Preparation Of Ethyl(R)-4-Chloro-3-Hydroxybutanoate By Enzymatic Asymmetric Reduction

Ethyl-(R)-4-Chloro-3-hydroxybutyrate((R)-CHBE)is an important chiral intermediate.Using carbonyl reductase to catalyze the asymmetric hydrogenation of ethyl-4-chloroacetoacetate(COBE)to prepare(R)-CHBE is a very promising synthetic method.However,the enzymes with high specific activity had been patented,and there are few reports on the molecular modification of carbonyl reductase to improve its catalytic activity.In this study,new enzyme with high catalytic activity and independent intellectual property rights was screened,molecular modification and expression level enhancement were carried out toward the carbonyl reductase.A double-enzymatic system was constructed to achieve coenzyme regeneration.(1)The carbonyl reductase enzyme library was constructed using gene mining,and a carbonyl reductase KY(from Rhodotorula mucilaginosa)with high catalytic activity for COBE was obtained.The crude enzyme activity was 3.60 U/mL,specific activity was 72.57 U/mg.(2)Using semi-rational design to carry out molecular modification of KY.Error-prone PCR was used to construct a random mutation library,and four positive mutants were obtained through high-throughput screening.Sequences analysis revealed that 157 and 213 were the key sites.Site-directed saturation mutagenesis was performed at 157 and 213,and 5 positive mutants were obtained.The activities were 1.23?1.82 times higher than wild KY.Finally,combined mutagenesis was applied based on the positive mutants of 157 and 213.The dominant mutant KY(T157S/G213P)was achieved with a crude enzyme activity of 7.10 U/mL and a specific activity of 138.42 U/mg,which was 1.91 times than that of wild KY.(3)The recombinant plasmid pET22b-KY(T157S/G213P,SKIK)was constructed by replacing the plasmid vector and adding fusion tags at the N-terminal.The crude enzyme activity was 24.51 U/mL.Afterwards,through optimization of culture condition and induction condition,the crude enzyme activity was increasing to 53.11 U/mL.(4)Carbonyl reductase and glucose dehydrogenase were used to build a double-enzymatic system for the regeneration of NADPH.At 35?,pH=7.5 and 200 rpm,when the system with 0.24 M COBE,0.24 M glucose and 0.01 mM NADP+,KY(T157S/G213P,SKIK)and BsGDH were added in the amount of 12 U/mL.Finally,the COBE could be completely converted after 14 h.