Asymmetrical synthesis of L-homophenylalanine using engineered Escherichia coli aspartate aminotransferase.
Site-directed mutagenesis was performed to change the substrate specificity of Escherichia coli aspartate aminotransferase (AAT). A double mutant, R292E/L18H, with a 12.9-fold increase in the specific activity toward L-lysine and 2-oxo-4-phenylbutanoic acid (OPBA) was identified. E. coli cells expressing this mutant enzyme could convert OPBA to L-homophenylalanine (L-HPA) with 97% yield and more than 99.9% ee using L-lysine as amino donor. The transamination product of L-lysine, 2-keto-6-aminocaproate, was cyclized nonenzymatically to form Delta(1)-piperideine 2-carboxylic acid in the reaction mixture. The low solubility of L-HPA and spontaneous cyclization of 2-keto-6-aminocaproate drove the reaction completely toward L-HPA production. This is the first aminotransferase process using L-lysine as inexpensive amino donor for the L-HPA production to be reported.
Lo
Hsueh-Hsia
HH
Institute of Molecular Biology and Instuitute of Biochemistry, National Chung-Hsing University, Taichung 402, Taiwan.
Hsu
Shih-Kuang
SK
Lin
Wei-De
WD
Chan
Nei-Li
NL
Hsu
Wen-Hwei
WH
D000613:Aminobutyrates / Q000138:chemical synthesis*; D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology*; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering
D000613:Aminobutyrates; C014328:2-amino-4-phenylbutyric acid; D001219:Aspartate Aminotransferases