英文文獻(xiàn)匯報(bào)課件

細(xì)胞色素P450酶系孫婉菊2014年12月17日TowardsPreparativeScaleSteroidHydroxylationwithCytochromeP450MonooxygenaseCYP106A21.CYP106A2fromBacillusmegateriumATCC13368,enablestheoxidationof3-keto-4-enesteroidsmainlyatposition15.2.Weexpressedthisenzymetogetherwiththeelectron-transferpartnersbovineadrenodoxinandadrenodoxinreductaseinEscherichiacoli.3.Anenzyme-coupledcofactorregenerationsystemwasimplementedbyexpressingalcoholdehydrogenasefromLactobacillusbrevis。文獻(xiàn)匯報(bào)CloningpBARTwin:bovineAdR、AdxpACYCFHH28:CYP106A2 pBARTripel:bovineAdR、AdX、LbADHSubstratesolubilityandtransportintothecellHighestactivitywasobservedinpresenceofeven2m(15.4vol%)propan-2-ol.Duringthesecondreactioncycleonly21%ofProgcouldbeconvertedinthepresenceofpropan-2-ol.Withoutthiscosolvent53%15b-Progwasformed;Thisindicatesthelimitedapplicabilityofpropan-2-olasacosolventinthissystem.Influenceofpropan-2-olontheactivity(A)andstability(B)ofthewhole-cellbiocatalyst21%53%Substratesolubilityandtransportintothecellbiotransformationwith“membrane-free”crudecellextract(CCE)andthewhole-cellFig.ConversionofprogesteronebyusingcrudecellextractofE.coliexpressingCYP106A2,AdR,AdxandLbADHasbiocatalystTheinitialreactionrateincreased:0.4U15?-Proggwcw-1to9.6U15?-Proggwcw-1glyophilisate-1incasesofCCE.

Alossinselectivitycomparedtorestingcells.20%sideproductsareformed.CofactordemandWhenpropan-2-olwasadded,theamountofNADPHdrasticallyincreasedto80%,duetothecofactorreductionbyLbADH.AfterstartingtheP450-catalyzedhydroxylationbyaddingProg,theamountofNADPHdroppedto70%.Mostofthecofactorispresentinreducedform.Whenthesteroidformationratedecreasedoverthereactiontime,theNADPHconcentrationincreasedagain.RedoxcofactorconcentrationduringbiotransformationBatchexperiments

15?-Progformation:45minreactionconversion:85%Tesconversion:30minconversion:nearlycompletearound20%SPsproductivity:3.7gL-1d-1for15?-Prog

5.5gL-1d-1for15?-Tesincreaseupto18-foldBatchexperimentsunderoptimizedconditionsforprogesterone(A)andtestosterone(B)conversion工作匯報(bào)1.發(fā)酵培養(yǎng)基中不同種類碳源對(duì)菌體生物量的影響碳源種類葡萄糖甘油第一批菌體量（g/L）17.518.9第二批菌體量（g/L）18.3212.靜息細(xì)胞轉(zhuǎn)化實(shí)驗(yàn)1.葡萄糖2.甘油3.甘油+乙醇4.葡萄糖+0.05VB5.葡萄糖+0.1VB6.葡萄糖+5乙酸鈉7.葡萄糖+10乙酸鈉8.葡萄糖+15乙酸鈉試驗(yàn)組0.2mg/