Akihiro Sakimae

Purification and characterization of recombinant esterase from Pseudomonas putida MR-2068 and its application to the optical resolution of dimethyl methylsuccinate

Abstract A thermostable esterase of Pseudomonas putida MR-2068, which catalyzes the stereoselective hydrolysis of methyl dl -β-acetylthioisobutyrate ( dl -ester) to give d -β-acetylthioisobutyrate (DAT) was cloned in Escherichia coli 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 yield 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°C. The enzyme was stable up to 60°C at pH 7.0 for 1 h and also stable from pH 6.0 to 8.0 at 4°C for 24 h. 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 α-methylalkanedicarboxylic acid dimethyl esters are also achieved.

Chemical Racemization of Methyl L-β-Acetylthioisobutyrate.

Methyl L-β-acetylthioisobutyrate was racemized with 1,8-diazabicyclo-[5.4.0]-undecene-7 as a catalyst. Methyl methacrylate and thioacetic acid were identified as the intermediates of the reaction. Thioacetic acid was relatively unstable and susceptible to decomposition during the racemization process. The addition of excess methyl mechacrylate to the reaction mixture prevented a decrease of the racemate. The racemized ester was confirmed to be usable as a substrate for the enzymatic production of D-β-acetylthioisobutyric acid.