Purification and properties of an extracellular lipase from Pythium ultimum

An extracellular triacylglycerol lipase (EC 3.1.1.3) from Pythium ultimum strain No. 144 was purified by ammonium sulfate precipitation, and by diethylaminoethyl Sepharose CL‐6B and Sephacryl S−200 chromatography. The purified enzyme preparation showed a prominent polypeptide band in polyacrylamide gel electrophoresis, associated with esterase activity according to activity staining. Molecular weight of the protein was estimated at 270 kD using gel filtration on Sephacryl S−200, and 68 kD by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis indicating that the enzyme may be a tetramer. The optimum pH and temperature for activity of the enzyme were 8.0 and 30°C, respectively. Activity was reduced by Co 2+ , Fe 2+ , Sn 2+ and Mn 2+ and stimulated by Ca 2+ , Mg 2+ , Na + , K + and surfactants such as taurocholic acid, Triton X−100, n ‐octyl glucoside, n ‐dodecyl‐β‐D‐maltoside, 3‐[(3‐cholamidopropyl) dimethylammonio]‐1‐propanesulfonate(CHAPS), and 3‐[‐cholamidopropyl)dimethylammonio]‐2‐hydroxy‐1‐propanesulfonate. The apparent maximum specific activity was 42 μmole/min/mg in the absence of CHAPS and 77 μmole/min/mg in its presence. The reaction rate was progressively higher with increasing number of double bonds in the substrate, and the enzyme showed a preference for triacylglycerols containing fatty acids having the cis double bond configuration.