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PublicationRSC Advances
Year2016
Volume6
Pages61707 - 61715
International

DEVELOPMENT OF SIMPLE PROTOCOLS TO SOLVE THE PROBLEMS OF ENZYME COIMMOBILIZATION. APPLICATION TO COIMMOBILIZE A LIPASE AND A β-GALACTOSIDASE.

Authors:Roberto Fernandez-Lafuente
Groups of research:Optimización de biocatalizadores y bioprocesos enzimáticos
Sara Peirce a,b, Jose J. Virgen-Ortíz a,   Veymar G. Tacias-Pascacio a,c, Nazzoly Rueda a,d, Rocio Bartolome-Cabrero a, Laura Fernandez-Lopez a , Maria Elena Russo e, Antonio Marzocchella b, Roberto Fernandez-Lafuente a*
 
a Departamento de Biocatálisis. Instituto de Catálisis-CSIC, Campus UAM-CSIC Madrid, Spain.
b Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale. Universita' degli Studi di Napoli Federico II, Italy.
c Unidad de Investigación y Desarrollo en Alimentos. Instituto Tecnológico de Veracruz, Calzada Miguel A. de Quevedo 2779, 91897 Veracruz, Mexico.
d Escuela de Química, Grupo de investigación en Bioquímica y Microbiología (GIBIM), Edificio Camilo Torres 210, Universidad Industrial de Santander, Bucaramanga, Colombia.
e Istituto di Ricerche sulla Combustione– Consiglio Nazionale delle Ricerche, Napoli, Italy.



This paper shows the coimmobilization of β-galactosidase from Aspergillus oryze (β-gal) and lipase B from Candida antarctica (CALB). The combi-biocatalyst was designed in a way that permits an optimal immobilization of CALB on octyl-agarose (OC) and the reuse of this enzyme after β-gal (an enzyme with lower stability and altogether not very stabilized by multipoint covalent attachment) inactivation, both of them serious problems in enzyme co-immobilization. To this goal, OC-CALB was coated with polyethylenimine (PEI) (this treatment did not affect the enzyme activity and even improved enzyme stability, mainly in organic medium). Then, β-gal was immobilized by ion exchange on the PEI coated support. We found that PEI can become weakly adsorbed on OC support, but the adsorption of PEI to CALB was quite strong. The immobilized β-gal can be desorbed by incubation in 300 mM NaCl. Fresh β-gal could be adsorbed afterwards, and this could be repeated for several cycles, but the amount of PEI showed a small decrease that made reincubation of the OC-CALB-PEI composite in PEI preferable in order to keep the amount of polymer. CALB activity remained unaltered under all these treatments. The combi-catalyst was submitted to inactivation at 60 ºC and pH 7, conditions where β-gal was rapidly inactivated while CALB maintained its activity unaltered. All β-gal activity could be removed by incubation in 300 mM NaCl, however, SDS analysis showed that part of the enzyme β-gal molecules remained immobilized on the OC-CALC-PEI composite, as the inactivated enzyme may become more strongly adsorbed on the ion exchanger. Full release of the β-gal after inactivation was achieved using 1 M NaCl and 40 ºC, conditions where CALB remained fully stable. This way, the proposed protocol permitted the reuse of the most stable enzyme after inactivation of the least stable one. It is compatible with any immobilization protocol of the first enzyme that does not involve ion exchange as only reason for enzyme immobilization.
Keywords:lipase interfacial activation, ion exchange, Enzyme coimmobilization, enzyme reuse, combi-biocatalysts, cascade reactions, PEI coating
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