Chen Ana, Li Yamei, Liu Xiuxia, Long Quan, Yang Yankun, Bai Zhonghu
National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China.
J Ind Microbiol Biotechnol. 2014 Dec;41(12):1803-10. doi: 10.1007/s10295-014-1523-3. Epub 2014 Oct 14.
Bacillus acidopullulyticus pullulanase (BaPul13A) is a widely used debranching enzyme in the starch industry. A few details have been reported on the heterologous expression of BaPul13A in Escherichia coli (E. coli). This study compares different E. coli expression systems to improve the soluble expression level of BaPul13A. When pET22b(+)/pET28a(+) was used as the expression vector, the soluble expression of BaPul13A can be achieved by tightly controlling basal expression, whereas pET-20b(+)/pGEX4T2 leads to insoluble inclusion bodies. An efficient process control strategy aimed at minimizing the formation of inclusion bodies and enhancing the production of pullulanase was developed by a step decrease of the temperature in a 5-L fermentor. The highest total enzyme activity of BaPul13A reached 1,156.32 U/mL. This work reveals that the T7 promoter with lac operator and lacI gene collectively contribute to the soluble expression of BaPul13A, whereas either a T7 promoter alone or combined with the lac operator and lacI gene results in poor solubility. Basal expression in the initial growth phase of the host significantly affects the solubility of BaPul13A in E. coli.
嗜酸普鲁兰芽孢杆菌普鲁兰酶（BaPul13A）是淀粉工业中广泛使用的脱支酶。关于BaPul13A在大肠杆菌（E. coli）中的异源表达已有一些报道。本研究比较了不同的大肠杆菌表达系统，以提高BaPul13A的可溶性表达水平。当使用pET22b(+)/pET28a(+)作为表达载体时，通过严格控制基础表达可实现BaPul13A的可溶性表达，而pET - 20b(+)/pGEX4T2则导致不溶性包涵体的形成。通过在5升发酵罐中逐步降低温度，开发了一种有效的过程控制策略，旨在最大限度地减少包涵体的形成并提高普鲁兰酶的产量。BaPul13A的最高总酶活性达到1156.32 U/mL。这项工作表明，带有lac操纵子和lacI基因的T7启动子共同促进了BaPul13A的可溶性表达，而单独的T7启动子或与lac操纵子和lacI基因组合则导致溶解性较差。宿主初始生长阶段的基础表达显著影响BaPul13A在大肠杆菌中的溶解性。
