:[phone];E2mail:[e-mail]21(,430071)2(BiochemistryDepartment,ImperialCollegeofScience,Technology&Medicine,London,SW72AY,UK),,,,,,,,TQ931C100023061(2002)0420401206,,,,,,,,,,,(Molecularenzymeengineering),,,3:;(Sitedirectedmutagenesis)(Invitromoleculardirectedevolution);(Fusionenzyme)13000,500,DNA350,100[1],,DNA,,,,Novozymes80%[2],,,,1,,,(Segregationalinstability)(Structuralinstability),,,,,;,,N2102030,N2;,2,:,lon-©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.;,T4Pin;1()Table1SomeofstudiesonenzymeexpressionTaqDNAThermusaquaticusT4DNA2Bacillusbrevis,,2,(Sitedirectedmuta2genesis)(Invitromoleculardirectedevolu2tion)[3],,,,(),,,211DNA,1Over2lapextensionPCR,CD[411](Glucoseisomerase),,[4],Pro138Gly138,,,1012,Pro138Gly138Gly138,,,,,,,,1Over2lapextensionPCRFig.1Sitedirectedmutagenesisprocedure20418©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.2DNAshufflingFig.2DNAshufflingprocedure212PCR(ErrorpronePCR)PCRPCR,dNTP(5%10%)dITPdNTPMn2+Mg2+DNA,(Subtilisin),ArnoldPCREdGTPdCTPdTTP1mmolPL,dATP011mmolPL,49CDNAPCR,(Dimethylformamide,DMF)D60N+Q103R+N218S85%DMF38[12];D60N+D97G+Q103R+G131D+E156G+N181S+S182G+S188P+N218S+T255A60%DMF256[13]213DNAshufflingDNAshuffling1994Stemmer[14],,DNAshuffling2,,,[1518]2Stemmer[19]DNAshuffling2DNAshuffling,16000D2(D2hydantoinase,HYD)N2(N2carbamylase,CAB)D2Kim[20]N2PD2,DNAshuf2flingDNAshuf2fling,F11,F1119,11(45,pH710),18hF1150%85%,5h,7h,D2,,F11D26DNAshuffling,Familyshuffling,4Familyshuffling,540,shuffling8[21],DNAshuf2flingStEP(Staggeredextensionprocess)[22](Random2priminginvitrorecombina2tion,RPR)[23](Randomchimeragenesisontransienttemplates,RACHITT)[24]StEPDNAshuffling,PCR,,,,,,RPR,DNA(,DNA),DNA,RPRDNAmRNARACHITTDNAshuffling,DNADNase,3[25]DNA,,,,311,(Affi2nitytail),3044:©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.,,,[26]2Table2ExamplesofcommercialsystemsusedintheproductionofrecombinantproteinsfusedtoaffinitytailsaFusionpartnerSizeLigandElutionconditionSupplier(s)ZZb14kDIgGLowpHPharmacia,SwedenHistail6210aaNi2+ImidazoleNovagen,USA;Invitrogen,USA;Qiagen,USAStrep2tag10aaStreptavidinIminobiotinBiometra,GermanyPinPointTM13kDcStreptavidindBiotinPromega,USAMBP40kDAmyloseMaltoseNewEnglandBiolabs,USAGST25kDGlutathioneReducingagentPharmacia,SwedenFlagTMpeptide8aaSpecificmAbLowcalciumIBIKodak,USAaAbbreviations:aa,aminoacids;His,histidine;GST,glutathione2S2transferase;MBP,maltosebindingprotein.bIgGbindingdomainofstaphylococcalproteinA.cEncodesadomainbiotinylatedinvivoinEscherichiacoli.dMonomericstreptavidin312(Greenfluorescentprotein,GFP),,[27]GFPNC,,Gordon[28](Glucoamylase,GA)2GFP,GFP,GA,GA(Organophosphorushydrolase,OPH)GFPOPH5,GFP2OPH,[29]IPTG,,OPHGFP,GFPOPH,OPH313,,,,[30],FMN:NAD(P)H(FMN:NAD(P)Hoxidoreductase)(Luciferase)Min[31](Biotincarboxycarrierprotein,BCCP)FMN:NAD(P)HN2,(Bio2tin)(Avidin),,NADH,8,,Hengsakul[32]C10(Streptag),EAP2Streptag(Streptavidin),(Carboxymethylateddextran,CMD),,(Link2er),,,814[33]314,(Sequenceenzymereaction),,[3437]D2D2(D2hydantoinase,HYD)N2(N2carbamylase,CAB),Kim[34]D2(HYDB.sthearothermophilusSD1,HYD1B.thermocatenulatusGH2)N2,CAB2HYDCAB2HYD1CAB2HYDCAB2HYD1D2N2,CAB2HYD1CAB2HYD,,,30%,,,,40418©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.[37](Glucoseoxidase,GOD)(Linker)(Glu2coamylase,GA)cDNA,GOD2Linker2GA,,,430kD,GODGA,GAGOD,GAGOD,GAGOD(,),,4,,,,,(Proteindenovodesign)[38],,,,,REFERENCES()[1]WhiteJS,WhiteDC.Sourcebookofenzymes.CRCPress,BocaRaton,NewYork,1997[2]JUNH().Thenewtrendinthestudiesofenzymeengineer2ingin21thcentury.IndustrialMicrobiology(),2001,31(1):3745[3]ZHANGXE().Molecularcontrolofenzymeusedinanaly2sis.JournalofWuxiUniversityofLightIndustry(),1999,18(5):118121[4]ZHUGP(),TENGMK()etal.MutationofG138PenhancedthethermostabilityofD2glucoseisomerase.ActaBiochimicaetBiophysicaSinica(),1998,30(6):607610[5]MaYF,EvansDEetal.Mutationsofbarleybeta2amylasethatim2provesubstrate2bindingaffinityandthermostability.MolGenetGenomics,2001,266(3):345352[6]HuangCY,ChangAKetal.Site2directedmutagenesisoftheioniz2ablegroupsintheactivesiteofZymomonasmobilispyruvatedecar2boxylase:effectonactivityandpHdependence.EurJBiochem,2001,268(12):35583565[7]WangSX,DunphyWG.ActivationofXenopusChk1bymutagene2sisofthreonine23771FEBSLett,2000,487(2):277281[8]ZhouH,WangHWetal.Themultiplerolesofconservedarginine286of12aminocyclopropane212carboxylatesynthase.Coenzymebind2ing,substratebinding,andbeyond.PlantPhysiol,1999,121(3):913919[9]AmidonWJ,PfeilJEetal.Modificationofluciferasetobeasub2strateforplantasparticproteinase.BiochemJ,1999,343Pt2:425433[10]TigueNJ,KayJ.Activesitemutantsofhumanherpesvirus26protei2nase.FEBSLett,1998,441(3):467469[11]AikawaJ,ParkYNetal.ReplacementsofaminoacidresiduesatsubsitesandtheireffectsonthecatalyticpropertiesofRhizomucorpu2silluspepsin,anasparticproteinasefromRhizomucorpusillus.JBio2chem(Tokyo),2001,129(5):791794[12]ChenKQ,ArnoldFH.Enzymeengineeringfornonaqueous