Overexpression and mutation of a novel lipase from Serratia marcescens L1 in Escherichia coli

A novel lipase gene (lipA) from Serratia marcescens L1 was cloned and expressed in Escherichia coli. LipA was shown to consist of 614 amino acid residues with seven residues that are different from the other nine reported lipases from S. marcescens. The activity of recombinant lipA based on a 50-mL culture scale approached 28 U/mL, which was more than that of the endogenous extracellular lipase from strain L1. The purified lipA fusion with a His-tag exhibited optimum activity at 30 degrees C and pH 8.0. Four effective mutants of lipase were obtained by site-directed mutation (SDM). The replacement of residues Arg552 and Gly2 in the enzyme molecule may play an important role in the catalysis characteristics and be responsible for the changes in thermostability and catalytic efficiency in the mutants. Mut G2P had the highest catalytic efficiency (k(cat)/K-m) which was enhanced by 2.4-fold compared with its wild-type predecessor. These results indicated that lipA from S. marcescens could be expressed at a high level in E. coli and could be optimized by SDM, which provides a feasible basis for further study of its application.