title: Purification and characterization of extracellular lipases from Ophiostoma piliferum.
abstract: Interest in lipases from microorganisms, animals, and plants has greatly increased in the past decade due to their applications in biotransformations and organic syntheses. We are reporting the purification and characterization of two lipases from the fungus, Ophiostoma piliferum, a saprophytic organism commonly found on wood. A major and a minor lipase have been co-purified by hydrophobic interaction chromatography on octyl sepharose FF, followed by ion exchange chromatography on Q sepharose FF. The lipases bound very tightly to octyl sepharose resulting in greater than 100-fold purification in this one step. The major lipase has a molecular weight of approximately 60 kDa, a pI of 3.79, and is glycosylated as determined by PAS staining. The minor lipase, which composes 10% of the total protein, has a pI of 3.6, and molecular weight of approximately 52 kDa and did not stain with the PAS reagent. Deglycosylation of the major lipase produced two proteins of lower molecular weight, a 55 kDa protein and a 52 kDa protein. The deglycosylated protein at 52 kDa co-migrates with the minor lipase on SDS-PAGE gels. N-terminal amino acid sequencing of the major and minor lipases indicated both lipases have the same N-termini and MALDI-TOF mass spectral analysis showed similar peptide patterns. Available data indicate that the lipases are derived from the same protein and appear to differ in their post-translational modification as evidenced by their pIs and molecular weight difference. The pH rate profile and thermal stability were determined for the purified O. piliferum lipase and were consistent with a mesophilic lipase. In aqueous solution, the lipases exhibited a higher rate of hydrolysis for p-nitrophenylbutyrate (C4) than for p-nitrophenylstearate (C18), which is an unexpected result.
authors.affiliation: Clariant Corporation, Biotechnology Research Division, Lexington, MA 02421, USA. theresa.brush@clariant.com
authors.forename: T S
authors.initials: TS
authors.lastname: Brush
authors.forename: R
authors.initials: R
authors.lastname: Chapman
authors.forename: R
authors.initials: R
authors.lastname: Kurzman
authors.forename: D P
authors.initials: DP
authors.lastname: Williams
mesh_terms: D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology* / Q000254:growth & development; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000201:enzymology; D005227:Fatty Acids / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D005227:Fatty Acids; D008049:Lipase