该研究通过信号肽筛选和启动子优化构建了一种在枯草芽孢杆菌中高效分泌嗜碱碱性卤杆菌蛋白酶的表达系统。结果表明,信号肽筛选和启动子优化的组合策略显著提高了该蛋白酶在枯草芽孢杆菌中的胞外表达量。在173种信号肽中,SPYdjm介导该蛋白酶分泌的效率最高,是原始信号肽SPaprE所介导酶活力的13.8倍。而串联启动子PHapII-Pylb对表达的促进作用最为显著,使酶活力进一步提升1.7倍。经发酵培养基适配,该表达系统在摇瓶中产生的蛋白酶活力为18 600 U/mL。进一步使用5 L发酵罐发酵后,酶活力达到34 050 U/mL。该表达体系有效的提升了嗜碱碱性卤杆菌蛋白酶的分泌量,为该酶的生产与应用提供了良好的基础。
The protease produced by Alkalihalobacillus alcalophilus performed high catalytic activity in alkaline and halophilic environments, which can be widely used in leather and detergent industry.In this study, a highly efficient expression system was constructed in Bacillus subtilis through signal peptide screening and promoter optimization.The result showed that these two strategies cumulatively enhanced the extracellular expression of the protease.Among the 173 signals peptide screened, SPYdjm significantly improved the secretion process, the protease activity was enhanced by 13.8 folds in comparison with the SPaprE.The dual promoter PHapII-Pylb further improved the expression by about 1.7 fold.After fermentation medium selection, the protease activity observed in the shake-flask supernatant is 18 600 U/mL.And in 5 L fermentation tank, the proteinolytic activity achieved 34 050 U/mL.This expression system effectively enhances the secretion of Alkalihalobacillus protease, providing a good foundation for the production and application of this enzyme.
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