Characteristics of alanine racemase in Lactobacillus sakeiZH-2 strain

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 K _m is 1.32 × 10^-3 (mol), with V _max 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.