A thermostable esterase of Pseudomonas putida MR-2068, which catalyzes the stereoselective hydrolysis of methyl DL-beta-acetylthioisobutyrate (DL-ester) to give D-beta-acetylthioisobutyrate (DAT) was cloned in Escherichia coil cells. The enzyme was purified to homogeneity by the methods including ammonium sulfate precipitation, ion exchange chromatography, and gel filtration chromatography. The enzyme was purified about 3.8-fold with a field of 57%. The purified enzyme had a molecular weight of about 29,000 Da and an isoelectric point of 3.9. The optimum pH was 7.0 and optimum temperature was 70 degrees C. The enzyme was stable up to 60 degrees C at pH 7.0 for Ih and also stable from pH 6.0 to 8.0 at 4 degrees C for 24h. This esterase also catalyses the hydrolysis of alkanedicarboxylic acid dimethyl esters to give exclusively pure monoesters. Hydrolytic activities were dependent on the carbon chain length of the substrates. Enantio- and regio-selective hydrolysis of alpha-methylalkanedicarboxylic acid dimethyl esters are also achieved.