Purification and Characterization of Hydantoin Racemase fromMicrobacterium liquefaciensAJ 3912

Abstract

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 electrophoretic 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 degrees C, and showed a chiral preference for L-5-benzyl- rather than D-5-benzyl-hydantoin.

Molecular Cloning and Expression of thehyuGenes fromMicrobacterium liquefaciensAJ 3912, Responsible for the Conversion of 5-Substituted Hydantoins to α-Amino Acids, inEscherichia coli

“…The estimated molecular mass (25,251 Da) agreed well with the apparent molecular mass of MHR as determined by SDS-PAGE in our previous study (27 kDa), 11) and was similar to those of known HRase proteins (25-27 kDa). [6][7][8][9][10] The deduced amino acid sequence of MHR showed homology with those of other HRase proteins (Fig.…”

“…The literature on AaHyuA notes the contribution of its cysteine residues to the expression of HRase activity and the existence of two highly conserved cysteine residues in the sequences of HRase proteins in general. 7) In the case of MHR, HRase activity was inhibited by cysteinemodifying reagents, 11) and the molecular subunit contains three cysteine residues at positions 77, 182, and 208. The cysteine residues at positions 77 and 182 were highly conserved among HRase proteins (Fig.…”

“…[6][7][8][9][10] Recently, we also isolated a new HRase protein from Microbacterium liquefaciens AJ 3912 (MHR). 11) This strain was first isolated as a potent producer of L-amino acids from the corresponding DL-5-substituted-hydantoins, and is also known to possess both a D,L-nonstereoselective hydantoinase (MHH) and a L-stereoselective N-carbamoylase (MCH). [12][13][14][15] In this paper, we describe the organization and heterologous expression of the genes encoding these three enzymes, i.e., hyuH (encoding MHH), hyuC (encoding MCH), and hyuA (encoding MHR).…”

New Enzymatic Method of Chiral Amino Acid Synthesis by Dynamic Kinetic Resolution of Amino Acid Amides: Use of Stereoselective Amino Acid Amidases in the Presence of α-Amino-ε-Caprolactam Racemase

“…L-cysteine is produced by hydrolases acting on amino-⌬ 2 -thiazoline-4-carboxylic acid in the presence of a racemase (23). Used in combination with D-specific hydantoinase (22,26), either with spontaneous racemization of hydantoin or with hydantoin racemase (21,24), N-carbamoyl-D-amino acid amidohydrolase catalyzes the hydrolysis of N-carbamoyl-D-amino acids to form D-amino acids (3,(16)(17)(18)(19)(20). N-acyl-D-amino acid amidohydrolase (27) and N-acyl amino acid racemase (25) are utilized together to form D-amino acids.…”

Use of Enzymes in the Synthesis of Amino Acids

“…Recently, more hydantoin racemases were characterized and cloned, for instance, from Agrobacterium tumefaciens [93], Sinorhizobium meliloti [94], and Microbacterium liquefaciens [95]. In particular, the new hydantoin racemase from an A. radiobacter strain has industrial potential because it does not suffer from substrate inhibition -a drawback of many other hydantoin racemases [96].…”