极耐热性β-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 β-glucuronidase from Thermotoga maritima

作　　者：王卓[1] 裴建军[1] 李华钟[1] 邵蔚蓝[1] 

机构地区：[1]江南大学生物工程学院,无锡214122

出　　处：《生物工程学报》2008年第8期1407-1412,共6页Chinese Journal of Biotechnology

基　　金：国家“973”计划(No.2004CB719600)资助~~

摘　　要：从海栖热袍菌克隆出编码热稳定性β-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,β-葡萄糖醛酸酶的最适反应温度为80oC,最适反应pH为5.0,pH5.8～8.2之间酶的稳定性较好,80oC的半衰期为2h,SDS-PAGE结果显示分子量为65.9kD,与理论推算值相吻合。以对硝基苯-β-葡萄糖醛酸苷(pNPG)为底物时,其动力学参数Km值0.18mmol/L,Vmax值为312u/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。The gene of β-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 β-glucuronidase was found at pH 5.0 and 80℃. The purified enzyme was stable over a pH range from 5.8 to 8.2 and had a half life of 2 h at 80℃. The kinetic experiments at 80℃ with p-nitrophenyl-β- 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.

关 键 词：Β-葡萄糖醛酸酶 甘草酸 甘草次酸 

分 类 号：Q78[生物学—分子生物学]