食品级高产亮氨酸氨肽酶重组Bacillus subtilis的构建和发酵优化

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

为解决枯草芽孢杆菌(Bacillus subtilis)在发酵产亮氨酸氨肽酶(leucine aminopeptidase,LAP)需额外添加抗生素的问题,该研究构建了1株食品级产LAP重组B.subtilis。首先通过Cre/lox系统敲除B.subtilis 168基因组中D-丙氨酸消旋酶基因(dal)得到缺陷型菌株BS168(dal)^-。其次使用高斯组装的方法构建以dal作为标记基因的质粒pMA5-lap-dal(Amp^R,Ori)-并转化至BS168(dal)^-中得到食品级重组菌BS168(dal)-/p MA5-lap-dal(Amp^R,Ori)^-。该工程菌发酵产LAP无需添加抗生素且不含抗性基因。对该菌株进行5 L发酵罐条件优化,在转速300 r/min、温度33℃、p H 7.0、分阶段补料的条件下,酶活最高达到302 U/m L。结果表明,构建的食品级重组B.subtilis产LAP具有潜在的工业应用前景。

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 p MA5-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(Amp^R,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.