| (R)-carbonyl reductase(RCR) from Candida parapsilosis CCTCC M203011 has high chemo-, region-, stereo-selectivity, but it requires coenzymes for chiral catalysis. The coenzymes are expensive, limiting the chiral biosynthesis by RCR. In this paper, we constructed a coupled system containing RCR and glucose dehydrogenase(GDH) to realize the cofactor regeneration using overlap extension technique. Since RCR gene has some rare codes, Escherichia coli RIL was used as host cell coexpressing RCR and GDH. Recombinant Escherichia coli RIL strain efficiently catalyzed the biotransformation of(R)-1-phenyl-1, 2-ethanediol((R)-PED) using 2-hydroxyacetophenon(2-HAP) as substrated and glucose as cosubstrate. Besides, a highthroughput screening method was established to screen RCR with high activity. The main results were described as follows:(1) The RCR and GDH genes were ligated with a Shine–Dalgarno and aligned spacing sequence as a linker between them through overlap-extension technique using pET28a-RCR and pET28a-GDH as template. The recombinant plasmid pET-R-SD-AS-G was constructed and then transformed into the competent E. coli BL21, E. coli RIL and E. coli Rosetta cells respectively, resulting three recombinant strains: E. coli BL21/pET-R-SD-AS-G, E. coli RIL/pET-R-SD-AS-G and E. coli Rosetta/pET-R-SD-AS-G. The expression level and biotransformation efficiency of recombinant protein in three above hosts were determined. The results suggested that E. coli RIL/pET-R-SD-AS-G showed the highest specific activity and biotransformation efficiency of(R)-PED. The introduction of GDH into E. coli RIL had almost no effect on cell-growth properties. Thus the recombinant E. coli RIL/pET-R-SD-AS-G was used for further experiments.(2) The protein expression conditions of recombinant RCR and GDH in E. coli RIL/pETR-SD-AS-G were optimized. The optimal expression conditions are follows: OD600 of the culture is 1.0, the final concentration of IPTG is 0.4 mmol·L-1, induce duration is 10 h, and the culture temperature is 37 °C after IPTG induction. The recombiant RCR abd GDH were both purified by one-step Ni-affinity chromatography. SDS-PAGE analysis showed that two apparent bands of 44 kDa and 30 kDa were observed in the cell-free extracts E. coli RIL/pETR-SD-AS-G, corresponding to the expected sizes of RCR and GDH. The purified proteins exhibited specific activity of 17.8 U·mg-1 for 2-HAP reduction, and 21.5 U·mg-1 for glucose oxidation. The kcat/KM values of RCR and GDH was 3.2 for the reduction of 2-HAP to release NAD+, and 3.9 for the oxidation of glucose to form NADH. The ratio of kcat/KM for RCR and GDH catalyzing 2-HAP and glucose was about 1.0.(3) The whole-biocatalyst biotransformation conditions of E. coli RIL/RCR-GDH were optimized. When the concentration of 2-HAP is 6 g·L-1, pH value is 7.5, temperature is 35°C, the reaction duration is 24 h, it produced(R)-PED with an optical purity and a yield both over 99.9%. Compared with E. coli BL21/pET-RCR, the optical purity and yield of products were obviously enhanced. And more importantly, the reaction time was reduced 24 h.(4) To screen RCR with higher enzyme activity, a quick detection method to determine 2-HAP concentration was established based on the color reaction between 2-HAP and 2, 4-dinitrophenylhydrazine. Though full-wave scanning phenylhydrazone derivative of 2-HAP and 2, 4-dinitrophenylhydrazine, the detection wavelength was determined in 500 nm. The absorbance presented good curve linearity when 2-HAP concentration was at a range of 0-5 g·L-1 in 500 nm. The linear equation was y=0.0123x+0.0641, in which correlation coefficient was 0.9973. The mutant library was established by error-prone PCR. The GeneMorph II EZClone Domain Mutagenesis Kit was used to screen the variants and the mutation rate was 0.25% by DNA sequencing. After screening, the mutant D154 C was obtained and the enzyme activity reached 3.10 U·mg-1. Compared with the RCR, enzyme activity increased nearly five times. |
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