Journal of Molecular Catalysis B: Enzymatic 32 (2005) 225–229 Activation in the family of Candida rugosa isolipases by polyethylene glycol Cristina Otero a,∗ ,M´ onica Fern´ andez-P´ erez a , Juan A. Hermoso b , Mart´ ın Mart´ ınez Ripoll b a Departamento de Biocat ´ alisis, Instituto de Cat ´ alisis, CSIC, Campus Universitario, Cantoblanco, 28049 Madrid, Spain b Grupo de Cristalograf´ ıa Macromolecular y Biolog´ ıa Estructural, Instituto “Rocasolano” CSIC, Serrano 119, 28006 Madrid, Spain Received 3 September 2004; accepted 2 December 2004 Available online 15 January 2005 Abstract We have investigated activation of two isoenzymes (lip1 and lip3) from Candida rugosa in polyethylene glycol (PEG) media. Aqueous solutions of PEG 8000 and 20,000 activate lip3 but not lip1 from C. rugosa. Maximum activation (260%) of lip3 requires 6 h of pre-incubation with PEG 8000 (4%, w/v). PEG seems to shift the equilibrium between the open and the closed forms of lip3 towards the active conformation. Inhibition experiments demonstrate that ligands have easier access to the lip3 active site than to the lip1 active site, both in the presence and the absence of PEG. The presence of PEG in the crystallization medium is responsible for reported differences in the crystal structures of lip1 and lip3.A comparative analysis of crystallographic models of lip1 and lip3 suggests a role for PEG in activation of lip3 and further stabilization of the activated/open form via dimerization in aqueous media. © 2004 Elsevier B.V. All rights reserved. Keywords: Lipase; Candida rugosa; Cholesterol esterase; Activation; Polyethylene glycol 1. Introduction Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) are en- zymes that “in vivo” hydrolyze the long chain aliphatic esters of triglycerides to produce fatty acids and glycerol. These en- zymes have received extensive attention for their potential use in biotechnology [1]. One can distinguish different modes for activation of li- pases at lipid–water interfaces [2]. The simplest case cor- responds to displacement of a hydrophobic lid rendering a pre-formed active site accessible to the substrate [3]. In other cases, the oxyanion hole is formed only after the activation process. In the most complex case, a lipase may require the Abbreviations: E600, Diethyl p-nitrophenyl phosphate; PEG, Polyethy- lene glycol; PNPB, p-Nitrophenyl butyrate; INH, Inhibitor ∗ Corresponding author. Tel.: +34 91 5854805; fax: +34 91 5854760. E-mail address: Cotero@icp.csic.es (C. Otero). simultaneous presence of a colipase [4]. However, enzymes with lipolytic activity have been identified that do not exhibit the interfacial activation phenomenon. Consequently, the lack of interfacial activation is not a sufficient criterion for distin- guishing lipases from esterases. A contemporary definition of lipases – carboxylesterases that are able to catalyze the hydrolysis of long-chain triacylglycerols [2,5] – includes (i) lipases with a lid covering the active site, for which the cor- responding oxyanion is formed only after activation [4]; (ii) lipases containing a preformed oxyanion but which do not contain lid, and which do not exhibit activation, for example, Candida antarctica B [6] and cutinase [7]; and (iii) lipases having a lid and which exhibit interfacial activation, for ex- ample, lipases from Candida rugosa. Interfacial activation of lipolytic enzymes is associated not only with the presence of one or more full-length loops cov- ering the active site, but also the presence of other structural elements that might stabilize closed or open conformations 1381-1177/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molcatb.2004.12.008