Mutant D-aminopeptidases from Ochrobactrum anthropi with increased thermal stability were obtained by random mutagenesis. One of the mutants, no. 65, was derived from E. coil cells transformed with DNA treated with sodium nitrite. The remaining activity of the purified mutant enzyme no. 65 after heat treatment at 52 degrees C for 10 min was 20% that of the untreated mutant enzyme no. 65, whereas the native enzyme showed 5% of the untreated native enzyme activity after the same treatment. The gene for the mutant enzyme no. 65 was sequenced and it was found that Gly155 and Gly279 in the native enzyme were replaced by Ser and Asp, respectively. Five mutants carrying one or two mutations were generated from the native gene by site-specific mutagenesis. The enhancement of the thermal stability of mutant enzyme no. 65 was attributed to the substitution of Gly155 to Ser.