A cold-adapted lipase of an Alaskan psychrotroph, Pseudomonas sp. strain B11-1: gene cloning and enzyme purification and characterization

Abstract

A psychrotrophic bacterium producing a cold-adapted lipase upon growth at low temperatures was isolated from Alaskan soil and identified as a Pseudomonas 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 Antarctic Moraxella TA144 (G. Feller, M. Thirty, 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 recombinant Escherichia coli C600 cells harboring a plasmid coding for the lipP 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 of p-nitrophenyl butyrate and p-nitrophenyl laurate were determined to be 11.2 and 7.7 kcal/mol, respectively, in the temperature range 5 to 35 degrees C. The enzyme was unstable at temperatures higher than 45 degrees C. The Km of the enzyme for p-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 bisnitrophenyl phosphate. Various water-miscible organic solvents, such as methanol and dimethyl sulfoxide, at concentrations of 0 to 30% (vol/vol) activated the enzyme.

FIG. 1

Polyacrylamide gel electrophoresis of LipP. Lane 1, molecular weight markers; lane 2, the preparation after ammonium sulfate precipitation (amount of protein loaded, 25 μg); lanes 3 and 4, the preparation after DEAE-Cellulofine chromatography (amount of protein loaded, 15 μg); lanes 5 and 6, the preparation after Gigapite chromatography (amount of protein loaded, 10 μg).

FIG. 2

Effect of pH on the stability (A) and activity (B) of LipP. (A) The enzyme was incubated at various pH values at 4°C for 24 h, and the activity was measured. The enzyme activity after treatment with Tris-HCl (pH 9.0) was taken as 100%. Buffers used (final concentration, 20 mM) were glycine-HCl (▪) (pH 2.2 to 3.6); sodium acetate (◊) (pH 3.6 to 5.6); Tris-malate (•) (pH 5.6 to 8.6); Tris-HCl (○) (pH 7.4 to 9.0); glycine-NaOH (□) (pH 8.6 to 10.6). (B) The enzyme was incubated with 100 mM tributyrin as a substrate in NaH₂PO₄-NaOH buffer at various pH values at 25°C for 10 min, and free fatty acid formed was titrated with 0.05 M NaOH.

FIG. 3

Effect of temperature on the activity of LipP. (A) The enzyme was incubated with a mixture containing 20 mM phosphate buffer (pH 7.25), 5% acetonitrile, and 0.5 mM p-nitrophenyl butyrate at various temperatures for 10 min, and p-nitrophenol formed was measured. The value obtained at 37°C was taken as 100%. (B) The logarithm of the specific activity (V) (in micromoles per milligram per minute) was plotted against the reciprocal of absolute temperature (T). The values shown are activation energy calculated from the linear part of the plot.

FIG. 4

Effect of temperature on the Michaelis constant of LipP. The enzyme was assayed with a mixture containing 0.1 M phosphate buffer (pH 7.25), 0.1 M NaCl, and various concentrations of p-nitrophenyl butyrate (0.005 to 0.5 mM) at 25°C. The K_m for p-nitrophenyl butyrate was obtained from a plot of velocity versus substrate concentration with KaleidaGraph software (Synergy Software, Reading, Pa.). The values of four separate experiments were averaged and plotted against temperature.