Kojima_2003_J.Biosci.Bioeng_96_242

The extracellular lipase gene (lipA) from Pseudomonas fluorescens HU380 was cloned from a genomic library constructed in pBluescript SK+. Nucleotide sequence analysis revealed an open reading frame of 1854 by encoding the lipase. Its deduced amino acid sequence included internal amino acid sequences of the lipase from this strain: The lipase showed significant sequence similarity to lipases of Serratia marcescens strains and P. fluorescens strains. In Escherichia coli, lipA was expressed in the form of inclusion bodies, which were subsequently solubilized by urea followed by dialysis. The refolded protein was soluble and biologically active. The lipase purified from the E. coli transformant by this denaturation-renaturation procedure followed by only two steps of column chromatographs exhibited the same electrophoretic mobility as did the enzyme purified from P. fluorescens HU380, and both enzymes were quite similar in physicochemical properties such as specific activity, suggesting that the recombinant lipase protein has an intrinsic folding capability in vitro. The function of its C-terminal region is also discussed.