长野芽孢杆菌普鲁兰酶基因在枯草芽孢杆菌中的表达及酶学性质研究  

Expression of Pullulanase Gene from Bacillus naganoensis in Bacillus subtilis and Enzymatic Properties

作　　者：张艳[1] 路福平[1] 刘逸寒[1] 李玉[1] 张春峰[1] 

机构地区：[1]天津科技大学生物工程学院、工业微生物教育部重点实验室,天津300457

出　　处：《生物技术通报》2012年第7期114-118,共5页Biotechnology Bulletin

基　　金："十二五"农村领域国家科技计划项目(2011AA100905-4);国家自然科学基金项目(31101219)

摘　　要：根据NCBI上报道的基因序列设计引物,以长野芽孢杆菌(Bacillus naganoensis)ATCC53909的染色体DNA为模板,PCR扩增普鲁兰酶编码基因pulB。将此基因与表达载体pWB980连接构建重组质粒pWB-pulB,并转化枯草芽孢杆菌WB600。SDS-PAGE结果显示,在100 kD处有特异性条带,经测定重组转化子粗酶液酶活力达10.94 U/mL。酶学性质分析表明,其最适反应温度为60℃,最适反应pH为5.0,且在温度30-60℃及pH4.0-6.0范围内稳定,适合淀粉加工行业的应用。Pullulanase gene from Bacillus naganoensis ATCC53909 was amplified with primes designed based on the NCBI database published sequence information. The gene was ligated to an expression vector pWB980, constructing the recombinant vector pWB-pulB, then transformed into Bacillus subtilis WB600 by electroporation. SDS-PAGE analysis showed a band with molecular weight of 100 kD, and the enzyme activity reached 10.94 U/mL. It was suggested that the recombinant protein had a maximum activity at 60℃, pH5.0. Moreover, it was able to keep stable at temperature 30-60℃ and pH4.0-6.0 which was suitable for starch industrial applications.

关 键 词：长野芽孢杆菌 普鲁兰酶 WB600表达 酶学性质 

分 类 号：Q93[生物学—微生物学]