Enantioselective synthesis of (S)‐2‐amino‐4‐phenylbutanoic acid by the hydantoinase method

Abstract

Biosynthesis of (S)-(+)-2-amino-4-phenylbutanoic acid (1) was performed by nonenantioselective hydantoinase and L-N-carbamoylase using racemic 5-[2-phenylethyl]-imidazolidine-2,4-dione (rac-2) as a substrate. The compounds involved in this biocatalysis process could be simultaneously resolved by high-performance liquid chromatography using Chirobiotic T column with a mobile phase of EtOH/H(2)O = 10/90 at pH 4.2-4.5. To our knowledge, this is the first report of the successful production of 1 by the combination of recombinant hydantoinase and L-N-carbamoylase.

Enantioselective Synthesis of l‐Homophenylalanine by Whole Cells of Recombinant Escherichia coli Expressing l‐Aminoacylase and N‐Acylamino Acid Racemase Genes from Deinococcus radiodurans BCRC12827

“…Since these processes are based on kinetic resolution, only 50% theoretical yield can be obtained in the reaction. In our previous study, the racemic HPAH can be converted to L-HPA with 100% ee by hydantoinase from B. caldolyticus and L-N-carbamoylase from B. kaustophilus (12). However, low activity of B. caldolyticus hydantoinase toward L-HPAH limits this process with industrial potential.…”

“…However, OPBA (substrate) inhibition is demonstrated in E. coli AAT activity. We have also found that the racemic homophenylalanylhydantoin (HPAH) can be converted to L-HPA by a nonstereoselective hydantoinase from Bacillus caldolyticus and L-N-carbamoylase from Bacillus kaustophilus (12). Since the B. caldolyticus hydantoinase has very low activity toward L-form HPAH, the space-time yield of the L-hydantoinase process for the desired 100% conversion of HPAH to L-HPA is rather low.…”

Rational re-design of the “double-racemase hydantoinase process” for optically pure production of natural and non-natural l-amino acids

“…However, for l-amino acid production, from these cheap racemic precursors, only the combination of a d-enantionselective hydantoinase and an l-enantioespecific N-carbamoylase produce 50% conversion of the d-isomer of the substrate into N-carbamoyld-amino acid without any formation of l-amino acid (see as an example of l-ABA production Fig. 3a) or with relatively low yield [43]. The strict d-enantioselectivity of the majority of hydantoinases, including AtDhyd, led us to tackle novel strategies other than enzyme design by molecular engineering.…”

Stereoselective synthesis of l-homophenylalanine using the carbamoylase method with in situ racemization via N-acylamino acid racemase

“…Such a process however is restricted in the real application due to the low solubility of NAc-HPA (about 20 mM in 50 mM Tris-HCl buffer at pH 8.0 and 50 8C). We also found that racemic homophenylalanylhydantoin can be converted to L-HPA with 99.9% ee by non-stereospecific hydantoinase from Bacillus caldolyticus and L-N-carbamolyase (LNCA) from Bacillus kaustophilus [10]. Since the B. caldolyticus hydantoinase displays high activity toward D-homophenylalanylhydantoin, a large amount of N-carbamoyl-D-homophenylalanine (NCa-D-HPA) is accumulated in the course of the reaction and only 49% yield of L-HPA can be obtained in the process.…”