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Immobilization of lipases on glyoxyl-octyl supports: improved stability and reactivation strategies

Authors:José Cleiton Sousa dos Santos, Roberto Fernandez-Lafuente
Groups of research:Optimization of biocatalysts and bioprocesses
Angélica Suescun a, +, Nazzoly Ruedaa,b,+,  Jose C. S. dos Santosb,c, John J.Castillo a, Claudia Ortizd, Rodrigo Torresa,e, Oveimar Barbosaf,* and Roberto Fernandez-Lafuenteb,*.
a: Escuela de Química, Grupo de investigación en Bioquímica y Microbiología (GIBIM), Edificio Camilo Torres 210, Universidad Industrial de Santander, CEP 680001 ,Bucaramanga, Colombia.
b:   ICP-CSIC. Campus UAM-CSIC. Cantoblanco. 28049 Madrid. Spain.
c: Departamento de Engenharia Química, Universidade Federal Do Ceará, Campus Do Pici, CEP 60455-760, Fortaleza, CE, Brazil.
d:  Escuela de Microbiología, Universidad Industrial de Santander, Bucaramanga, Colombia
e: Current address: Laboratorio de Biotecnología, Instituto Colombiano del Petróleo-Ecopetrol, Piedecuesta, Bucaramanga, Colombia.
f: Departamento de Química, Facultad de Ciencias. Universidad del Tolima, Ibagué, Colombia

Lipases from Candida rugosa (CRL) and from C. antarctica (isoform A) (CALA) have been successfully immobilized on octyl-glyoxyl agarose (OCGLX) beads and compared to the octyl-agarose (OC) or glyoxyl (GLX) beads immobilized counterparts. Immobilization on OCGLX gave similar hyperactivations than those found for the immobilization on OC supports, although the incubation at pH 10.0 for 4 h decreased the activity of both enzymes by 25%. After reduction, more than 95% of the enzyme activity was covalently attached to the support. The fraction not covalently attached was desorbed by washing with detergent. These biocatalysts were more stable than the octyl counterparts in thermal or organic solvent inactivation. More interestingly, the irreversible immobilization permitted the reactivation of CALA biocatalysts inactivated by incubation in organic solvent, after unfolding in the presence of guanidine and refolding in aqueous buffer (around 55% of the activity could be recovered during 3 successive cycles of inactivation/reactivation). GLX-CALA permitted to recover 75% of the activity, but the thermal stability and activity was much lower, and this strategy could not be applied to CRL. Neither the enzyme immobilized on cyanogen bromide nor the enzyme immobilized on OCGLX exhibited significant activity after the unfolding/refolding strategy.
Keywords:covalent immobilization, Enzyme stabilization, immobilization of lipases via interfacial activation, enzyme reactivation, unfolding/refolding
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