Terao Y, Miyamoto K, Ohta H
Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan.
Appl Microbiol Biotechnol. 2006 Dec;73(3):647-53. doi: 10.1007/s00253-006-0518-z. Epub 2006 Jul 25.
Arylmalonate decarboxylase (EC 4.1.1.76) catalyzes enantioselective decarboxylation of alpha-aryl-alpha-methylmalonates to give optically pure alpha-arylpropionates. Recently, we have succeeded in creating a double mutant enzyme that gave opposite enantionmer as the product. Unfortunately, however, the activity of the mutant decreased far lower than that of the native enzyme. Thus, we performed the directed evolution of the mutant via the random mutagenesis method employing the mutator strain Escherichia coli XL1-Red. About 50,000 mutants were screened on color assay plate, and one mutant with higher activity was obtained. Gene analysis of this mutant indicated that the obtained enzyme had an S36N mutation in addition to its original G74C/C188S mutations. The activity of the triple mutant enzyme was tenfold higher than that of the starting doubly mutated enzyme.
芳基丙二酸脱羧酶（EC 4.1.1.76）催化α-芳基-α-甲基丙二酸的对映选择性脱羧反应，生成光学纯的α-芳基丙酸。最近，我们成功构建了一种双突变酶，其产物的对映体与原来相反。然而，不幸的是，该突变体的活性比天然酶低得多。因此，我们通过使用诱变菌株大肠杆菌XL1-Red的随机诱变方法对该突变体进行定向进化。在显色分析平板上筛选了约50,000个突变体，获得了一个活性较高的突变体。对该突变体的基因分析表明，所获得的酶除了原来的G74C/C188S突变外，还发生了S36N突变。三突变酶的活性比起始双突变酶高十倍。
