Steroselective Hydrolysis of DL‐Beta‐Acetylthioisobutyramide Catalyzed by Genetically Engineered E. Coli Immobilized on Celite 580 in a Packed Bed Bioreactor

Abstract

AbstractPseudomonas 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.