为了实现来源于嗜热脂肪芽孢杆菌的β-半乳糖苷酶在枯草芽孢杆菌中的分泌表达,建立一种较简便的方法筛选能够分泌该酶的信号肽,即针对11种信号肤构建信号肽编码序列库,采用随机筛选方法得到合适的信号肽,构建相应的分泌表达载体和重组表达菌株.结果表明,通过信号肽随机筛选方法获得的枯草芽孢杆菌中性蛋白酶NprE信号肽能够有效引导该酶的细胞外分泌,重组菌株摇瓶培养16h后,上清液中积累的耐热β-半乳糖苷酶活力达到64.02 U/mL,占该酶所表达的总酶活力的29.6％.该信号肽筛选方法可以为微生物蛋白质分泌过程中的信号肽快速选择和优化提供参考.%In order to achieve the extracellular secretion of the thermostable β-galactosidase from Geobacillus stearothermophilus, a simple and rapid method was developed in this study for screening of the signal peptide that can direct the secretion of the target enzyme. A signal peptide coding sequence library was constructed according to 11 signal peptides, and the proper signal peptide coding sequences were obtained by random selection. The secretion plasmids and the recombinant strains were then constructed. Results showed that the signal peptide of NprE which obtained by random selection can secret the target enzyme properly. After cultivation for 16 h, the p-galacto-sidase accumulated in the supernatant reached 64. 02 U/mL, which accounted for 29. 6% of the total target enzyme synthesized. The signal peptide screening method developed in this study can be applied for the rapid selection and optimization of signal peptides in other microbial protein secretion systems.
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