Purification and characterization of a novel thermostable lipase from Pseudomonas cepacia.
A thermostable lipase from Pseudomonas cepacia has been purified to homogeneity as judged by SDS-PAGE and isoelectric focusing. The purification included treatment of the culture supernatant with acrinol, hydrophobic interaction chromatography, and gel filtration. The enzyme was a monomeric protein with M(r) of 36,500 and pI of 5.1. The optimal pH at 50 degrees C and optimal temperature at pH 6.5 were 5.5-6.5 and 55-60 degrees C, respectively, when olive oil was used as the substrate. Simple triglycerides of short and middle chain fatty acids (C < or = 12) were the preferred substrates over those of long chain fatty acids. The enzyme cleaved all the ester bonds of triolein, with some preference for the 1,3-ester bonds. The enzyme retained all its activity even after incubation at 75 degrees C (pH 6.5) for 30 min. Further, the activity was not impaired during 21 h storage at pH 6.5 in 40% water-miscible solvents including methanol, ethanol, acetone, acetonitrile, dimethylformamide, dimethylsulfoxide, and dioxane. The addition of dimethylsulfoxide or acetone to the assay mixture in the range of 0-35% stimulated the enzyme, whereas benzene or n-hexane had an inhibitory effect. These properties together with the N-terminal amino acid sequence confirmed that the enzyme differs from the known Pseudomonas sp. lipases.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D016956:Burkholderia cepacia / Q000201:enzymology*; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008049:Lipase / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D013379:Substrate Specificity
D000596:Amino Acids; D008049:Lipase