title: Purification and characterization of a regiospecific lipase from Aspergillus terreus.
abstract: Aspergillus terreus lipase was purified to homogeneity with 18.0% yield. The specific activity of the enzyme increased from 20.80 to 250 U/mg of protein. Ion exchange on Q-Sepharose was highly effective in the purification process. The molecular mass of the purified enzyme was 41+/-1 kDa as determined by SDS/PAGE. The purified lipase showed excellent temperature tolerance (15-90 degreesC) and was highly thermostable, retaining 100% activity at 60 degreesC for 24 h. It showed good pH tolerance (3.0-12.0) and was stable over a pH range of 4.0-10.0 for 24 h. The activity of the enzyme was inhibited by ionic detergents, whereas non-ionic detergents stimulated enzyme activity. Mg2+ and Ca2+ ions stimulated lipase activity, whereas Co2+, Cu2+, Ni2+ and Fe3+ ions caused inhibition. The enzyme was unaffected by the metal chelator EDTA or by 2-mercaptoethanol and potassium ferrocyanide. At a concentration of 100 microM, 3,4-dichloroisocoumarin caused weak inhibition with 40% loss of activity, but diethyl p-nitrophenyl phosphate at the same concentration strongly inhibited enzyme activity (98.12% loss of activity), confirming that the A. terreus lipase is a serine hydrolase. The lipase was highly active on pig fat (151% relative activity) and groundnut oil (103% relative activity) and least active on kusum oil (18% relative activity). Extensive dialysis did not affect enzyme activity up to 168 h, suggesting the absence of any dialysable cofactor in the enzyme. The A. terreus lipase retained significant activity on freeze-drying and had a shelf-life of more than 6 months at room temperature. The A. terreus lipase exhibited 1,3-regiospecificity and was stable in various organic solvents.
authors.affiliation: Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India.
authors.forename: R P
authors.initials: RP
authors.lastname: Yadav
authors.forename: R K
authors.initials: RK
authors.lastname: Saxena
authors.forename: R
authors.initials: R
authors.lastname: Gupta
authors.forename: W S
authors.initials: WS
authors.lastname: Davidson
mesh_terms: D001230:Aspergillus / Q000201:enzymology*; D002412:Cations; D003902:Detergents / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry; D008049:Lipase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D008970:Molecular Weight; D013379:Substrate Specificity
chemical_list: D002412:Cations; D003902:Detergents; D004791:Enzyme Inhibitors; D004492:Edetic Acid; D008049:Lipase