To address the challenge of cofactor regeneration,a dual-plasmid co-expression system was developed by constructing carbonyl reductase gene CatS126A/R129Q/V194A and glucose dehydrogenase gene GDH in single plasmids.Three single plasmid co-expression vectors were successfully designed,with the optimal vector identified as pETDUET.Through a detailed analysis of the dual-promoter and dual-expression vector,the crucial enzymes Cats126A/R129Q/V194A were prioritized.Ultimately,E.coli BL21(DE3)/pETDUET-GDH-CatS126A/R129Q/V194A was chosen as the biocatalyst.The expression conditions of this system were systematically optimized.The results indicated that the optimal induction temperature for E.coli BL21(DE3)/pETDUET-GDH-CatS126A/R129Q/V194A was 25 ℃,with an optimal IPTG concentration of 0.1 mmol/L and an optimal induction OD600 of 3.0.After optimization,the specific enzyme activity reached 20.8 U/mg(wet cells),representing a 64％improvement compared to pre-optimization levels.Subsequently,a 100 mL reaction system was investigated using the recombinant bacteria cultured under the optimized co-expression conditions as the catalyst,and ethyl 8-chloro-6-oxooctanoate(ECOO)as the substrate.The results demonstrated that 100 g/L of recombinant bacteria efficiently reduced 220 g/L of ECOO to(R)-8-chloro-6-hydroxy ethyl octanoate((R)-ECHO)within 6 hours.The process exhibited a high yield of 96％and an excellent enantiomeric excess(ee)of 98％.
biocatalysisco-expressionoptimization of expression conditions
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