| Ethyl(S)-4-chloro-3-hydroxybutanoate((S)-CHBE)is one of the key chiral intermediates for synthesis side chain of the cholesterol-lowering drug atorvastatin,which is also widely applied in the synthesis of marine bromopyrrole alkaloids,1,4-dihydropyridine-type p-blocker and HIV protease inhibitor(S)-3-hydroxytetrahydrofuran.Now,the biosynthesis of(S)-CHBE has been drawn extensive attentions,but there is still no any reports about its production in industrial scale.It is very necessary to study biosynthesis process of(S)-CHBE and enhance its productivity.In this paper,the carbonyl reductase and glucose dehydrogenase genes were cloned into pCDFDute-1 and the recombinant plasmid was introduced into E.coli BL21(DE3)and the recombinant E.coli BL21(DE3)/pCDFDuet-l-GDH6-6a(ZJB12065)was successfully con-structed.Then,the recombinant E.coli catalyzed the asymmetric reduc-tion of ethyl 4-chloroacetoacetate(COBE),the product was identified as(S)-CHBE with e.e.of 99%.Cultivation conditions of recombinant E.coli coexpressing carbonyl reductase and glucose dehydrogenase were investigated by single-factor method.The optimized fermentation medium was as follows:peptone 15,0 g/L,yeast extract 12.0 g/L,NaCl 10.0 g/L,(NH4)2SO4 5 g/L,glycer-ol 15 g/L,KH2PO4 1.36 g/L,K2HPO4·3H2O 2.28 g/L,MgSO4·7H2O 0.375 g/L and initial pH 7.0.50?g/mL streptomycin sulfate was added into medium after sterilization.The optimal fementation conditions were following:fermentation temperature was 37 C,flask volumn was 100 mL in 500 mL flask,inoculum size was 2%,and cultured time was 2 h at 150 rpm respectively.10 g/L lactose was added to induce enzymes ex-pressed for 12 h at 28?.Under the optimal conditions,the enzyme ac-tivity and biomass increased to 9093.78 U/L and 4.07 g DCW/L which was 1.64-and 1.75-fold as compared to before optimazation.In the 50 L and 500L bioreactors,the biomass and enzyme activity increased to 3.24 and 2.60 folds compared with that in the flask,respectively,under the op-timal condition obtained in this study,culture process in 5 t bioreactor was subsequently designed.In this study,the asymmetric reduction from COBE to(S)-CHBE was studied.In the aqurous/organic diphase system,the optimized reac-tion mixture of 100 mL in 100 mM KH2PO4-K2HPO4 buffer(pH 7.0)concluded the same volumn of butyl acetate,600 mM COBE,870 mM glucose and 15 g DCW/L cells.The reaction was carried out at 35'C,350 rpm in the reactor.The pH of the reaction mixture was controlled at 7.0 by 2 M NaOH.60 mM COBE and 87 mM glucose was added to the mix-ture at 30 min while 50 mM COBE and 72.5 mM glucose were added at 90 min.All COBE was completely transformed to(S)-CHBE at 4 h.The concentration of(S)-CHBE and the yield reached 1652.71 mM and 97%with a space time yield of 413.17 mM/h and 27.55 mM/h/g DCW which was higher than any reports.The optical purity of(S)-CHBE was greater than 99%of e.e..Finally,the extraction process of(S)-CHBE was studied.2 g/L acti-vated carbon was added to the reaction mixture and then it was filtrated after stirring 30 min at 100 rpm.The(S)-CHBE in aqurous phase of fil-trate was extracted by ethyl acetate,the volume ratio of ethyl acetate to the aqurous phase was 0.2 and the extraction stage was 5,and then the ethyl acetate was used to extract the activated carbon residue.Amber product was obtained by vacuum distillation from the organic phase after washed by NaHCO3 or NaCl saturated solution,and subsequently dried by anhydrous Na2SO4.The yield of(S)-CHBE was 82%,and the product purity was 95%... |
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