泰山土壤宏基因组DNA中耐热β-半乳糖苷酶基因克隆、表.PDF

微生物学通报 NOV 20, 2008, 35(11): 1686~1690 Microbiology © 2008 by Institute of Microbiology, CAS tongbao@ 研究报告 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 * 魏 萍 宋文刚 郝岗平 ( 271000) 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O N PG 的米氏常数K m 为 0.83 mmol/L 。 关键词: β-半乳糖苷酶, 宏基因组, pwtsA , 基因表达 Clone, Expression and Characterization of the Thermostable β-Galactosidase from Soil Metagenomic DNA of Taishan Mountain * WEI Ping SONG Wen-Gang HAO Gang-Ping (Department of Basic Medicine, Taishan Medical University, Tai’an 271000) Abstract: 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 in- duction, with a molecular mass of 57 kD analyzed by SDS. 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 tempera