Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (4): 253-260.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0755

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Effects of Different Integration Sites on the Expression of Exogenous Alkaline Protease in Bacillus amyloliquefaciens

NIU Xin1(), ZHANG Ying1, WANG Mao-jun1, LIU Wen-long2, LU Fu-ping1, LI Yu1()   

  1. 1. Key Laboratory of Industrial Fermentation Microbiology,Ministry of Education,College of Biotechnology,Tianjin University of Science & Technology,Tianjin 300457
    2. Shandong Longkete Enzyme Preparation Co. Ltd.,Yishui 276400
  • Received:2021-06-10 Online:2022-04-26 Published:2022-05-06
  • Contact: LI Yu E-mail:niuxin0968@outlook.com;liyu@tust.edu.cn

Abstract:

In order to explore the effects of different gene integration sites on the expression of exogenous alkaline protease in the genome of Bacillus amyloliquefaciens,based on the genomic information of B. amyloliquefaciens TCCC 111018,the integration site of yaah gene was determined by predicting the replication initiation site OriC on the genome. In addition,the effects of 6 extracellular protease genes(epr,vpr,mpr,apr,bpr and nprE)after deleted individually on the activity of exogenous alkaline protease were analyzed,and the main secreted proteins in the fermentation supernatant were analyzed and identified by mass spectrometry. It is determined that the positions of the nprE and amyE genes are the other two integration sites. The alkaline protease gene aprE,from Bacillus clausii was integrated at three integration sites,then the alkaline protease activity of the integrated strain was compared and analyzed. The results showed that the alkaline protease activity of the integrated strain B.amyloliquefaciens 18-ΔY∷aprE and 18-ΔN∷aprE reached 784.36 U/mL and 1 289.09 U/mL,respectively,which were 15% and 88.9% higher than that of the original strain. Real-time fluorescent quantitative PCR and SDS-PAGE gel electrophoresis indicated that the transcription level and expression level of the alkaline protease of 18-ΔN∷aprE were the highest.The results showed that the integration of alkaline protease gene at nprE gene site effectively improved the expression of alkaline protease quantity,which laid a foundation for the further construction of industrial enzyme production strain.

Key words: Bacillus amyloliquefaciens, integration sites, alkaline protease, heterologous expression