Construction and fermentation optimization of food-grade recombinant Bacillus subtilis for the production of leucine aminopeptidase

  • ZHANG Dawei ,
  • LIU Dehua ,
  • HUANG Qinqin ,
  • TIAN Yaping
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  • (Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China)

Received date: 2020-01-14

  Online published: 2020-05-20

Abstract

To solve the problem that antibiotics has to be added during the production of leucine aminopeptidase (LAP) using Bacillus subtilis fermentation, a food-grade recombinant B. subtilis strain was constructed. The D-alanine racemase gene (dal) in the B. subtilis 168 genome was knocked out with the Cre/lox recombination system to obtain a defective strain BS168 (dal)-. By employing the Gibson assembly method, the plasmid pMA5-lap-dal (AmpR, Ori)- with the dal as a selection marker was introduced into the auxotrophic strain BS168 (dal)- to obtain food-grade recombinant strain BS168 (dal)-/pMA5-lap-dal (AmpR, Ori)-. This strain contained no resistance gene and did not need adding antibiotics during LAP fermentation. The condition of 5 L bioreactor was optimized for this strain. The LAP activity reached 302 U/mL under the following conditions: agitation speed 300 r/min; temperature 33°C; pH 7.0 and fed-batch. The results show that the food-grade LAP recombinant B. subtilis constructed in this study has potential industrial application.

Cite this article

ZHANG Dawei , LIU Dehua , HUANG Qinqin , TIAN Yaping . Construction and fermentation optimization of food-grade recombinant Bacillus subtilis for the production of leucine aminopeptidase[J]. Food and Fermentation Industries, 2020 , 46(8) : 1 -6 . DOI: 10.13995/j.cnki.11-1802/ts.023374

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