Catalytic Performance Analysis Of Stereoselective Oxidoreductases And Enzyme-coupled Deracemization Of Chiral Alcohols

Biocatalytic preparation of optically active alcohols has the advantages of high efficiency, specificity, stereoselectivity and mild reaction conditions. Dozens of stereoselective oxidoreductases from various microorganisms were collected and the recombinant Escherichia coli was used for overexpressing these oxidoreductases. Several prochiral aryl ketones, aliphatic ketones and ketoesters were applied as substrates to conduct asymmetric reduction to investigate the properties of the biocatalysts. In order to build a deracemization system with（R,S）-1-phenyl-1,2-ethanediol（PED） as the model substrate, the suitable candidates were obtained to construct the enzymatic deracemization system involving cofactor self-recycling, through evaluation of available stereoselective oxidoreductases on activity, selectivity, and cofactor dependency. Based on the above research, a deracemization system with（R,S）-N,N-dimethyl-3-hydroxy-（2-thienyl）-1-propanamine（DHTP） as substrate was built and used for the production of（S）-DHTP. The concrete contents include:（1） 20 stereoselective oxidoreductases from 8 kinds of microorganisms were collected. The protein expression of the stereoselective oxidoreductases was optimized through the research of the expression condition of recombinant proteins. The results indicated that the suitable induction temperature of SCR and CPAR1 was 30 ℃, the suitable induction temperature of other recombinant enzymes was 17℃. The optimal lactose concentration was 2% for CPAR5 and CPAR8. The optimal IPTG concentration was 0.5 mmol×L^-1 for CR4, KRD, SCR. The optimal IPTG concentration was1 mmol for other enzymes. The optimized stereoselective oxidoreductases were both purified by one-step Ni-affinity chromatography, and used for the subsequent experiments.（2） The capability of recombinant enzymes for catalyzing reduction of carbonyl compounds was investigated towards 14 kinds of substrates including prochiral aryl ketones, aliphatic ketones and ketoesters. The substrate specificity, kinetic parameters and stereoselectivity were measured. The results indicated that each stereoselective oxidoreductases had activity toward a variety of prochiral substrates. For the selected substrates, 20 kinds of stereoselective redox enzymes showed a larger difference, which had higher catalytic activity toward 2-hydroxyacetophenone, 2’-chloroacetophenone, 3’-chloroacetophenone, 4’-bromo acetophenone, 2-octanone, methyl acetoacetate than 2’-hydroxyacetophenone, 2-heptanone. The stereoselective oxidoreductase toolbox was outstanding with broad substrate spectrum, high catalytic efficiency and excellent stereoselectivity, which had high efficiency to catalyze asymmetric transformation.（3） For deracemizing（R,S）-PED to（R）-PED, a facile one-pot system was established by combination of two stereoselective oxidoreductases, the stereospecific carbonyl reductase 1（SCR1） and the ketoreductase（KRD）. To rebalance the activities and catalytic functions of different enzymes involved in the multi-enzyme system, the reaction conditions of SCR1-catalyzed oxidation and KRD-mediated reduction were optimized, respectively. Consequently, the deracemization system involving cofactor self-recycling was built to produce（R）-PED with the optical purity of 95.50%e.e. and the yield of 91.62% from the corresponding racemate, under the optimal reaction conditions including activity ratio of SCR1/KRD 1:4, molar ratio of NADP+/NADPH 3:1, 1 mmol×L^-1 ZnSO4, 30℃, and pH 7.0. Therefore, the developed strategy would be useful to construct the multi-enzyme deracemization system based on systematic evaluation of enzyme features.（4） According to the strategy to construct the multi-enzyme deracemization system, a deracemization system was established by selective oxidation and asymmetric reduction, for deracemizing（R,S）-DHTP to（S）-enantiomer. The stereospecific carbonyl reductase 3（SCR3） and the carbonyl reductase（CR2） catalyzed selective oxidation and asymmetric reduction, respectively. The deracemization of racemic（R,S）-DHTP was performed with the condition of triethanolamine buffer（pH 8.0）, 30℃, 1 mmol×L^-1 ZnSO4, by optimizing the catalytic conditions for selected enzymes. Consequently, the deracemization system involving cofactor self-recycling was built to produce（S）-DHTP with the optical purity of 91.71%e.e. and the yield of 89.45% from the corresponding racemate, under the optimal reaction conditions including activity ratio of SCR3/CR2 1:2, molar ratio of NADP+/NADPH 3:1. When the substrate concentration was increased to 4 g×L^-1, the yield and optical purity of the product（S）-DHTP was 78.29% and 86.79%e.e., respectively.