SCREENING AND SELECTION OF A MICROBIAL LIPASE FOR THE STEREOSPECIFIC HYDROLYSIS OF VERLUKAST

A search. was implemented for a microbial lipase capable of bioconverting a diester (dimethyl 5-(3-(2-(7-chloroquinolin-2-yl)ethyl)phenyl)4,6-dithianonanedioate) to its S-ester acid, an intermediate in the production of Verlukast (a leukotriene receptor antagonist). Required properties of the sought-after enzyme included a high enantiomeric selectivity (e.e. > 98%), the formation of only trace amounts of diacid and a high bioconversion rate. This search yielded 57 lipase-producing microorganisms, 18 of which presented detectable bioconversion activity. Thirteen of these microbes were selected for further study based upon their lipase production level and enzyme stability at harvest. Despite their common enzymatic property, namely the hydrolysis of triglycerides, these lipase preparations presented diverse ester acid specific synthesis rates (from <0.01 mug/unit/h to 0.98 mug/unit/h) and diacid formation levels (from 0% to 35%). One of these microbes, identified as Pseudomonas aeruginosa (strain MB 5001), was found to produce a lipase having all of the above-listed required properties. The initial fermentation process developed in shake flasks was rapidly and successfully scaled up in 23-liter laboratory bioreactors, achieving a maximum production of 35 units/ml of lipase after 48 h of cultivation.