信号肽对Bacillus subtilis表达环糊精葡萄糖基转移酶的影响

将来源于作者所在实验室构建保存E.coli pET-20b（+）/CGT?驻E-CBMAmy的CGT⊿E-CBMAmy基因插入6种含有不同信号肽（estA、bpr、vpr、yncM、yvgO和ywbN）的穿梭质粒pMA0911-SPs中，构建了6种重组表达载体并将其转入表达宿主B. subtilis WB600中，获得了6株重组菌。在相同的发酵条件下，estA信号肽介导的分泌效果最好，L-抗坏血酸2-葡萄苷产量达到0.81 g/L，明显高于其他5株重组菌。选取WBpMB/CGT?驻E-CBMAmy作为出发菌株，进行了摇瓶发酵条件优化，实现了CGT?驻E-CBMAmy酶的高效表达，最终使AA-2G产量提高了2.16倍，VC转化率达到21.9%。
CGT⊿ECBMAmy gene from E. coli pET-20b（+）/CGT?驻E-CBMAmy was cloned and inserted into the expression plasmid pMA0911-SPs containing six different signal peptides（estA，bpr，vpr，yncM，yvgO and ywbN ），which were further transformed into B. subtilis WB600 for overexpression to obtain six recombinant strains. The results showed that estA was the optimal signal peptide among all the signal peptides under the same culture conditions，the yields of AA-2G was up to 0.81 g/L，obvious higher than that of the other five strains. Therefore，WBpMB/CGT?驻E-CBMAmy was chosen as the starting strain and the optimum conditions for extracellular expression of CGT?驻E-CBMAmy were investigated in shaking flask. Finally，the high expression of CGT?驻E-CBMAmy enzyme was achieved，and the yields of AA-2G was about 2.16 times higher than that before optimization and the conversion rate of VC reached 21.9%