Trichoderma sp. SKW-36 exhibiting D-aminoacylase activity was isolated from soil. D-Aminoacylase from Trichoderma sp. SKW-36 was purified to homogeneity by ammonium sulfate fractionation, followed by DEAE-Toyopearl 650, Butyl-Toyopearl 650, and Sephacryl S-200 gel filtration chromatography. The purified D-aminoacylase (specific activity: 42.1 U mg(-1) protein) was a monomer and both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography confirmed it to have a molecular mass of 55 kDa. The enzyme was not produced inductively by any D-amino acids and N-acetyl-D-amino acids tested. This enzyme was constitutive and this is different from bacterial enzymes in protein expression. The enzyme was specific to the D-isomer and hydrolyzed N-acetyl derivatives of D-leucine, D-methionine, D-phenylalanine, and D-valine preferably. The K-m for N-acetyl-D-methionine and N-acetyl-D-luecine was 1.89 and 2.04 mM, respectively. The optimum pH and temperature for enzyme activity was at pH 8.0 and 50 degreesC, respectively. The enzyme was fully stable between pH 7.0 and 9.0 and at temperatures up to 40 degreesC, but inactivated at 60 degreesC. The isoelectric point was 4.5. The N-terminal amino acid sequence was NH2-Ala-Val-Asp-Ile-Leu-Phe-Gln-Ser-Pro-Thr-Val-Ile-Thr. The N-terminal amino acid sequence showed relatively high similarity (43-56%) with that reported for D-aminoacylases from bacteria. (C) 2003 Elsevier Ltd. All rights reserved.