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Asymmetrical Synthesis of l ‐Homophenylalanine Using Engineered Escherichia coli Aspartate Aminotransferase
Author(s) -
Lo HsuehHsia,
Hsu ShihKuang,
Lin WeiDe,
Chan NeiLi,
Hsu WenHwei
Publication year - 2008
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp049756i
Subject(s) - transamination , lysine , escherichia coli , chemistry , yield (engineering) , mutant , mutagenesis , amino acid , enzyme , biochemistry , substrate (aquarium) , directed evolution , solubility , stereochemistry , organic chemistry , biology , gene , ecology , materials science , metallurgy
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 Δ 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.