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Biotechnology and Applied Biochemistry (1998) 28, (243–249) (Printed in Great Britain)

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Purification and characterization of a regiospecific lipase from Aspergillus terreus

Raman P. Yadav, Rajendra K. Saxena¹, Rani Gupta and W. Sheba Davidson

Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India

¹ To whom correspondence should be addressed.

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!ER2771p124!1 kDa as determined by SDS/PAGE. The purified lipase showed excellent temperature tolerance (15–90 °C) and was highly thermostable, retaining 100% activity at 60 °C 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 µM, 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.

Received 12 August 1997/16 March 1998; accepted 9 June 1998

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