Ma Yingfang, Shen Wei, Chen Xianzhong, Liu Long, Zhou Zhemin, Xu Fei, Yang Haiquan
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122 China.
The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122 China.
J Biol Eng. 2016 Oct 17;10:13. doi: 10.1186/s13036-016-0035-2. eCollection 2016.
Alkaline amylase has significant potential for applications in the textile, paper and detergent industries, however, low yield of which cannot meet the requirement of industrial application. In this work, a novel ARTP mutagenesis-screening method and fermentation optimization strategies were used to significantly improve the expression level of recombinant alkaline amylase in 168.
The activity of alkaline amylase in mutant 168 mut-16# strain was 1.34-fold greater than that in the wild-type, and the highest specific production rate was improved from 1.31 U/(mg·h) in the wild-type strain to 1.57 U/(mg·h) in the mutant strain. Meanwhile, the growth of was significantly enhanced by ARTP mutagenesis. When the agitation speed was 550 rpm, the highest activity of recombinant alkaline amylase was 1.16- and 1.25-fold of the activities at 450 and 650 rpm, respectively. When the concentration of soluble starch and soy peptone in the initial fermentation medium was doubled, alkaline amylase activity was increased 1.29-fold. Feeding hydrolyzed starch and soy peptone mixture or glucose significantly improved cell growth, but inhibited the alkaline amylase production in 168 mut-16#. The highest alkaline amylase activity by feeding hydrolyzed starch reached 591.4 U/mL, which was 1.51-fold the activity by feeding hydrolyzed starch and soy peptone mixture. Single pulse feeding-based batch feeding at 10 h favored the production of alkaline amylase in 168 mut-16#.
The results indicated that this novel ARTP mutagenesis-screening method could significantly improve the yield of recombinant proteins in . Meanwhile, fermentation optimization strategies efficiently promoted expression of recombinant alkaline amylase in 168 mut-16#. These findings have great potential for facilitating the industrial-scale production of alkaline amylase and other enzymes, using cultures as microbial cell factories.
碱性淀粉酶在纺织、造纸和洗涤剂工业中具有重要的应用潜力，然而其低产量无法满足工业应用的需求。在本研究中，采用了一种新型的常压室温等离子体（ARTP）诱变筛选方法和发酵优化策略，以显著提高重组碱性淀粉酶在枯草芽孢杆菌168中的表达水平。
突变体168 mut-16#菌株中碱性淀粉酶的活性比野生型高1.34倍，最高比生产率从野生型菌株的1.31 U/(mg·h)提高到突变体菌株的1.57 U/(mg·h)。同时，ARTP诱变显著增强了枯草芽孢杆菌168的生长。当搅拌速度为550 rpm时，重组碱性淀粉酶的最高活性分别是450和650 rpm时活性的1.16倍和1.25倍。当初始发酵培养基中可溶性淀粉和大豆蛋白胨的浓度加倍时，碱性淀粉酶活性提高了1.29倍。补料水解淀粉和大豆蛋白胨混合物或葡萄糖显著促进了枯草芽孢杆菌168 mut-16#的细胞生长，但抑制了碱性淀粉酶的产生。补料水解淀粉时碱性淀粉酶的最高活性达到591.4 U/mL，是补料水解淀粉和大豆蛋白胨混合物时活性的1.51倍。在10 h进行基于单次脉冲补料的分批补料有利于枯草芽孢杆菌168 mut-16#中碱性淀粉酶的产生。
结果表明，这种新型的ARTP诱变筛选方法可以显著提高枯草芽孢杆菌中重组蛋白的产量。同时，发酵优化策略有效地促进了重组碱性淀粉酶在枯草芽孢杆菌168 mut-16#中的表达。这些发现对于利用枯草芽孢杆菌培养物作为微生物细胞工厂促进碱性淀粉酶和其他酶的工业化规模生产具有巨大潜力。
