Screening,Cloning And Expression Of An Aldo-keto Reductase And Its Application In Chiral Alcohol Synthesis

t-Butyl 6-cyano-?3R,5R?-dihydroxyhexanoate??3R,5R?-CDHHB?is one of the widely applicable chirality intermediates,which is a crucial building block and pharmacophore of atorvastatin.The aldo-keto reductase?AKR?has highly stereoselective towards carbonyl compound in biocatalytic asymmetric reduction.We make an attempt to obtain enantiomerically pure?3R,5R?-CDHHB by means of the asymmetric reduction of?3R,5R?-CDHHB with the AKR,and establish the research process.In this paper,the AKR gene Caakr was cloned into the expression vector pET-28a?+?and the recombinant plasmid was introduced into E.coliBL21?DE3?.Through colony PCR,sequence analysis and SDS-PAGE,the AKR gene Caakr was successfully express in E.coliBL21?DE3?.Unfortunately,the catalytic activity was comparatively low.The recombinant E.coliBL21?DE3?/p ET-28a-Caakr?Ca-7?cells were cultured in optimized fermentation conditions containing 10.0 g/L peptone,yeast extract 5.0 g/L,10.0 g/L NaCl,5.0 g/L glycerol,5.0 g/L Na₂HPO₄,p H 7.0.They were cultured at 37?C for inoculation time 2-3 h.For induction the gene,9 g/L lactose was added to the medium with induced temperature25?C for 10 h.Under these optimal conditions,the AKR activity has 1.48times higher than the initial numerus.The recombination AKR was purified and characterized.First the AKR was purified by metal chelate affinity chromatography.The purified enzyme was approximately 38.6 kDa with a single subunit.The enzyme was higher activity at a pH range of 6.0-8.0.The optimal reaction temperature was 30?C.Then,substrate specificity of the AKR was also studied.The AKR showed high activity and enantioselectivity with all carbonyl compound reductions.The kinetics parameters of NADH and?3R,5R?-CDHHB were determined.Last but not least,the homology structure modeling was using by Discovery Studio 2.5.,and this work will lay the foundation for subsequent genetic modification.