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-degrees-C, respectively. Activity was reduced by Co2+, Fe2+, Sn2+ and Mn2+ and stimulated by Ca2+, Mg2+, Na+, K+ and surfactants such as taurocholic acid, Triton X-100, n-octyl glucoside, n-dodecyl-beta-D-maltoside, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate(CHAPS), and 3-[-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate. The apparent maximum specific activity was 42 mumole/min/mg in the absence of CHAPS and 77 mumole/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.