Suzuki Yoichi, Miyamoto Kenji, Ohta Hiromichi
Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan.
FEMS Microbiol Lett. 2004 Jul 1;236(1):97-102. doi: 10.1016/j.femsle.2004.05.026.
We have characterized an esterase expressed from the putative esterase gene (ST0071) selected from the total genome analysis from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7. The ORF was cloned and expressed as a fusion protein in Escherichia coli. The protein was purified with heat treatment, affinity column chromatography, and size exclusion filtration. The optimum activity for ester cleavage against p-nitrophenyl esters was observed at around 70 degrees C and pH 7.5-8.0. The enzyme exhibited high thermostability and also showed activity in a mixture of a buffer and water-miscible organic solvents, such as acetonitrile and dimethyl sulfoxide. From the kinetic analysis, p-nitrophenyl butyrate was found to be a better substrate than caproate and caprylate.
我们对从嗜热嗜酸古菌硫磺矿硫化叶菌7株的全基因组分析中筛选出的假定酯酶基因（ST0071）所表达的酯酶进行了表征。该开放阅读框被克隆并在大肠杆菌中作为融合蛋白表达。通过热处理、亲和柱层析和尺寸排阻过滤对该蛋白进行了纯化。观察到对硝基苯酯的酯裂解最佳活性在70℃左右以及pH 7.5 - 8.0。该酶表现出高热稳定性，并且在缓冲液与可与水混溶的有机溶剂（如乙腈和二甲基亚砜）的混合物中也显示出活性。通过动力学分析发现，对硝基苯丁酸酯是比对己酸酯和辛酸酯更好的底物。
