β-1,3-葡聚糖酶在枯草芽孢杆菌中的表达与性质  

Expression and Characterization of β-1,3-Glucanase in Bacillus subtilis

作　　者：张佳妮 徐岩 喻晓蔚[1,2] ZHANG Jiani;XU Yan;YU Xiaowei(Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学工业生物技术教育部重点实验室,江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与生物技术学报》2022年第2期29-36,共8页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目(31671799)。

摘　　要：藤黄节杆菌(Athrobacter luteu)β-1,3-葡聚糖酶(βglⅠ)能够特异性的作用于β-葡聚糖中的β-1,3-糖苷键,在酵母裂解活性上具有突出优势,但是βglⅠ在生产上存在表达低、生产成本高的问题。作者利用无缝克隆技术构建表达载体,实现了βglⅠ在枯草芽孢杆菌中的分泌表达,并通过比较不同Tat类型和Sec类型信号肽、RBS、5′UTR等表达元件来提高βglⅠ的胞外产量。结果表明,信号肽SPLipA使得βglⅠ表达水平提高了0.4倍,在此信号肽基础上继续比较各RBS和5′UTR序列,其中cry3A 5′UTR使βglⅠ产量高出出发菌株1.2倍。此外,利用Ni2+亲和层析柱纯化βglⅠ并对其酶学性质进行研究,结果表明该酶的最适反应温度为50℃,最适反应pH为6.0,在0~45℃、pH 4.0~9.0的范围内稳定性较好,比活为973 U/mg。Athrobacter luteu β-1,3-glucanase(βglⅠ) specifically acts on β-1,3-glycosidic bonds inβ-glucan, which has outstanding advantages in yeast lytic activity. However, the expression level ofβglⅠis low resulting in high cost of its production. This study achieved the secretory expression ofβgl Ⅰ by using seamless cloning technology in Bacillus subtilis, and improved the extracellular production of βglⅠ by optimizing expression elements such as Tat and Sec signal peptides, RBS and5′UTR. The results showed that the signal peptide SPLipA increased the expression level of βglⅠ by0.4 times. Then, RBS and 5′UTR were compared on the basis of this signal peptide, and the yield ofβglⅠincreased by cry3 A 5′UTR was 1.2 times higher than that of the parent strain. In addition, a Ni2+affinity chromatography column was used to purify βglⅠ and its enzymatic properties were studied.The results showed that the optimum temperature and pH was 50 ℃ and pH 6.0, respectively. βglⅠwas more stable in the range of 0~45 ℃ and pH 4.0~9.0, and the specific activity of βglⅠ was973 U/mg.

关 键 词：βglⅠ 枯草芽孢杆菌 分泌表达 信号肽 RBS 5′UTR 

分 类 号：Q814.4[生物学—生物工程]