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RevistaRSC Advances
Año2015
Volumen5
Páginas55588-55594
Internacional

REACTIVATION OF LIPASES VIA UNFOLDING/REFOLDING OF COVALENTLY IMMOBILIZED BIOCATALYSTS

Autores:José Cleiton Sousa dos Santos, Roberto Fernandez-Lafuente
Grupos de investigación:Optimización de biocatalizadores y bioprocesos enzimáticos
Nazzoly Ruedaa,b,+,  Jose C. S. dos Santosa,c.+, Rodrigo Torresb,d, Oveimar Barbosae,
Claudia Ortizf,*, and Roberto Fernandez-Lafuentea,*.

a: ICP-CSIC. Campus UAM-CSIC. Cantoblanco. 28049 Madrid. Spain.
b:   Escuela de Química, Grupo de investigación en Bioquímica y Microbiología (GIBIM), Edificio Camilo Torres 210, Universidad Industrial de Santander, CEP 680001 ,Bucaramanga, Colombia.
c: Departamento de Engenharia Química, Universidade Federal Do Ceará, Campus Do Pici, CEP 60455-760, Fortaleza, CE, Brazil.
d: Current address: Laboratorio de Biotecnología, Instituto Colombiano del Petróleo-Ecopetrol, Piedecuesta, Bucaramanga, Colombia.
e: Grupo de investigación en productos naturales (GIPRONUT) Departamento de Química, Facultad de Ciencias. Universidad del Tolima, Ibagué, Colombia
f:  Escuela de Microbiología, Universidad Industrial de Santander, Bucaramanga, Colombia



            Lipases from Candida antarctica (isoform B) (CALB) and Thermomyces lanuginosus (TLL) have been immobilized covalently or via interfacial activation versus octyl support followed by covalent attachment via glyoxyl groups using octyl-glyoxyl agarose beads (OCGLX). These biocatalysts have been submitted to successive cycles of unfolding by incubation in 9 M guanidine and refolding by incubation in aqueous 100 mM phosphate buffer at pH 7, before and after total inactivation in the presence of organic solvents. The four preparations may be reactivated in some extension using this strategy, but results depended on the preparation. Glyoxyl immobilized CALB may recover 100% of activity versus the p-nitrophenyl butyrate, but after solvent inactivation this activity recovery was only 95%. The pure covalent TLL preparation permitted to recover around 80% of activity after or before solvent inactivation. Both enzymes offer lower activity recoveries using OCGLX, as may be expected from the hydrophobic nature of the groups in the support (60% for CALB and 45% for TLL). Moreover, using previously solvent inactivated enzymes, the results decreased in a 5-10%. These values were maintained along three successive cycles. However, using R and S methyl mandelate, it is clear that the activity recovery decreased along the reactivation cycles. Altogether, the unfolding/refolding strategy may be used to obtain part of the enzyme activity and that is relevant from an applied point of view, as this may permit to use the enzyme preparations for longer times. However, to reach the same enzyme structure in each reactivation cycle it is necessary to perform further studies that may involve from use of other supports to improve  the unfolding and refolding steps of this strategy.
Palabras clave:enzyme reactivation, operation stability, enzyme solvent inactivation, enzyme unfolding, enzyme refolding, octyl-glyoxyl supports, heterofunctional support.
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