Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Taichung 406, Taiwan.
Biotechnol Prog. 2009 Nov-Dec;25(6):1637-42. doi: 10.1002/btpr.272.
L-Homophenylalanine (L-HPA) and N(6)-protected-2-oxo-6-amino-hexanoic acid (N(6)-protected-OAHA) can be used as building blocks for the manufacture of angiotensin-converting enzyme inhibitors. To synthesize L-HPA and N(6)-protected-OAHA simultaneously from 2-oxo-4-phenylbutanoic acid (OPBA) and N(6)-protected-L-lysine, several variants of Escherichia coli aspartate aminotransferase (AAT) were developed by site-directed mutagenesis and their catalytic activities were investigated. Three kinds of N(6)-protected-L-lysine were tested as potential amino donors for the bioconversion process. AAT variants of R292E/L18H and R292E/L18T exhibited specific activities of 0.70+/-0.01 U/mg protein and 0.67+/-0.02 U/mg protein to 2-amino-6-tert-butoxycarbonylamino-hexanoic acid (BOC-lysine) and 2-amino-6-(2,2,2-trifluoro-acetylamino)-hexanoic acid, respectively. E. coli cells expressing R292E/L18H variant were able to convert OPBA and BOC-lysine to L-HPA and 2-oxo-6-tert-butoxycarbonylamino-hexanoic acid (BOC-OAHA) with 96.2% yield in 8 h. This is the first report demonstrating a process for the simultaneous production of two useful building blocks, L-HPA and BOC-OAHA.
L-高苯丙氨酸（L-HPA）和 N（6）-保护的-2-氧代-6-氨基己酸（N（6）-保护-OAHA）可用作制造血管紧张素转换酶抑制剂的构建块。为了从 2-氧代-4-苯基丁酸（OPBA）和 N（6）-保护的 L-赖氨酸同时合成 L-HPA 和 N（6）-保护-OAHA，通过定点突变开发了几种大肠杆菌天冬氨酸氨基转移酶（AAT）变体，并研究了它们的催化活性。三种 N（6）-保护的 L-赖氨酸被测试为生物转化过程的潜在氨基供体。R292E/L18H 和 R292E/L18T 的 AAT 变体对 2-氨基-6-叔丁氧羰基氨基-己酸（BOC-赖氨酸）和 2-氨基-6-（2,2,2-三氟乙酰氨基）-己酸的比活性分别为 0.70+/-0.01 U/mg 蛋白和 0.67+/-0.02 U/mg 蛋白。表达 R292E/L18H 变体的大肠杆菌细胞能够将 OPBA 和 BOC-赖氨酸转化为 L-HPA 和 2-氧代-6-叔丁氧羰基氨基-己酸（BOC-OAHA），在 8 小时内产率为 96.2%。这是首次报道同时生产两种有用构建块 L-HPA 和 BOC-OAHA 的过程。
