极耐热性b-葡萄糖醛酸酶的高效表达和酶学性质及其 应用

Expression, Characterization and Application of Thermostable b-glucuronidase from Thermotoga maritima

摘要:

从海栖热袍菌克隆出编码热稳定性b-葡萄糖醛酸酶基因, 以热激载体pHsh为表达质粒, 在大肠杆菌中得到高效表达。基因表达产物通过一步热处理后, 酶纯度达电泳均一。纯化重组酶酶学性质研究表明, b-葡萄糖醛酸酶的最适反应温度为80oC, 最适反应pH为5.0, pH 5.8~ 8.2之间酶的稳定性较好, 80oC的半衰期为2 h, SDS-PAGE结果显示分子量为65.9 kD, 与理论推算值相吻合。以对硝基苯-b-葡萄糖醛酸苷(pNPG)为底物时, 其动力学参数Km值0.18 mmol/L, Vmax值为312 u/mg。初步的应用分析表明, 该重组酶能催化甘草酸转化为甘草次酸。

Abstract:

The gene of b-glucuronidase from Thermotoga maritima was cloned into the plasmid pHsh, and expressed in Escherichia coli JM109. The recombinant protein was purified to homogeneity by a simple step, heat treatment. The recombinant enzyme had a molecular mass of 65.9 kD. The optimal activity of b-glucuronidase was found at pH 5.0 and 80oC. The purified enzyme was stable over a pH range from 5.8 to 8.2 and had a half life of 2 h at 80oC. The kinetic experiments at 80oC with p-nitrophenyl-b- glucuronide as substrate gave a Km and Vmax of 0.18 mmol/L and 312 u per mg of protein. The purified enzyme could transform glycyrrhizin to glycyrrhetinic acid.