Study On Asymmetric Bioreduction Of Tert-butyl(R)-6-cyano-5-hydroxy-3-carbonylhexanoate

t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate is the key chiral intermediate in the enantioselective synthesis of statins,an effective medicine for the treatment of hypercholesterolemia.Therefore,a good method to synthetic t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate is of great economic and social benefits.Because of its mild reaction conditions,high conversion rate and outstanding stereochemical specificity,biocatalysis has become the most promising and preferred method in the asymmetric synthesis.In this work,the cultivation conditions of E.coli BL21/pET28a-cr3 carrying carbonyl reductase gene and E.coli BL21/pET28a-gdh4 carrying glucose dehydrogenase gene were optimized in detail.In order to solve the problem of coenzyme regeneration,a two-enzyme system in which glucose dehydrogenase was coupled with carbonyl reductase was constructed.The two-enzyme system was studied for asymmetric reduction of t-butyl(R)-6-cyano-5-hydroxy-3-carbonylhexanoate,as well as the kinetics.First,the cultivation conditions of E.coli BL21(DE3)-pET28a-cr3 were optimized and given as follows:seed age 7 h,inoculum size 3.0%,culture at 37 ? for 2 h,then induced by 9.0 g·L-1 lactose for 9.0 h at 28 ?.Under the optimal cultivation conditions,the specific activity reached 2622.3 U·g-1,with the biomass of 3.6 g·L-1.The optimal cultivation conditions of E.coli BL21(DE3)-pET28a-gdh4 were listed as follows:seed age 7 h,inoculum size 4.0%,culture at 37 ? for 2 h,then induced by 9.0 g·L-1 lactose for 9 h at 28 ?.Under the optimal cultivation conditions,the specific activity reached 3410.4 U·g-1,with the biomass of 3.5 g·L-1.t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate was synthesized via asymmetric reduction of t-butyl(R)-6-cyano-5-hydroxy-3-carbonylhexanoate catalyzed by carbonyl reductase coupled with glucose dehydrogenase.Under the optimal biocatalytic conditions,which were t-butyl(R)-6-cyano-5-hydroxy-3-carbonylhexanoate 147.0 g·L-1,glucose 128.2 g·L-1,cell loading 15.0 g·L-1,and the pH6.5,temperature 30 ?,t-butyl(R)-6-cyano-5-hydroxy-3-carbonylhexanoate was completely converted in 6 h without exogenous addition of NADP(H),with t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate d.e.value over 99.5%.In addition,the lyophilization greatly prolonged the preservation time of biocatalysts.The freeze-dried cells can be stored for 3 weeks at 4 ?.t-Butyl(R)-6-cyano-5-hydroxy-3-carbonylhexanoate asymmetric reduction can be completed in 7?8 h under the same condition by taking the fresh cell with the d.e.value over 99.5%.The kinetics of CR as well as GDH catalyzed reaction,in which two substrates participated was studied.The kinetic parameters were obtained by the initial-rate analysis of the CR and GDH catalyzed reaction,the kinetic equations of CR and GDH were obtained as follows.Product inhibition studies revealed that the CR follows an ordered bi-bi mechanism with NADPH binding first to CR.These equations were well fitted to the two-enzyme coupled system.