枯草芽孢杆菌普鲁兰酶基因的分泌表达及其在粉丝制作中的应用

Secretory Expression of Pullulanase Gene from Bacillus subtilis and Its Application to Vermicelli Production

作者旨在研究枯草芽孢杆菌普鲁兰酶的分泌表达及表达产物的应用性能。枯草芽孢杆菌(Bacillus subtilis)168菌株的普鲁兰酶编码基因BsP分别在大肠杆菌(Escherichia coli)和枯草芽孢杆菌中进行表达,重组酶在两种宿主中均能利用自身结构分泌到细胞外。重组酶分泌到枯草芽孢杆菌细胞外的产物的酶学性质与积累在大肠杆菌胞质内的产物有明显的差异,分泌到枯草芽孢杆菌细胞外的重组酶BsBsP的比酶活为153.7 U/mg,是大肠杆菌胞质内产物的5.5倍。重组酶BsBsP最适pH为6.0,最适温度为45℃。重组酶BsBsP对直链淀粉无降解作用,为I型普鲁兰酶。在以土豆淀粉为原料的粉丝制作工艺中,以重组酶BsBsP水解芡糊淀粉可以有效降低粉丝断条率。枯草芽孢杆菌普鲁兰酶可利用自身结构实现高效分泌表达,重组酶适用于以淀粉为原料的粉丝制作。

This study aimed to investigate the secretory expression of the pullulanase gene from Bacillus subtilis and the application of the recombinant enzyme. The BsP gene encoding pullulanase in B. subtilis strain 168 was expressed in Escherichia coli and B. subtilis, respectively. The recombinant enzymes expressed in both hosts could be secreted to the outside of cell via its own structure. The enzymatic properties of the expression products secreted to the culture medium significantly differed from those accumulated in the cytoplasm of Escherichia coli. The specific enzyme activity of recombinant enzyme BsBsP expressed in B. subtilis and secreted to the culture medium was 153.7 U/mg, which was 5.5 times higher than that of the products accumulated in the cytoplasm of E. coli. The optimum pH and temperature of BsBsP were 6.0 and 45 ℃, respectively.The recombinant enzyme BsBsP could not hydrolyze amylose, and it was considered as a type I pullulanase. Hydrolysis of starch paste with the recombinant enzyme BsBsP significantlyreduced the broken rate of vermicelli in the processing of potato starch vermicelli. B. subtilis pullulanase could use its own structure to achieve high-efficiency secretory expression, and the recombinant enzyme Bs BsP is suitable for starch-based vermicelli production.