Purification, Immobilization, and Characterization of a Specific Lipase from Staphylococcus warneri EX17 by Enzyme Fractionating via Adsorption on Different Hydrophobic Supports Giandra Volpato Departamento de Biocata ´lisis, Instituto de Cata ´lisis-CSIC, Campus UAM, Cantoblanco, Madrid 28049, Spain Instituto Federal de Educac ¸a ˜o, Cie ˆncia e Tecnologia do Rio Grande do Sul, Rua Ramiro Barcelos, 2777, ZC 90035-007, Porto Alegre, RS, Brazil Marco Filice and Blanca de las Rivas Departamento de Biocata ´lisis, Instituto de Cata ´lisis-CSIC, Campus UAM, Cantoblanco, Madrid 28049, Spain Rafael C. Rodrigues Departamento de Biocata ´lisis, Instituto de Cata ´lisis-CSIC, Campus UAM, Cantoblanco, Madrid 28049, Spain Biotechnology and Biochemical Engineering Laboratory-BiotecLab, Federal University of Rio Grande do Sul State, Av. Bento Gonc ¸alves, 9500, PO Box 15090, ZC 91501-970, Porto Alegre, RS, Brazil Ju ´lio X. Heck Instituto Federal de Educac ¸a ˜o, Cie ˆncia e Tecnologia do Rio Grande do Sul, Rua Ramiro Barcelos, 2777, ZC 90035-007, Porto Alegre, RS, Brazil Roberto Fernandez-Lafuente, Jose M. Guisan, and Cesar Mateo Departamento de Biocata ´lisis, Instituto de Cata ´lisis-CSIC, Campus UAM, Cantoblanco, Madrid 28049, Spain Marco Anto ˆnio Za ´chia Ayub Biotechnology and Biochemical Engineering Laboratory-BiotecLab, Federal University of Rio Grande do Sul State, Av. Bento Gonc ¸alves, 9500, PO Box 15090, ZC 91501-970, Porto Alegre, RS, Brazil DOI 10.1002/btpr.601 Published online April 20, 2011 in Wiley Online Library (wileyonlinelibrary.com). 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 adsorp- tion of SWL-45 on octyl-Sepharose increased the enzyme activity by 60%, but the other li- pases 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 incuba- tion 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 concentra- tions. Optimal reaction conditions and the effect of other additives on the enzyme activity were also investigated. V V C 2011 American Institute of Chemical Engineers Biotechnol. Prog., 27: 717–723, 2011 Keywords: Staphylococcus warneri lipase, enzyme purification, selective adsorption, hydrophobic immobilization supports, interfacial activation Introduction Lipases are among the most widely used enzymes in bio- catalysis due to their very broad range of substrates, very of- ten accompanied with a high regio- or enantioselectivity. 1,2 Correspondence concerning this article should be addressed to M. A. Z. Ayub at mazayub@ufrgs.br. V V C 2011 American Institute of Chemical Engineers 717