Cloning And Expressing Of Aldo-Keto Reductase TdAKR And Its Application In The Systhesis Of (S)-4-Chloro-3-Oxobutanoate

Chrial alcohols are building blocks in the synthesis of important chrial pharmaceuticals.?S?-4-chloro-3-hydroxybutanoate??S?-CHBE?is a versatile chrial intermediate,especially as a crucial intermediate of atrovastatin calcium that is a cholesterol-lowering drug.Producing?S?-CHBE by using biocatalysis technology has received increasing attentions due to the following advantages,such as mild reaction condition,predominant enantioselectivity,and fewer by-product,environmentally friendly.This thesis reported an aldo-keto reductase was cloned,heterogously expressed and applied in the synthesis of?S?-CHBE.1.A novel aldo-keto redutase Td AKR has been cloned and heterogously expressed in E.coli BL21?DE3?from Torulaspora delbrueckii ZJB15080.The recombinant enzyme was purified and its enzymatic characteritics were investigated.Td AKR has a molecular weight about 38.6 kDa and is active at 30?and pH 7.0.It is stable at temperatures blow 35?,acidic condition.The maximal reaction rate(v_max)to ethyl 4-chloro-3-oxobutanoate?COBE?,apparent Michaelis-Menten constant?K_m?for NADPH and COBE and catalytic number(k_cat)were47.51 U·mg^-1,0.07 mM,1.19 mM and 30.56 s^-1.2.The cultivation condition was optimized for E.coli BL21?DE3?/pET-28a-tdakr.the optimized cultivation condition was as folloes:2%?v/v?inoculation size was preferred;lactose was added into media at a final concentration of 9 g·L^-11 after cultivated for 2 h at37?.After induced at 28?for 12 h,the enzyme activity reached 1932.6U·g^-1,while biomass approached 1.92 g·L^-1?DCW?.Adding 10 g·L^-1sucrose and 5 g·L^-1 NaNO₃ into medium can improve enzyme activity to2374.35 U·g^-1,while biomass is 2.1 g·L^-1?DCW?.3.The process of COBE reduction to?S?-CHBE by TdAKR coupled with EsGDH for cofactor regeneration was studied.At 50 g·L^-1 COBE,the yield of?S?-CHBE reached 91.5%,with e.e.valueof 95.6%and space-time yield of 549 g·L^-1·d^-1.At 75 g·L^-1 COBE,the reaction reached the equilibrium after 3 h conversion,in a yield of 90.3%,e.e.95.7%and space-time yield of 541.8 g·L^-1·d^-1.While the concentration of COBE was raised 100 g·L^-1,E.coli BL21?DE3?/pET-28a-tdakr still displayed good catalytic performance,with a yield of 87.2%,e.e.value 95.7%,but the space-time yield was lowered to 380.7 g·L^-1·d^-11 and the yield decreased to64.8%when at 125 g·L^-1 COBE.It was demonstratd that Td AKR is qualified for industrial application.