title: Immobilization of lipases on hydrophobic supports involves the open form of the enzyme.
abstract: The lipases from Thermomyces lanuginosus and Pseudomonas cepacia have been immobilized on octyl and cyanogen bromide (CNBr) agarose beads. The immobilization on octyl-agarose is slowed with increasing ionic strength, while the immobilization on CNBr is not significantly affected by the ionic strength. The inhibition of the immobilized preparations with diethyl p-nitrophenylphosphate (D-pNPP) was analyzed. The inhibition was more rapid using octyl-lipase preparations than using covalent preparations, and the covalent preparations were much more sensitive to the reaction medium. The addition of detergent increased the inhibition rate of the covalent preparation while an increase on the ionic strength produced a slowdown of the inhibition rate by D-pNPP for both lipases. The effect of the medium on the activity versus fully soluble substrate (methyl mandelate) was in the same direction. The octyl preparations presented a slight decrease in activity when comparing the results using different concentrations of sodium phosphate buffer (between 0.025 and 1M), while the CNBr preparations suffered drastic drops in its activity at high ionic strength. The results confirm that the lipases immobilized on octyl agarose presented their open form stabilized while the covalent preparation maintains a closing/opening equilibrium that may be modulated by altering the medium.
authors.affiliation: Universidade Federal do Rio de Janeiro, Faculdade de Farmácia, Departamento de Biotecnologia Farmacêutica, Centro de Ciências e Saúde, Bloco B, 1 andar (1 floor), Laboratório Multidisciplinar de Pesquisas em Biotecnologia, CEP 21941902, Rio de Janeiro, Brazil; Department of Biocatalysis, ICP-CSIC, Campus UAM-CSIC, Cantoblanco, 28049 Madrid, Spain.
authors.forename: Evelin A
authors.initials: EA
authors.lastname: Manoel
authors.affiliation: Department of Biocatalysis, ICP-CSIC, Campus UAM-CSIC, Cantoblanco, 28049 Madrid, Spain; Departamento de Engenharia Química, Universidade Federal do Ceará, Campus do Pici, CEP 60455-760, Fortaleza, CE, Brazil.
authors.forename: José C S
authors.initials: JC
authors.lastname: Dos Santos
authors.affiliation: Programa de Pós-Graduação em Bioquímica, Departamento de Bioquímica, Brazil.
authors.forename: Denise M G
authors.initials: DM
authors.lastname: Freire
authors.affiliation: Escuela de Química, Grupo de investigación en Bioquímica y Microbiología (GIBIM), Edificio Camilo Torres 210, Universidad Industrial de Santander, Bucaramanga, Colombia.
authors.forename: Nazzoly
authors.initials: N
authors.lastname: Rueda
authors.affiliation: Department of Biocatalysis, ICP-CSIC, Campus UAM-CSIC, Cantoblanco, 28049 Madrid, Spain. Electronic address: rfl@icp.csic.es.
authors.forename: Roberto
authors.initials: R
authors.lastname: Fernandez-Lafuente
mesh_terms: D001203:Ascomycota / Q000201:enzymology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D016956:Burkholderia cepacia / Q000201:enzymology; D003488:Cyanogen Bromide; D004800:Enzymes, Immobilized / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008049:Lipase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D009994:Osmolar Concentration; D011487:Protein Conformation; D012685:Sepharose / Q000031:analogs & derivatives
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D005656:Fungal Proteins; C027176:octyl-sepharose CL-4B; D012685:Sepharose; D008049:Lipase; D003488:Cyanogen Bromide
keywords: Detergents; Interfacial activation; Ionic strength; Irreversible inhibition; Lipase immobilization; Octyl-agarose