Enzymatic resolution of diltiazem intermediate by Serratia marcescens lipase: molecular mechanism of lipase secretion and its industrial application

A lipase from Serratia marcescens was selected as an asymmetric hydrolytic enzyme for trans -3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM], a key intermediate in the synthesis of diltiazem hydrochloride that is useful as a coronary vasodilator. This lipase has high enantioselectivity ( E =135) and was applied to the industrial production of the optically active intermediate of diltiazem using two-phase reaction system of organic solvent–water. Introduction of enzymatic reaction into the chemical synthetic route of diltiazem reduces the number of processes from nine to five. Analyses of the secretion mechanism of the lipase from S. marcescens cell membrane revealed that lipase (LipA), metalloprotease (PrtA), cell surface protein (SlaA) and flagellin are secreted via ABC-transporter, which is a common secreting mechanism in Gram-negative bacteria other than N-terminal signal peptide-dependent secreting mechanism. Molecular cloning of both the lipA gene, which codes the lipase protein, and lipBCD genes, which code the secretion device proteins, enable the production of the lipase by the self-cloning strain 140-fold as compared to the wild type strain. Immobilization of the lipase on a hollow fiber type membrane reactor contributes to the repeated use of enzyme and to efficient separation of the reaction product. Thus, enzymatic reaction and product separation are achieved simultaneously.