Asymmetric Synthesis For Chiral Alcohols With Recombinant Escherichia Coli

Biocatalytic asymmetric reduction potential chiral carbonyl compounds are effective approach for the preparation of chiral alcohols.In the process,the substrate inhibition and/or product inhibition tend to affect the biological catalysis efficiency to a certain extent.In this paper,a recombinant E.coli was used as biocatalyst,which was successfully constructed by our research group previous,the biocatalytic asymmetric reduction 3,5-bis?trifluoromethyl?phenyl ethanone?BTAP?to?R?-3,5-bis?trifluoromethyl?phenyl alcohol??R?-BTPE?and ethyl acetoacetate?EAA?to?R?-ethyl-3-hydroxybutyrate??R?-EHB?were used as model reaction.In order to enhance recombinant E.coli catalytic efficiency,some methods of strengthening the reaction process and protein molecule modification technology were applied in this study.The main contents were as follow:In this study,substrate fed-batch strategy was explored in recombinant E.coli catalytic asymmetric reduction BTAP to?R?-BTPE,the concentration of BTAP was raised to 2 M from 1 M by 2times-addition,the yield was 65.5%in 24 h;the space-time yield per gram of biomass of this asymmetric reduction was 4.3 mmolL^-1h^-1g^-1_DCW,then by introducing SP207 adsorption resin to further improve the reaction efficiency,at the final with adding 100 g/L resin at 9 h,the yield and product ee value were 75.5%and 99%,respectively,the reaction time was shortened to 20 h.In the recombinant E.coli catalytic EAA asymmetric synthesis for?R?-EHB,an organic solvent/buffer biphasic system was established.n-Hexane was selected as optimum non aqueous medium.And the effects of diverse reaction conditions were investigated systematacially.The obtained optimized conditions for the bioreduction were as follows:10%?v/v?n-hexane,buffer pH 7.5?0.2 M Na₂HPO₄-NaH₂PO₄?,2 M of EAA,15 g/L?DCW?cells as biocatalyst,30oC and 180 rpm,under the optimization conditions,the yield of 91%in 10 h and>99%of product e.e.were obtained,respectively.Lastly,the preliminary study of saturated mutations was carried out based on the homology modeling,molecular docking and the analysis of enzyme conservative sequence to select key active site residues of LXCAR-S154Y.The mutant LXCAR-S154Y-D200Q showed better performance in catalyzing asymmetric reduction EAA to?R?-EHB,the result was EAA concentration of 2 M,?R?-EHB with yield of 90.5%and e.e.of 99%was obtained after 6 h reaction.