泰山土壤宏基因组DNA中耐热b-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

摘要:

从泰山土壤宏基因组文库中发现可能的b-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b-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。

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 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.