不同启动子调控羰基还原酶基因在枯草芽胞杆菌中的表达水平

Expression of carbonyl reductase in Bacillus subtilis with different promoters

为了实现羰基还原酶基因mldh在枯草芽胞杆菌中的高效表达,以摩氏摩根菌Morganella morganii CMCC(B)49208染色体DNA为模板,PCR扩增得到目的基因mldh,分别与启动子PQ和启动子p43进行连接,构建不同启动子组合的表达载体PHY-p43-mldh、PHY-PQ-mldh、PHY-p43-p43-mldh和PHY-p43-PQ-mldh,化学法转化B.subtilis Wb600后对重组菌细胞破碎液进行SDS-PAGE分析及全细胞生物转化反应实验发现,4种重组菌的转化能力差异显著,其中重组菌B.subtilis Wb600(PHY-p43-p43-mldh)进行全细胞转化反应,转化液中d-伪麻黄碱的浓度最高,达到142.1 mg/L,底物转化率为78.25%,成功实现了羰基还原酶基因mldh在枯草芽胞杆菌中的高效表达。

In order to achieve its high expression in Bacillus subtilis, carbonyl reductase gene mldh was amplified by PCR with the template of chromosome DNA from Morganella morganii CMCC （ B ） 49208 and connected with promoter PQ and promoter p43 to obtain different expression plasmids PHY-p43- mldh, PHY-PQ-mldh, PHY-p43-p43-mldh, and PHY-p43-PQ-mldh. Subsequently, these recombined vectors were transformed into B. subtilis Wb600. SDS-PAGE analysis of recombinants. And whole cellular biotransformation showed that there was distinctive difference among the four recombinants, in which the B. subtilis Wb600 （PHY-p43-1M3-mldh） could obtain the highest concentration （ 142. 1 mg/L） of d- pseudoephedrine in conversion solution and the mole ratio of substrate was 78.25%.