Purification and characterization of hydantoin racemase from Microbacterium liquefaciens AJ 3912

• Published in: Bioscience, biotechnology, and biochemistry Vol. 69; no. 3; pp. 530 - 536
• Main Authors: Suzuki, S. (Ajinomoto Co. Inc., Kawasaki, Kanagawa (Japan). Central Research Labs.), Onishi, N, Yokozeki, K
• Format: Journal Article
• Language: English
• Published: Tokyo Japan Society for Bioscience, Biotechnology, and Agrochemistry 01.03.2005; Japan Society for Bioscience Biotechnology and Agrochemistry; Oxford University Press
• Subjects: amino acid production; Amino Acid Sequence; AMINO ACIDS; BACTERIA; Biological and medical sciences; CATALYTIC ACTIVITY; CHROMATOGRAPHY; Chromatography, Gel; ELECTROPHORESIS; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; hydantoin; hydantoin racemase; Hydrogen-Ion Concentration; Kinetics; LIGASES; Microbacterium; Microbacterium liquefaciens; Micrococcaceae - enzymology; Molecular Sequence Data; MOLECULAR WEIGHT; PURIFICATION; Racemases and Epimerases - chemistry; Racemases and Epimerases - isolation & purification; Racemases and Epimerases - metabolism; Characterization; Purification; amino acid production; hydantoin; Aminoacid; Production; Microbacterium liquefaciens; hydantoin racemase; Hydantoins
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.69.530

A hydantoin racemase that catalyzed the racemization of 5-benzyl-hydantoin was detected in a cell-free extract of Microbacterium liquefaciens AJ 3912, a bacterial strain known to produce L-amino acids from their corresponding DL-5-substituted-hydantoins. This hydantoin racemase was purified 658-fold to electro-phoretic homogeneity by serial chromatography. The N-terminal amino acid sequence of the enzyme showed homology with known hydantoin racemases from other microorganisms. The apparent molecular mass of the purified enzyme was 27 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 117 kDa on gel-filtration in the purification conditions, indicating a homotetrameric structure. The purified enzyme exhibited optimal activity at pH 8.2 and 55 deg C, and showed a chiral preference for L-5-benzyl- rather than D-5-benzyl-hydantoin.