β-葡萄糖苷酶基因和内切葡聚糖酶基因在枯草芽孢杆菌中的表达  

Expression of Endoglucanase Gene and β-Glucosidase Genes in Bacillus subtilis

作　　者：王远[1] 高秋强[1] 辛秀娟[1] 鲍杰[1] 

机构地区：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

出　　处：《应用与环境生物学报》2013年第6期990-996,共7页Chinese Journal of Applied and Environmental Biology

基　　金：国家"973计划"项目(2011CB707406);"863计划"项目(2012AA022301);中国博士后基金(2011M500742/2012T50380/2012M520850);中央高校基本科研业务费专项资金(WF0913005/1114054/1214025);上海市重点学科建设项目(B505)资助~~

摘　　要：为了实现多种纤维素酶基因的分泌型共表达,利用含有枯草芽孢杆菌中性蛋白酶NprB信号肽的穿梭质粒pP43JM2作为表达载体,将来源于热纤梭菌的内切葡聚糖酶基因celA与来源于多粘芽孢杆菌的β-葡萄糖苷酶基因bglA和bglB在枯草芽孢杆菌WB800中进行了分泌型的单独表达及共表达.结果表明,内切葡聚糖酶基因celA与β-葡萄糖苷酶基因bglA和bglB均能够在枯草芽孢杆菌中实现分泌表达,而且检测到了c elA和bglB基因在枯草芽孢杆菌中的分泌型共表达,其中,共表达菌株的胞外酶液分别与PAS C和纤维二糖底物反应时,释放出674 mg/L和24 mg/L的葡萄糖.而bglA基因只检测到在胞内表达,其胞内酶液以纤维二糖为底物时生成938.7 mg/L的葡萄糖.本研究为实现在枯草芽孢杆菌中纤维素酶多组分人工组装并为集成生物工艺菌种的构建奠定了一定的实验基础.In order to archive the secretory coexpression of cellulase genes, this study used the pP43JM2 shuttle vector carrying NprB signal peptide of the Bacillus subtilis neutral protease as expression vector to express in B. subtilis WB800 the celA gene from Clostridium thermocellum DSM 1237 encoding endoglucanase and bglA and bglB genes from B. polymyxa encoding β-glucosidases. The results showed that the endoglucanase encoded by celA and the β-glucosidases encoded by bglB were secreted into the culture broth successfully; the two cellulase genes could be coexpressed and secreted; the extracellular enzyme solution reacted with PASC and cellobiose, producing 674 and 24 mg/L glucose, respectively; on the contrary, the β-glucosidases encoded by bglA did not react with PASC, only with cellobiose, its intracellular enzyme solution producing 938.7 mg/L glucose. The current work suggested a possible method for multiple cellulase secretion in B. subtilis and may provide a way of economical construction of bioprocessing strains for bulk chemical production.

关 键 词：枯草芽孢杆菌 Β-葡萄糖苷酶 内切葡聚糖酶 胞外分泌 共表达 

分 类 号：Q786[生物学—分子生物学] Q814