title: Purification, immobilization, and characterization of a specific lipase from Staphylococcus warneri EX17 by enzyme fractionating via adsorption on different hydrophobic supports.
abstract: Staphylococcus warneri strain EX17 produces three lipases with different molecular weights of 28, 30, and 45 kDa. The 45 kDa fraction (SWL-45) has been purified from crude protein extracts by one chromatographic step based on the selective adsorption of this lipase by interfacial activation on different hydrophobic supports at low ionic strength. The adsorption of SWL-45 on octyl-Sepharose increased the enzyme activity by 60%, but the other lipases were also adsorbed on this support. Using butyl-Toyopearl, which is a lesser hydrophobic support, the purification factor was close to 20, and the only protein band detected on the sodium dodecyl sulfate-polyacrylamide electrophoresis analysis gel was that corresponding to the SWL-45, which could be easily desorbed from the support by incubation with triton X-100, producing a purified enzyme. SWL-45 was immobilized under very mild conditions on cyanogen bromide Sepharose, showing similar activities and stability as for its soluble form but without intermolecular interaction. The effects of different detergents over the activity of the immobilized SWL-45 were analyzed, which was hyperactivated by factors of 1.3 and 2.5 with 0.01% Tween 80 and 0.1% Triton X-100, respectively, while ionic detergents produced detrimental effects on the enzyme activity even at very low concentrations. Optimal reaction conditions and the effect of other additives on the enzyme activity were also investigated.
authors.affiliation: Departamento de Biocatálisis, Instituto de Catálisis-CSIC, Campus UAM, Cantoblanco, Madrid 28049, Spain.
authors.forename: Giandra
authors.initials: G
authors.lastname: Volpato
authors.forename: Marco
authors.initials: M
authors.lastname: Filice
authors.forename: Blanca
authors.initials: B
authors.lastname: de las Rivas
authors.forename: Rafael C
authors.initials: RC
authors.lastname: Rodrigues
authors.forename: Júlio X
authors.initials: JX
authors.lastname: Heck
authors.forename: Roberto
authors.initials: R
authors.lastname: Fernandez-Lafuente
authors.forename: Jose M
authors.initials: JM
authors.lastname: Guisan
authors.forename: Cesar
authors.initials: C
authors.lastname: Mateo
authors.forename: Marco Antônio Záchia
authors.initials: MA
authors.lastname: Ayub
mesh_terms: D000327:Adsorption; D002845:Chromatography / Q000379:methods*; D003902:Detergents / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000302:isolation & purification*; D057927:Hydrophobic and Hydrophilic Interactions; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013210:Staphylococcus / Q000201:enzymology*
chemical_list: D003902:Detergents; D004800:Enzymes, Immobilized; D008049:Lipase