Purification and Some Properties of a Thermostable Lipase fromHumicola lanuginosaNo. 3

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Abstract
A thermostable lipase from Humicola lanuginosa No. 3 was purified by means of acetone precipitation and successive chromatographies on Sephadex G-75, DEAE Sepharose Cl-6B and hydroxyapatite columns. The enzyme was purified about 150-fold with a yield of 15.0% and a specific activity of about 3000 U/mg protein. The purified enzyme showed a single protein band on polyacrylamide gel electrophoresis. Its molecular weight was estimated to be 39,000. on both sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration on Sephadex G-100, suggesting that the enzyme was a monomer. Its isoelectric point was pH 6.6. The optimum pH and temperature were 7.0 and- 45°C, respectively. The enzyme was stable over a pH range of 5 to 9 (45°C, 24 hr), and its activity was maintained at 60°C for about 20 hr. The activity was inhibited by Co2 +, Cu2 +, Ni2 +, Hg2+ and Sn2 +, and slightly affected by Zn2 +, Mg2 +, EDTA and sodium dodecyl sulfate. The Km value for trilaurin (45°C) was 14.2 mg/ml. The enzyme was specific for substrates containing a C12 fatty acid component. Analysis of hydrolyzates of triolein and olive oil after the lipase reaction revealed that Humicola lanuginosa No. 3 produced a 1,3 positional specific lipolytic enzyme.

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