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Purification and properties of an extracellular lipase from Pythium ultimum
Author(s) -
Mozaffar Zahid,
Weete John D.
Publication year - 1993
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/bf02535933
Subject(s) - chemistry , chromatography , sodium dodecyl sulfate , lipase , polyacrylamide gel electrophoresis , gel electrophoresis , chaps , pythium ultimum , enzyme assay , enzyme , biochemistry , biology , ecology , biological pest control
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.