Cloning, expression and characterization of an organic solvent tolerant lipase from Pseudomonas fluorescens JCM5963

A novel lipase gene from an organic solvent degradable strain Pseudomonas fluorescens JCM5963 was cloned, sequenced. and overexpressed as an N-terminus His-tag fusion protein in E coli. The alignment of amino acid sequences revealed that the protein contained a lipase motif and shared a medium or high similarity with lipases from other Pseudomonas strains. It could be defined as a member of subfamily 1.1 lipase. Most of the recombinant proteins expressed as enzymatically active aggregates soluble in 20mM Tris-HCl buffer (pH 8.0) containing sodium deoxycholate are remarkably different from most subfamily 1.1 and 1.2 members of Pseudomonas lipases expressed as inactive inclusion body formerly described in E coli. The recombinant lipase (rPFL) was purified to homogeneity by Ni-NTA affinity chromatography and Sephacryl S-200 gel filtration chromatography. The purified lipase was stable in broad ranges of temperatures and pH values, with the optimal temperature and pH value being 55 degrees C and 9.0. respectively. Its activity was found to increase in the presence of metal ions such as Cal(2+), Sn2+ and some non-ionic surfactants. In addition, rPFL was activated by and remained stable in a series of water-miscible organic solvents solutions and highly tolerant to some water-immiscible organic solvents. These features render this novel lipase attraction for biotechnological applications in the field of organic synthesis and detergent additives. (C) 2008 Elsevier B.V. All rights reserved.