The lipase isolated from Serratia marcescens (LipA) is a useful biocatalyst for kinetic resolution of a pharmaceutically relevant epoxyester, (+/-)-3-(4'-methoxyphenyl) glycidic acid methyl ester [(+/-)-MPGM], to afford optically pure (-)-MPGM, a key intermediate for the synthesis of diltiazem hydrochloride. Two mutants, LipA(L315S) and LipA(S271F), were identified from the combinatorial saturation mutation library of 14 amino acid residues lining the substrate-binding pocket. LipA(L315S), LipA(S271F), and their combination LipA(L315S/S271F) showed 2.6-, 2.2-, and 4.6-fold improvements in their specific activities towards para-nitrophenyl butyrate (pNPB), respectively. Among these positive mutants, LipA(S271F) displayed a 3.5-fold higher specific activity towards the pharmaco substrate (+/-)-MPGM. Kinetic study showed that the improvement in catalytic efficiency of LipA(S271F) against (+/-)-MPGM was mainly resulted from the enhanced affinity between substrate and enzyme, as indicated by the decrease of K (m). Furthermore, to address the insoluble expression issue in Escherichia coli, the homologous expression of LipA gene in S. marcescens was achieved by introducing it into an expression vector pUC18, resulting in ca. 20-fold higher lipase production. The significantly improved volumeric production and specific activity of S. marcescens lipase make it very attractive as a new-generation biocatalyst for more efficient and economical manufacturing of (-)-MPGM.