Cloning Its Useand Expression of Pseudomonas putida Esterase Gene in in Enzymatic Production of D - ll - Acetylthioisobutyric Acidt

DL-fi-acetylthioisobutyrate (DL-ester).'} A newly isolated strain, Pseudomonas puticin MR-2068, had been selected as the best DAT producer because of its cnzymatic characterization.2} The enzyme produced by the strain has enhanced thermostability as compared to previously available enzymes. There are many advantages in using thermostable enzymes in industrial processes as compared to thermolabile enzymes.3'`) However, for industry, a microorganism with more esterase activity is needed. One of the promising strategies is to construct a hyperproducing strain of a particular esterase by cloning the esterase gene and introducing it into an appropriate host under the control of a strong promoter. In this report, we describe the cloning and expression of a gene for the esterase from Pseudomonas puticla MR-2068 in llscherichia coli and its use for enzymatic production of DAT, Chromosomal DNA of Pseudomonas putitla MR-2068 was partially digested with EcoRI. The cloning vector, pUC19. was cleaved with EcoRI, The ligation and transformation to E. coli JMI09 were done by the standard methods. The transforrnants were spread on LB agar plates containing 100 ugfml of ampicillin, 1 mg!ml of isopropyl6-D-thiogalactopyranosicle (IPTG), and 20"glml of 5bromo-4-chloro-3-indolyl-fi-D-galactopyranoside (X-gal), (first selecting plates). Esterase activity ofthe transformants was detected as fo11ows. Small portions of the white colonies on the first plates were put on the second selecting plates containing O,1% (wfv) DI=ester, O.Ol% (wfy) bromocresol