Abstract
Isoleucine 2-epimerase (ILEP) is a novel branched-chain amino acid racemase isolated from Lactobacillus buchneri. In this study, we examined production of free d-branched-chain amino acids such as d-valine, d-leucine, and d-allo-isoleucine, using lactic acid bacteria carrying homologs to ILEP. Twelve selected strains of lactic acid bacteria were grown at optimal growth temperatures and accumulation of d-branched-chain amino acids in the medium was monitored in exponential, early stationary, and stationary phases. To analyze the d-branched-chain amino acids, enantiomers in the medium were initially converted into diastereomers using pre-column derivatization with o-phthaldialdehyde plus N-isobutyryl-l-cysteine. The resultant fluorescent isoindole derivatives were analyzed on an octadecylsilyl stationary phase using ultra-high performance liquid chromatography. The analyses revealed that the seven following lactic acid bacteria carrying homologs showing 53–60% amino acid sequence identity to the L. buchneri ILEP accumulate d-branched-chain amino acids: Lactobacillus fermentum and Weissella paramesenteroides produce d-valine, d-leucine, and d-allo-isoleucine; Lactobacillus reuteri, Leuconostoc mesenteroides subsp. mesenteroides, and Leuconostoc gelidum subsp. gasicomitatum accumulate d-leucine and d-allo-isoleucine; and Lactobacillus vaginalis and Leuconostoc pseudomesenteroides produce d-allo-isoleucine. These results suggest that d-branched-chain amino acids are produced by a variety of lactic acid bacteria species, particularly those carrying homologs to the ILEP.
Highlights
D-Amino acids, enantiomers of L-amino acids, play key roles as components of the peptidoglycan cell wall of bacteria
Each of 12 strains of lactic acid bacteria carrying an L. buchneri isoleucine 2epimerase (ILEP) homologous gene was cultured until the stationary phase, and accumulation of D-branched-chain amino acids (D-BCAAs) in the culture medium was investigated
The production of D-BCAAs such as D-Val, Dallo-Ile, and D-Leu by 12 strains of lactic acid bacteria carrying a homologous gene to L. buchneri ILEP was investigated
Summary
D-Amino acids, enantiomers of L-amino acids, play key roles as components of the peptidoglycan cell wall of bacteria. In some lactic acid bacteria, which have a peptidoglycan type A4α or A4β, the peptidoglycans contain D-aspartate (D-Asp) in addition to D-Ala and D-Glu (Schleifer and Kandler, 1972; Bellais et al, 2006; Veiga et al, 2006) These D-amino acids are primarily produced from the corresponding. On the basis of the N-terminal amino acid sequence of the purified enzyme, a gene encoding a homolog of this racemase from L. otakiensis was identified in the genome of Lactobacillus buchneri (Mutaguchi et al, 2013b). This gene is annotated as a γ -aminobutyrate aminotransferase (GABA-AT), the gene product enzyme expressed in Escherichia coli showed BCAAR activity, but not GABA-AT activity. We investigated the levels of D-BCAAs in growth media and the ILEP activity in cells of 12 strains of lactic acid bacteria carrying the ILEP homologous gene
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