A Cold-Adapted Lipase of an Alaskan Psychrotroph, Pseudomonas sp. Strain B11-1: Gene Cloning and Enzyme Purification and Characterization

A psychrotrophic bacterium producing a cold-adapted lipase upon growth at low temperatures was isolated from Alaskan soil and identified as aPseudomonas strain. The lipase gene (lipP ) was cloned from the strain and sequenced. The amino acid sequence deduced from the nucleotide sequence of the gene (924 bp) corresponded to a protein of 308 amino acid residues with a molecular weight of 33,714. LipP also has consensus motifs conserved in other cold-adapted lipases, i.e., Lipase 2 from AntarcticMoraxella TA144 (G. Feller, M. Thiry, J. L. Arpigny, and C. Gerday, DNA Cell Biol. 10:381–388, 1991) and the mammalian hormone-sensitive lipase (D. Langin, H. Laurell, L. S. Holst, P. Belfrage, and C. Holm, Proc. Natl. Acad. Sci. USA 90:4897–4901, 1993): a pentapeptide, GDSAG, containing the putative active-site serine and an HG dipeptide. LipP was purified from an extract of recombinantEscherichia coli C600 cells harboring a plasmid coding for thelipP gene. The enzyme showed a 1,3-positional specificity toward triolein.p -Nitrophenyl esters of fatty acids with short to medium chains (C4 and C6 ) served as good substrates. The enzyme was stable between pH 6 and 9, and the optimal pH for the enzymatic hydrolysis of tributyrin was around 8. The activation energies for the hydrolysis ofp -nitrophenyl butyrate andp -nitrophenyl laurate were determined to be 11.2 and 7.7 kcal/mol, respectively, in the temperature range 5 to 35°C. The enzyme was unstable at temperatures higher than 45°C. TheKm of the enzyme forp -nitrophenyl butyrate increased with increases in the assay temperature. The enzyme was strongly inhibited by Zn2+ , Cu2+ , Fe3+ , and Hg2+ but was not affected by phenylmethylsulfonyl fluoride and bis-nitrophenyl phosphate. Various water-miscible organic solvents, such as methanol and dimethyl sulfoxide, at concentrations of 0 to 30% (vol/vol) activated the enzyme.