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PublicationEnzyme and Microbial Technology

Immobilization of lipases on hydrophobic supports involves the open form of the enzyme

Authors:José Cleiton Sousa dos Santos, Roberto Fernandez-Lafuente
Groups of research:Optimization of biocatalysts and bioprocesses
Evelin A. Manoela,b*, José C. S. dos Santosb,c*,  Denise M. G. Freired, Nazzoly Ruedae, and Roberto Fernandez-Lafuente b
a Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 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.
b Department  of biocatalysis. ICP-CSIC. Campus UAM-CSIC. Cantoblanco. 28049 Madrid. Spain
cDepartamento de Engenharia Química, Universidade Federal do Ceará, Campus do Pici, CEP 60455-760, Fortaleza, CE, Brazil.
dPrograma de Pós-Graduação em Bioquímica, Departamento de Bioquímica, Brazil;
eEscuela de Química, Grupo de investigación en Bioquímica y Microbiología (GIBIM), Edificio Camilo Torres 210, Universidad Industrial de Santander, Bucaramanga, Colombia.

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 0.025-1M), 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.
Keywords:Lipase immobilization, interfacial activation, detergents, octyl-agarose, cyanogen bromide, irreversible inhibition, ionic strength
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