title: Significantly enhancing recombinant alkaline amylase production inBacillus subtilisby integration of a novel mutagenesis-screening strategy with systems-level fermentation optimization.
abstract: BACKGROUND
  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 inB. subtilis168.
  
  
  RESULTS
  The activity of alkaline amylase in mutantB. subtilis168 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 ofB. subtiliswas 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 inB. subtilis168 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 inB. subtilis168 mut-16#.
  
  
  CONCLUSIONS
  The results indicated that this novel ARTP mutagenesis-screening method could significantly improve the yield of recombinant proteins inB. subtilis. Meanwhile, fermentation optimization strategies efficiently promoted expression of recombinant alkaline amylase inB. subtilis168 mut-16#. These findings have great potential for facilitating the industrial-scale production of alkaline amylase and other enzymes, usingB. subtiliscultures as microbial cell factories.
authors: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122 China.
authors: Yingfang
authors: Y
authors: Ma
authors: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122 China.
authors: Wei
authors: W
authors: Shen
authors: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122 China.
authors: Xianzhong
authors: X
authors: Chen
authors: The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122 China.
authors: Long
authors: L
authors: Liu
authors: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122 China.
authors: Zhemin
authors: Z
authors: Zhou
authors: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122 China.
authors: Fei
authors: F
authors: Xu
authors: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122 China.
authors: Haiquan
authors: H
authors: Yang
keywords: ARTP mutagenesis; Alkaline amylase; B. subtilis; Fed-batch; Fermentation optimization; Overexpression