title: Characterization of Lactobacillus salivarius alanine racemase: short-chain carboxylate-activation and the role of A131.
abstract: Many strains of lactic acid bacteria produce high concentrations of d-amino acids. Among them, Lactobacillus salivarius UCC 118 produces d-alanine at a relative concentration much greater than 50 % of the total d, l-alanine (100d/d, l-alanine). We characterized the L. salivarius alanine racemase (ALR) likely responsible for this d-alanine production and found that the enzyme was activated by carboxylates, which is an unique characteristic among ALRs. In addition, alignment of the amino acid sequences of several ALRs revealed that A131 of L. salivarius ALR is likely involved in the activation. To confirm that finding, an L. salivarius ALR variant with an A131K (ALR(A131K)) substitution was prepared, and its properties were compared with those of ALR. The activity of ALR(A131K) was about three times greater than that of ALR. In addition, whereas L. salivarius ALR was strongly activated by low concentrations (e.g., 1 mM) of short chain carboxylates, and was inhibited at higher concentrations (e.g., 10 mM), ALR(A131K) was clearly inhibited at all carboxylate concentrations tested (1-40 mM). Acetate also increased the stability of ALR such that maximum activity was observed at 35 °C and pH 8.0 without acetate, but at 50 °C in the presence of 1 mM acetate. On the other hand, maximum ALR(A131K) activity was observed at 45 °C and around pH 9.0 with or without acetate. It thus appears that A131 mediates the activation and stabilization of L. salivarius ALR by short chain carboxylates.
authors.affiliation: Microbial Genetics Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan.
authors.forename: Jyumpei
authors.initials: J
authors.lastname: Kobayashi
authors.affiliation: Applied Molecular Microbiology and Biomass Chemistry Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan.
authors.forename: Jotaro
authors.initials: J
authors.lastname: Yukimoto
authors.affiliation: Microbial Genetics Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Shimizu
authors.affiliation: Department of Biomedical Engineering, Faculty of Engineering, Osaka Institute of Technology, 5-16-1 Omiya, Asahi-ku, Osaka, 535-8585 Japan.
authors.forename: Taketo
authors.initials: T
authors.lastname: Ohmori
authors.affiliation: Functional Genomics of Extremophiles, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, 812-8581 Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Suzuki
authors.affiliation: Department of Biomedical Engineering, Faculty of Engineering, Osaka Institute of Technology, 5-16-1 Ohmiya, Asahi-ku, Osaka, 535-8585 Japan.
authors.forename: Katsumi
authors.initials: K
authors.lastname: Doi
authors.affiliation: Microbial Genetics Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan ; Department of Biomedical Engineering, Faculty of Engineering, Osaka Institute of Technology, 5-16-1 Ohmiya, Asahi-ku, Osaka, 535-8585 Japan.
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Ohshima
keywords: Alanine racemase; Lactic acid bacteria; Lactobacillus salivarius; Short-chain carboxylate; d-amino acid