极耐热性b-葡萄糖醛酸酶的高效表达与酶学性质及其.docVIP

PAGE 2 ISSN1000-3061 CN11-1998/Q Chin J Biotech January 25,2007 Vol.24 No.1 眉题 PAGE 2 J J 生物工程学报 Chin J Biotech 2008, August 25; 24(8): 1407-1412 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ ? 2008 Institute of Microbiology, CAS CSM, All rights reserved 研究报告 研究报告 Received: December 21, 2007; Accepted: March 14, 2008 Supported by: 973 Program of China (No. 2004CB719600). Corresponding author: Weilan Shao. Tel/Fax: +86-25 E-mail: wlshao@ #These authors contribute equally to this publication. 国家“973”计划 (No. 2004CB719600)资助。 极耐热性?-葡萄糖醛酸酶的高效表达和酶学性质及其 应用 王卓1#, 裴建军1#, 李华钟1, 邵蔚蓝1,2 1 江南大学生物工程学院, 无锡 2141222 南京师范大学生命科学学院, 南京 210046 摘 要: 从海栖热袍菌克隆出编码热稳定性?-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, ?-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-?-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。 关键词: ?-葡萄糖醛酸酶, 甘草酸, 甘草次酸 Expression, Characterization and Application of Thermostable ?-glucuronidase from Thermotoga maritima Zhuo Wang1#, Jianjun Pei1#, Huazhong Li1, and Weilan Shao1,2 1 School of Biotechnology, Jiangnan University, Wuxi 214122, China 2 Department of Life Sciences, Nanjing Normal University, Nanjing 210046, China Abstract: The gene of ?-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 ?-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-?-