首页|双酶共表达的条件优化及其硫辛酸中间体制备

双酶共表达的条件优化及其硫辛酸中间体制备

Optimization of co-expression conditions of two enzymes and preparation of lipoic acid intermediate

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为解决酶法制备硫辛酸关键手性中间体(R)-8-氯-6羟基辛酸乙酯中的辅酶循环再生问题,将羰基还原酶基因CatS126A/R129Q/V194A和葡萄糖脱氢酶GDH基因构建在同一质粒中,成功构建了 3种单质粒共表达载体,筛选出最佳单质粒共表达载体pETDUET.研究了 2个酶在双启动子双表达载体中的顺序,考察了反应中的关键酶CatS126A/R129Q/V194A,选择E.coli BL21(DE3)/pETDUET-GDH-CatS126A/R129Q/V194A作为生物催化剂.优化该表达系统的表达条件,结果表明:E.coli BL21(DE3)/pETDUET-GDH-CatS126A/R129Q/V194A的最适诱导温度为25 ℃,最适诱导剂IPTG浓度为0.1 mmol/L,最适诱导OD600为3.0.优化后的比酶活达20.8 U/mg(湿细胞),较优化前提高了64%.在此基础上,以共表达优化条件下培养的重组菌为催化剂,以8-氯-6-氧代辛酸乙酯(ECOO)为底物,开展100 mL反应体系研究.研究结果表明:质量浓度为100 g/L的重组菌可以在6 h内将220 g/L的ECOO还原成(R)-8-氯-6-羟基辛酸乙酯((R)-ECHO),收率达96%,ee为98%.
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

魏婷、姜灵、杨胜利

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浙江工业大学药学院,浙江湖州 313200

浙江工业大学绿色制药协同创新中心,浙江湖州 313200

生物催化 共表达 表达条件优化

2024

发酵科技通讯
中国发酵工业协会,杭州西湖味精集团有限公司

发酵科技通讯

影响因子:0.311
ISSN:1674-2214
年,卷(期):2024.53(4)