DEVELOPMENT OF A NEW BACILLUS CARBOXYL ESTERASE FOR USE IN THE RESOLUTION OF CHIRAL DRUGS

We have screened a new enzyme for the resolution of R, S-naproxen enan tiomers. The enzyme is free of lipase activity, and possesses a Very h igh stereospecificity on S-naproxen [2-(6-methoxy-2-naphthyl)propionic acid] esters and esters of related drugs. The primary structure of th e enzyme, determined from the nucleotide sequence, shows limited homol ogy with the catalytic site of lipases. The gene coding for the stereo selective carboxylesterase has been cloned and expressed in Bacillus S ubtilis. Using a multicopy Vector and an additional strong promoter an efficient production process was developed. The enzyme was shown to b e sensitive to very high concentrations of the products formed during the reaction it catalyses. To increase the resistance of the enzyme, l ysine residues thought to be responsible for this phenomenon were repl aced through site-directed mutagenesis. Enzymes with improved stabilit y were obtained. An explanation is given in terms of a model in which a reaction of the acid moiety of naproxen with free lysine NH2 groups is a major cause of inactivation.