Characterization of Lactobacillus salivarius alanine racemase: short-chain carboxylate-activation and the role of A131

Jyumpei Kobayashi, Jotaro Yukimoto, Yasuhiro Shimizu, Taketo Ohmori, Hirokazu Suzuki, Katsumi Doi, Toshihisa Ohshima

Research output: Contribution to journalArticle

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.

Original languageEnglish
Article number639
JournalSpringerPlus
Volume4
Issue number1
DOIs
Publication statusPublished - Dec 1 2015
Externally publishedYes

Fingerprint

Lactobacillus salivarius
alanine
acetates

All Science Journal Classification (ASJC) codes

  • General

Cite this

Characterization of Lactobacillus salivarius alanine racemase : short-chain carboxylate-activation and the role of A131. / Kobayashi, Jyumpei; Yukimoto, Jotaro; Shimizu, Yasuhiro; Ohmori, Taketo; Suzuki, Hirokazu; Doi, Katsumi; Ohshima, Toshihisa.

In: SpringerPlus, Vol. 4, No. 1, 639, 01.12.2015.

Research output: Contribution to journalArticle

Kobayashi, Jyumpei ; Yukimoto, Jotaro ; Shimizu, Yasuhiro ; Ohmori, Taketo ; Suzuki, Hirokazu ; Doi, Katsumi ; Ohshima, Toshihisa. / Characterization of Lactobacillus salivarius alanine racemase : short-chain carboxylate-activation and the role of A131. In: SpringerPlus. 2015 ; Vol. 4, No. 1.
@article{cac2703d694a4a9ea12e983572090d06,
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.",
author = "Jyumpei Kobayashi and Jotaro Yukimoto and Yasuhiro Shimizu and Taketo Ohmori and Hirokazu Suzuki and Katsumi Doi and Toshihisa Ohshima",
year = "2015",
month = "12",
day = "1",
doi = "10.1186/s40064-015-1335-6",
language = "English",
volume = "4",
journal = "SpringerPlus",
issn = "2193-1801",
publisher = "Springer Science and Business Media Deutschland GmbH",
number = "1",

}

TY - JOUR

T1 - Characterization of Lactobacillus salivarius alanine racemase

T2 - short-chain carboxylate-activation and the role of A131

AU - Kobayashi, Jyumpei

AU - Yukimoto, Jotaro

AU - Shimizu, Yasuhiro

AU - Ohmori, Taketo

AU - Suzuki, Hirokazu

AU - Doi, Katsumi

AU - Ohshima, Toshihisa

PY - 2015/12/1

Y1 - 2015/12/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84945143975&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84945143975&partnerID=8YFLogxK

U2 - 10.1186/s40064-015-1335-6

DO - 10.1186/s40064-015-1335-6

M3 - Article

AN - SCOPUS:84945143975

VL - 4

JO - SpringerPlus

JF - SpringerPlus

SN - 2193-1801

IS - 1

M1 - 639

ER -