Purification and some properties of a thermostable lipase from Humicola lanuginosa No.3.

A thermostable lipase from Humicola lanuginosa No. 3 was purified by means of acetone precipitation and successive chromatographies on Sephadex G-75, DEAESepharose 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, 24hr), and its activity was maintained at 60°C for about 20hr.The activity was inhibited by Co2+, Cu2+, Ni2+, Hg2+ and Sn2+, and slightly affected by Zn2+, Mg2+, EDTAand sodium dodecyl sulfate.The Kmvalue for trilaurin (45°C) was 14.2mg/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.