Development of an enzymatic screening method for d-aspartate-producing lactic acid bacteria

d-Aspartate (d-Asp) is an important intermediate for synthetic penicillin and an endogenous amino acid that plays important roles in the endocrine and nervous systems in animals including humans. Lactic acid bacteria (LABs) have been used as probiotics in humans, and some LAB species produce d-Asp as a component of cell wall peptidoglycan. LAB strains with greater d-Asp production would therefore be valuable for industrial d-Asp production. In this study, we developed an enzymatic screening method for d-Asp-producing LABs and isolated a strain with high d-Asp production. The d-Asp concentration in the culture medium was colorimetrically estimated up to 4 mM using d-aspartate oxidase (ChDDO) from the yeast Cryptococcus humicola strain UJ1 coupled with horseradish peroxidase, although a more accurate determination required correction because of interference by the medium component Mn²⁺. We isolated 628 LAB strains from various foods and screened them for d-Asp production using the enzymatic d-Asp assay method. The screening identified 13 d-Asp-producing LAB strains, which were suggested to belong to the genera Latilactobacillus, Levilactobacillus, Lactococcus, and Enterococcus. d-Asp production ability was likely to widely differ among the strains in the same genera and species. One strain, named strain WDN19, produced much higher d-Asp levels (1.84 mM), and it was closely related to Latilactobacillus curvatus. These results indicated that the enzymatic screening method was useful for identifying and isolating d-Asp-producing LABs rapidly and easily, and it might provide novel findings regarding d-Asp production by LABs.