Properties and substrate specificities of an extracellular lipase purified from Ophiostoma piceae |
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Authors: | Gao Y. Breuil C. |
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Affiliation: | (1) Forest Products Biotechnology, Department of Wood Science, Faculty of Forestry, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada; fax: ( |
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Abstract: | An extracellular lipase produced by the sapstaining fungus Ophiostoma piceae 387N in a liquid medium was purified to homogeneity using ammonium sulphate and acetone fractionation, hydrophobic interaction and anion exchange chromatography. The overall purification based on lipase activity was 5200-fold with a yield of 26%. The molecular mass of the lipase was 35kDa, as determined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), and 37 kDa, as measured by size exclusion chromatography. The purified enzyme was resolved as three bands at pI values of 4.3, 4.1 and 3.8 in IEF (isoelectric focusing) gels. Lipolytic stain demonstrated that all three bands were lipolytically active. The N-terminal amino acid sequence was determined asD1-V2-S3-V4-T5-T6-T7-D8-I9-D10-A11-L12-A13-F14-F15-T16-Q17-W18-A19-G20 . The lipase was shown to be glycosylated, containing 10.1% carbohydrate. The lipase was stable between pH 4 and pH 8 and at temperatures below 40°C. The lipase activity had a pH optimum of approximately 5 and a temperature optimum of 30°C. The enzyme activity was not influenced by N-ethylmaleimide, -mercaptoethanol or dithiothreitol, was enhanced by Ca2+ or Mn2+, but was severely inhibited by Hg2+, Fe3+, butyric acid, caproic acid, diethyl pyrocarbonate, and diethyl p-nitrophenyl phosphate. The lipase hydrolysed mainly triglycerides, although some activity was measured on waxes and cholesteryl esters. It belongs to a group of 1 (3) positional specific lipases. It showed little activity for substrates with short chain fatty acids (C2–C6), but demonstrated high specificity for substrates with intermediate and long chain fatty acid residues (C10–C18). |
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Keywords: | Characterization lipase Ophiostoma piceae purification sapstain substrate specificity |
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