Screening of DCase Producing Microorganisms

The screening and isolation methods used for soil microorganisms were as follows (12). For the isolation of microorganisms that can hydrolyze A-carbamoyl-D-amino acids to D-amino acids, soil samples were suspended in saline and the supernatants were inoculated into growth medium containing A-carbamoyl-D-amino acids as sole nitrogen sources for enrichment culture. After aerobic cultivation, the reduction of A-carbamoyl-D-amino acids and the production of D-amino acids were detected by silica gel thin-layer chromatography (TLC) with a solvent system of n-butanol:acetic acid:water (4:1:1). A-Carbamoyl-D-amino acids and D-amino acids were detected with p-dimethylam-inobenzaldehyde in a 6 M HCl solution and ninhydrin, respectively. From the culture broth from which A-

carbamoyl-D-amino acids disappeared or in which D-amino acids accumulated, microorganisms were isolated on agar plates.

The substrate specificities of these DCases were examined by means of the resting cell reaction with detection by the TLC method (Table 1). We screened two good me-sophile strains producing a lot of enzymes that were classified as Agrobacterium sp. or Rhizobium sp. (12). A similar enzyme-producing strain, classified as Agrobacterium ra-diobacter, was also found by Olivieri et al. (4) and characterized. We also screened thermotolerant strains by means of enrichment culture at 45 °C and obtained some Pseudomonas sp. strains (Table 1) (13). The strain isolated by Yokozeki and Kubota was classified as Pseudomonas sp. (5) but was not a thermotolerant strain.

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