PURIFICATION AND PROPERTIES OF AN EXTRACELLULAR LIPASE FROM PYTHIUM-ULTIMUM

An extracellular triacylglycerol lipase (EC 3.1.1.3) from Pythium ulti mum strain No. 144 was purified by ammonium sulfate precipitation, and by diethylaminoethyl Sepharose CL-6B and Sephacryl S-200 chromatograp hy. The purified enzyme preparation showed a prominent polypeptide ban d in polyacrylamide gel electrophoresis, associated with esterase acti vity according to activity staining. Molecular weight of the protein w as estimated at 270 kD using gel filtration on Sephacryl S-200, and 68 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indic ating that the enzyme may be a tetramer. The optimum pH and temperatur e 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 Ca 2+, Mg2+, Na+, K+ and surfactants such as taurocholic acid, Triton X-1 00, n-octyl glucoside, n-dodecyl-beta-D-maltoside, 3-[(3-cholamidoprop yl) dimethylammonio]-1-propanesulfonate(CHAPS), and pyl)dimethylammoni o]-2-hydroxy-1-propanesulfonate. The apparent maximum specific activit y 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 increas ing number of double bonds in the substrate, and the enzyme showed a p reference for triacylglycerols containing fatty acids having the cis d ouble bond configuration.