Quax_1994_Appl.Microbiol.Biotechnol_41_425

We have screened a new enzyme for the resolution of R, S-naproxen enantiomers. The enzyme is free of lipase activity, and possesses a very high sterospecificity on S-naproxen [2-(6-methoxy-2-naphthyl)-propionic acid] esters and esters of related drugs. The primary structure of the enzyme, determined from the nucleotide sequence, shows limited homology with the catalytic site of lipases. The gene coding for the steroselective carboxylesterase has been cloned and expressed in Bacillus subtilis. Using a multicopy vector and an additional strong promoter an efficient production process was developed. The enzyme was shown to be sensitive to very high concentrations of the products formed during the reaction it catalyses. To increase the resistance of the enzyme, lysine residues thought to be responsible for this phenomnon were replaced through site-directed mutagenesis. Enzymes with improved stability 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.