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Asymmetric synthesis of L ‐homophenylalanine by equilibrium‐shift using recombinant aromatic L ‐amino acid transaminase
Author(s) -
Cho ByungKwan,
Seo JooHyun,
Kang TaeWon,
Kim ByungGee
Publication year - 2003
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.10661
Subject(s) - transaminase , substrate (aquarium) , chemistry , enantioselective synthesis , amino acid , recombinant dna , biochemistry , stereochemistry , enzyme , biology , catalysis , gene , ecology
Abstract L ‐Homophenylalanine ( L ‐HPA) was asymmetrically synthesized from 2‐oxo‐4‐phenylbutyric acid (2‐OPBA) and L ‐aspartate using a recombinant aromatic amino acid transaminase (AroAT). To screen microorganisms having such an L ‐specific AroAT with a relaxed substrate inhibition in the asymmetric synthesis of unnatural amino acids, enrichment cultures were performed in a minimal media containing 50 mM L ‐HPA as a sole nitrogen source. To reduce the intracellular background synthetic activity by amino acid pools in the cells, a two‐step screening method was used. The putative AroAT (i.e., AroAT Es ) from the screened Enterobacter sp. BK2K‐1 was cloned, sequenced, and overexpressed in E. coli cells. The activity of the overexpressed AroAT Es was 314‐fold higher than that of the wild‐type cell. The substrate specificities of the enzyme and homology search revealed that the cloned transaminase is true AroAT. The AroAT Es showed a substrate inhibition by 2‐OPBA from 40 mM in the asymmetric synthesis, which made it difficult to perform batch asymmetric synthesis of L ‐HPA at high concentrations of 2‐OPBA. To avoid the substrate inhibition by 2‐OPBA, intermittent addition of the solid‐state substrate was attempted to obtain a high concentration of L ‐HPA. By using the cell extract (75 U) obtained from the recombinant E. coli harboring the AroAT Es gene, the asymmetric synthesis of L ‐HPA at 840 mM of 2‐OPBA resulted in >94% of conversion yield and >99% ee of L ‐HPA of optical purity. Due to the low solubility (<2 mM) of L ‐HPA in the reaction buffer, synthesized L ‐HPA was continuously precipitated in the reaction media, which drives the reaction equilibrium towards the product formation. After full completion of the reaction, L ‐HPA of high purity (>99% ee) was easily recovered by simple pH shift of the reaction media. This method can permit very efficient asymmetric synthesis of other unnatural amino acids using a single transaminase reaction. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 226–234, 2003.