Purification and characterization of an extracellular lipase from Geotrichum marinum.
An extracellular lipase (EC 3.1.1.3) from Geotrichum marinum was purified 76-fold with 46% recovery using Octyl Sepharose 4 Fast Flow and Bio-Gel A 1.5 m chromatography. The purified enzyme showed a prominent band on SDS-PAGE and a single band on native PAGE based on the activity staining. The molecular mass of the lipase was estimated to be 62 kDa using SDS-PAGE and Bio-Gel A chromatography, indicating that the lipase likely functions as a monomer. The pl of the lipase was determined to be 4.54. The apparent V(max) and Km were 1000 micromol/min/mg protein and 11.5 mM, respectively, using olive oil emulsified with taurocholic acid as substrate. The lipase demonstrated a pH optimum at pH 8.0 and a temperature optimum at 40 degrees C. At 6 mM, Na+, K+, Ca2+, and Mg2+ stimulated activity, but Na+ and K+ at 500 mM and Fe2+ and Mn2+ at 6 mM reduced lipase activity. The anionic surfactant, taurocholic acid, and the zwitterionic surfactant, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, enhanced the activity at 0.1 mM. Other anionic surfactants such as SDS and sodium dioctyl sulfosuccinate, the cationic surfactants methylbenzethonium bromide and cetyltriethylammonium bromide, and the nonionic surfactants Tween-20 and Triton X-100 inhibited the lipase activity to different extents. The lipase was found to have a preference for TG containing cis double bonds in their FA side chains, and the reaction rate increased with an increasing number of double bonds in the side chain. The lipase had a preference for ester bonds at the sn-1 and sn-3 positions over the ester bond at the sn-2 position.
Huang
Youliang
Y
Department of Biological Sciences, Auburn University, Auburn, Alabama 36849, USA. huangyo@mail.auburn.edu
Locy
Robert
R
Weete
John D
JD
D005848:Geotrichum / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008066:Lipolysis; D008670:Metals / Q000737:chemistry; D008970:Molecular Weight; D013379:Substrate Specificity; D014304:Triolein / Q000737:chemistry
D007477:Ions; D008670:Metals; D014304:Triolein; D008049:Lipase