羰基还原酶基因与葡萄糖脱氢酶基因在大肠杆菌中的共表达及其在不对称还原产麻黄碱中的初步应用

Co-expression of glucose dehydrogenase and carbonyl reductase in Escherichia coli and its application in asymmetric synthesis of d-Ψ-ephedrine

通过羰基还原酶基因与葡萄糖脱氢酶基因在大肠杆菌中的共表达,解决羰基还原酶在催化底物过程中的辅酶再生的问题。以枯草芽孢杆菌基因组为模板,采用PCR的手段扩增得到葡萄糖脱氢酶基因gdh与已构建好的pKK223-3-mldh连接,转化E.coli JM109获得重组菌E.coli pKK223-3-gdh-mldh。SDS-PAGE结果表明羰基还原酶及葡萄糖脱氢酶均有表达其相对分子质量分别为43 kD和31 kD。液相检测重组菌细胞破碎液在不添加外源的葡萄糖脱氢酶的情况下能专一性转化1-苯基-2-甲氨基丙酮简称MAK为d-伪麻黄碱。全细胞转化实验表明0.1 g湿菌体与0.15 mg MAK及6 mg葡萄糖30℃反应10 h生成0.091 mg d-伪麻黄碱,MAK的摩尔转化率为67.4%。

In order to get hydrogen sources continouesly which was necessary to perform the reduction reaction for ephedrine production, a recombinant Escherichia coli with carbonyl reductase coupled with glucose dehydrogenase was constructed. The glucose dehydrogenase gene gdh was amplified from Bacillus subtilis by PGR technique. Then the purified PCR products were inserted into plasmid pKK223-3-m/dh being constructed previously to construct plasmid pKK223-3-gdh-mldh. The positive plasmid was transformed into E. coli JM109, and a recombinant strain E. coli pKK223-3-gdh-m/dh was obtained. The results showed by SDS-PAGE analysis that two enzymes were expressed simultaneously and the molecular weights were 43 kid and 31 kD respectively. It was illustrated by HPLC assay that the recombinant strain could be used to catalyze MAK （1-phenyl-1-keto-2-methylaminopropane） to d-ψ-ephedrine without adding glucose dehydrogenase. In further whole-cell transformation test, 0.1 g thallus incubated with 0.15 mg MAK and 6 mg glucose could produce 0.09 mg d-ψ-ephedrine at 30 C for 10 h, and the transformation rate of MAK reached 67.4%.