Deng Yi, Nie Yao, Zhang Yu, Wang Yue, Xu Yan
School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China.
School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China.
Protein Expr Purif. 2018 Aug;148:9-15. doi: 10.1016/j.pep.2018.03.012. Epub 2018 Mar 27.
Pullulanase is crucial to the specific hydrolysis of branch points in amylopectin and is generally employed as an important enzyme in the starch-processing industry. Recombinant Bacillus subtilis that employs an inducible promoter would be a suitable candidate for pullulanase expression because of its safety and controllable production, but its level of pullulanase activity is relatively low. In this study, we investigated the effect of the enhancers DegQ, DegU, and DegS on pullulanase expression in a recombinant B. subtilis inducible system. The genes degQ, degU, and degS were introduced to the recombinant plasmid pMA0911-P-pul harboring the promoter P, signal peptide LipA, and gene encoding pullulanase. The regulatory effects of the enhancers involved in recombinant plasmids on pullulanase expression level were evaluated in B. subtilis WB600 and WB800, respectively. The positive regulation of DegQ toward pullulanase expression was detected from B. subtilis WB800, leading to a 60% increase in enzyme activity. In addition, enzyme activity was further enhanced by inserting the degQ gene to the position closer to the promoter P. Consequently, pullulanase activity reached 26.5 U ml from the B. subtilis WB800/pMA0911-P- pul-degQ(N) after expression optimization, which was a 5.9-fold increase compared to that of the original strain B. subtilis WB800/pMA0911-P-pul. Hence, the inducible expression of the enzyme was efficiently enhanced by regulating the enhancer DegQ from recombinant B. subtilis WB800.
普鲁兰酶对于支链淀粉中分支点的特异性水解至关重要，并且在淀粉加工行业中通常用作一种重要的酶。采用诱导型启动子的重组枯草芽孢杆菌因其安全性和可控制的生产而将是普鲁兰酶表达的合适候选者，但其普鲁兰酶活性水平相对较低。在本研究中，我们研究了增强子DegQ、DegU和DegS对重组枯草芽孢杆菌诱导系统中普鲁兰酶表达的影响。将基因degQ、degU和degS引入携带启动子P、信号肽LipA和编码普鲁兰酶基因的重组质粒pMA0911-P-pul中。分别在枯草芽孢杆菌WB600和WB800中评估了重组质粒中涉及的增强子对普鲁兰酶表达水平的调节作用。从枯草芽孢杆菌WB800中检测到DegQ对普鲁兰酶表达的正向调节，导致酶活性增加60%。此外，通过将degQ基因插入更靠近启动子P的位置，酶活性进一步增强。因此，经过表达优化后，枯草芽孢杆菌WB800/pMA0911-P-pul-degQ(N)的普鲁兰酶活性达到26.5 U/ml，与原始菌株枯草芽孢杆菌WB800/pMA0911-P-pul相比增加了5.9倍。因此，通过调节重组枯草芽孢杆菌WB800中的增强子DegQ，有效地增强了该酶的诱导表达。
