基于易错PCR技术定向进化枯草芽孢杆菌β-葡聚糖酶  

Directed evolution of β-glucanase from Bacillus subtilis by Error-prone PCR

作　　者：邵敏[1] 李长福[2] 葛正龙[2] 周鹤峰[1] 

机构地区：[1]遵义医学院珠海校区生物工程系,珠海519041 [2]遵义医学院生物化学教研室,遵义563003

出　　处：《生物技术通报》2013年第12期141-145,共5页Biotechnology Bulletin

基　　金：贵州省科技厅基金项目(黔科合J字LKZ[2010]42号)

摘　　要：利用易错PCR技术对来自枯草芽孢杆菌的β-葡聚糖酶基因(bgls)进行改造,并对野生酶和突变酶的酶学性质进行研究。以枯草芽孢杆菌基因组DNA为模板,通过易错PCR技术得到突变基因产物,将其重组于表达载体pET32a(+),构建β-葡聚糖酶基因的突变体文库,通过刚果红平板染色法进行高通量筛选,最终获得两株突变酶,其最适作用温度比野生酶都提高了15℃,最适pH与野生酶基本相同,野生酶酶活力为84.2 U/mL,突变酶酶活力分别为247.3 U/mL和125.1 U/mL,是野生酶酶活的2.9倍和1.5倍。试验获得了具有耐高温高酶活的β-葡聚糖酶,为β-葡聚糖酶的工业化应用奠定基础。In order to obtain β-glucanase with higher thermostability,error-prone PCR was used to evolve the β-glucanase from Bacillus subtilis ,and properties of intact and mutant enzymes were analysed .Using the total DNA of Bacillus subtilis as template,the gene of β-glucanase（bgls）was amplified by error-prone PCR,the gene was cloned into pET32a（＋）expression vector,and a mutant library was constructed,and positive clones were screened by Congo red staining,two mutant enzymes were obtained .Comparing to intact enzyme,the optimal temperature of two mutant enzymes were both increased by 15℃,while the optimal pH of mutant enzymes were nearly unchanged.The catalytic activity of intact enzyme was 84.2 U/mL,the catalytic activities of mutant enzyme were 247.3 U/mL and 125.1U/mL respectively,the catalytic activities of two mutants were 2.9 and 1.5 fold higher than that of the intact enzyme respectively.These results have provided a base for further industrialized application of β-glucanase.

关 键 词：枯草芽孢杆菌 Β-葡聚糖酶 易错PCR 定向进化 

分 类 号：Q93[生物学—微生物学]