Characteristics of alanine racemase in Lactobacillus sakei ZH‐2 strain

Abstract

Abstract Some d‐amino acid functions for food production are widely known: d‐alanine improves sensory evaluations of sake, beer, and fermented foods. Therefore, for the application of d‐amino acids, alanine racemase (ALRase) in Lactobacillus sakei ZH‐2, which has strong racemization, was analyzed using molecular biological methods. It had been hypothesized that ALRase coding DNA, alr, in ZH‐2 strain differs from those of other Lactobacillus sakei strains. However, complete genome sequencing by the National Center for Biotechnology (NCBI) revealed the amino acid sequence of alr in ZH‐2 strain to have homology of 99.4% similarity with the alr in Lactobacillus sakei 23K strain. However, it is considered that the sequence of alr was a unique amino acid sequence in the lactic acid bacteria group. DNA “alr” of ZH‐2 strain has a 1140 bp DNA base with 41 kDa molecular mass. Its molecular mass was inferred as approximately 38.0 kDa using SDS‐PAGE. Its optimum conditions are pH 9.0 at 30–40°C, showing stability at pH 9.0–10.0 and 4–40°C. Its cofactor is pyridoxal phosphate. Its activity is activated more by copper and zinc ions than by the lack of a metal ion. Additionally, its Km is 1.32 × 10−3 (mol), with Vmax of 4.27 × 10−5 (μmol−1 min−1). ALRase reacted against alanine most strongly in other substrates such as amino acids. The enzyme against serine was found to have 40% activity against alanine. The enzyme converted up to 54.5% of d‐alanine from l‐alanine ZH‐2 strain.

Keywords

• alanine racemase
• enzyme
• Lactobacillus sakei