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 strainBS168 (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.
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