Pseudomonas putida IFO12996 catalyzes the stereoselective hydrolysis of methyl DL-β-acetylthioisobutyramide (DL-ATIA) to form D-β- acetylthioisobutyric acid (DAT), a key intermediate for synthesis of a series of angiotensin converting enzyme inhibitors. The esterase gene of Pseudomonas putida IFO12996 was cloned and expressed in Escherichia coli which was further immobilized and retained on a packed bed bioreactor filled with Celite 580. The packed bed bioreactor was used to conduct the stereoselective hydrolysis of DL-ATIA and to give DAT with a yield of 34.5%, enantiometric excess value of 97% and enantioselectivity value > 150. The optimal pH and temperature for the reaction were 9.0 and 57 °C ∼ 67 °C, respectively. The kinetic constants (Km and Vmax) of immobilized cells were found to be 372.5 mM and 285.7 μmol min-1 (g cell)-1, respectively. The immobilized cells retained over 60% of the initial catalytic activity after 5 batch cycles of production. This paper presents a simple, practical and economical process of immobilization of genetically engineered E. coli on a novel packed bed bioreactor for production of DAT.
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