泰山土壤宏基因组DNA中耐热β-半乳糖苷酶基因的克隆、表达及性质  

Clone, Expression and Characterization of the Thermostable β-Galactosidase from Soil Metagenomic DNA of Taishan Mountain

作　　者：魏萍[1] 宋文刚[1] 郝岗平[1] 

机构地区：[1]泰山医学院基础医学部,泰安271000

出　　处：《微生物学通报》2008年第11期1686-1690,共5页Microbiology China

基　　金：山东省医药卫生科技发展计划项目资助(No.2007HZ036)

摘　　要：从泰山土壤宏基因组文库中发现可能的β-半乳糖苷酶基因pwtsA,将其克隆到表达载体pET30a,转化E.coli BL21(DE3)。工程菌在IPTG诱导下高效表达可溶性的重组蛋白PWTSA,分子量为57 kD,与预期大小一致。PWTSA能够水解ONPG产生o-硝基酚,酶活力为13.6 U/mg,确证了重组蛋白为β-半乳糖苷酶。PWTSA的最适反应温度在85°C～95°C之间,最适pH值为6.5,对90°C左右的高温有很好的耐受力。在标准反应条件下,酶作用于底物ONPG的米氏常数Km为0.83 mmol/L。A possible β-Galactosidase gene（pwtsA） was discovered from soil metagenomic DNA of Taishan Mountain.PwtsA gene was inserted into the expression vector pET30a and transferred into E.coli BL21（DE3）.Recombinant protein PWTSA was expressed as a soluble form at high level through IPTG induction,with a molecular mass of 57 kD analyzed by SDS-PAGE.PWTSA can produce o-nitrophenol from o-nitrophenol-β-D-galactopyranoside（ONPG）,and its specific activity was determined as 13.6 U/mg.The enzymatic studies demonstrated that the recombinant protein PWTSA was a thermostable β-Galactosidase,its optimum temperature and pH were 85°C～95°C and 6.5 respectively.In standard assays,the Km for ONPG was 0.83 mmol/L.

关 键 词：Β-半乳糖苷酶 宏基因组 pwtsA 基因表达 

分 类 号：Q78[生物学—分子生物学]