英文文獻匯報課件

細胞色素P450酶系孫婉菊2014年12月17日TowardsPreparativeScaleSteroidHydroxylationwithCytochromeP450MonooxygenaseCYP106A21.CYP106A2fromBacillusmegateriumATCC13368,enablestheoxidationof3-keto-4-enesteroidsmainlyatposition15.2.Weexpressedthisenzymetogetherwiththeelectron-transferpartnersbovineadrenodoxinandadrenodoxinreductaseinEscherichiacoli.3.Anenzyme-coupledcofactorregenerationsystemwasimplementedbyexpressingalcoholdehydrogenasefromLactobacillusbrevis。文獻匯報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1.發(fā)酵培養(yǎng)基中不同種類碳源對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.靜息細胞轉(zhuǎn)化實驗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乙酸鈉試驗組0.2mg/