Identification, purification, and characterization of a novel amino acid racemase, isoleucine 2-epimerase, from Lactobacillus species

Abstract

Accumulation of d-leucine, d-allo-isoleucine, and d-valine was observed in the growth medium of a lactic acid bacterium, Lactobacillus otakiensis JCM 15040, and the racemase responsible was purified from the cells and identified. The N-terminal amino acid sequence of the purified enzyme was GKLDKASKLI, which is consistent with that of a putative γ-aminobutyrate aminotransferase from Lactobacillus buchneri. The putative γ-aminobutyrate aminotransferase gene from L. buchneri JCM 1115 was expressed in recombinant Escherichia coli and then purified to homogeneity. The enzyme catalyzed the racemization of a broad spectrum of nonpolar amino acids. In particular, it catalyzed at high rates the epimerization of l-isoleucine to d-allo-isoleucine and d-allo-isoleucine to l-isoleucine. In contrast, the enzyme showed no γ-aminobutyrate aminotransferase activity. The relative molecular masses of the subunit and native enzyme were estimated to be about 49 kDa and 200 kDa, respectively, indicating that the enzyme was composed of four subunits of equal molecular masses. The Km and Vmax values of the enzyme for l-isoleucine were 5.00 mM and 153 μmol·min(-1)·mg(-1), respectively, and those for d-allo-isoleucine were 13.2 mM and 286 μmol·min(-1)·mg(-1), respectively. Hydroxylamine and other inhibitors of pyridoxal 5'-phosphate-dependent enzymes completely blocked the enzyme activity, indicating the enzyme requires pyridoxal 5'-phosphate as a coenzyme. This is the first evidence of an amino acid racemase that specifically catalyzes racemization of nonpolar amino acids at the C-2 position.

Fig 1

Time course of D-BCAA accumulation in culture media of L. otakiensis JCM 15040 (A) and L. buchneri JCM 1115 (B). Each of the Lactobacillus strains was cultured in MRS medium, and the time-dependent accumulation of d-Leu (solid circles), d-allo-Ile (solid triangles), and d-Val (solid squares) in the culture medium was monitored using a UPLC system. The cell concentration was assayed at OD₆₀₀ (open circles).

Fig 2

Time courses of the isoleucine 2-epimerase reaction with l-Ile (A) or d-allo-Ile (B) as the substrate. The data for l-Ile and d-allo-Ile are depicted as solid circles and open circles, respectively.

Fig 3

Effect of pH on the isoleucine 2-epimerase activity with l-Ile (A) and d-allo-Ile (B) as the substrates. The buffers used were sodium citrate (pH 3.5 to 6.0) (open circles), MES (morpholineethanesulfonic acid)-NaOH (pH 6.0 to 7.0) (solid circles), HEPES-NaOH (pH 7.0 to 8.0) (open squares), TAPS {3-([2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-amino)-1-propanesulfonic acid}-NaOH (pH 8.0 to 9.0) (solid squares), and CHES (N-cyclohexyl-2-aminoethanesulfonic acid)-NaOH (pH 9.0 to 10.0) (open triangles). Enzyme activities are expressed as percentages relative to 156 μmol·mg⁻¹·min⁻¹ (A) and 233 μmol·mg⁻¹·min⁻¹ (B), which are shown as 100%. All values were obtained through repeated measurements (n = 3).

Fig 4

Effect of temperature on the isoleucine 2-epimerase activity with l-Ile (open circles) and d-allo-Ile (solid circles) as the substrates. Enzyme activities are expressed as percentages relative to 178 μmol·mg⁻¹·min⁻¹ (l-Ile as the substrate) and 267 μmol·mg⁻¹·min⁻¹ (d-allo-Ile as the substrate), which are shown as 100%. All values were obtained through repeated measurements (n = 3).

Fig 5

Effects of pH (A) and temperature (B) on the stability of the recombinant enzyme. The buffers used were sodium citrate (pH 3.0 to 6.0) (open circles), MES-NaOH (pH 6.0 to 7.0) (solid circles), HEPES-NaOH (pH 7.0 to 8.0) (open squares), TAPS-NaOH (pH 8.0 to 9.0) (solid squares), CHES-NaOH (pH 9.0 to 10.0) (open triangles), CAPS (N-cyclohexyl-3-aminopropanesulfonic acid)-NaOH (pH 10.0 to 11.0) (solid triangles), and Na₂HPO₄-NaOH (pH 11.0 to 12.0) (open diamonds). Enzyme activities are expressed as percentages relative to 149 μmol·mg⁻¹·min⁻¹, which is shown as 100%. The values were obtained through repeated measurements (n = 3).