National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China.
Appl Microbiol Biotechnol. 2013 Mar;97(6):2433-41. doi: 10.1007/s00253-012-4102-4. Epub 2012 May 3.
A highly enantioselective cis-epoxysuccinic acid hydrolase from Nocardia tartaricans was purified to electrophoretic homogeneity. The enzyme was purified 184-fold with a yield of 18.8 %. The purified cis-epoxysuccinic acid hydrolase had a monomeric molecular weight of 28 kDa, and its optimum conditions were 37 °C and pH 7-9. With sodium cis-epoxysuccinate as the substrate, Michaelis-Menten enzyme kinetics analysis gave a Km value of 35.71 mM and a Vmax of 2.65 mM min(-1). The enzyme was activated by Ni(2+) and Al(3+), while strongly inhibited by Fe(3+), Fe(2+), Cu(2+), and Ag(+). The cis-epoxysuccinic acid hydrolase gene was cloned, and its open reading frame sequence predicted a protein composed of 253 amino acids. A pET11a expression plasmid carrying the gene under the control of the T7 promoter was introduced into Escherichia coli, and the cis-epoxysuccinic acid hydrolase gene was successfully expressed in the recombinant strains.
从土曲霉中纯化出一种高度对映选择性的顺式-环氧琥珀酸水解酶，达到电泳纯。该酶经纯化 184 倍，产率为 18.8%。纯化的顺式-环氧琥珀酸水解酶的单体分子量为 28 kDa，其最适条件为 37°C 和 pH 7-9。以顺式-环氧琥珀酸钠为底物，米氏酶动力学分析得出 Km 值为 35.71 mM，Vmax 值为 2.65 mM·min(-1)。该酶被 Ni(2+)和 Al(3+)激活，而被 Fe(3+)、Fe(2+)、Cu(2+)和 Ag(+)强烈抑制。克隆了顺式-环氧琥珀酸水解酶基因，其开放阅读框序列预测该蛋白由 253 个氨基酸组成。携带受 T7 启动子控制的基因的 pET11a 表达质粒被引入大肠杆菌，重组菌株中成功表达了顺式-环氧琥珀酸水解酶基因。
