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PublicationPROCESS BIOCHEMISTRY
Year2014
Volume49
Pages604-616
International

TUNING OF LECITASE FEATURES VIA SOLID-PHASE CHEMICAL MODIFICATION: EFFECT OF THE IMMOBILIZATION PROTOCOL

Authors:Cristina Garcia-Galan, José Cleiton Sousa dos Santos, V.C: Corberan, Roberto Fernandez-Lafuente
Groups of research:Optimización de biocatalizadores y bioprocesos enzimáticos, Selective oxidation catalysts and processes (SOCP)
Cristina Garcia-Galana,+, Jose C. S. dos Santosa,b, +, Oveimar Barbosac, Rodrigo Torresc, Ernandes B. Pereirad, Vicente Cortes  Corberana, Luciana R.B. Gonçalvesb and Roberto Fernandez-Lafuentea*.
 
a: Instituto de Catálisis-CSIC. Campus UAM-CSIC. Cantoblanco. 28049 Madrid. Spain.
b: Departamento de Engenharia Química, Universidade Federal Do Ceará, Campus Do Pici, CEP 60455-760, Fortaleza, CE, Brazil 
c: Escuela de Química, Grupo de investigación en Bioquímica y Microbiología (GIBIM), Edificio Camilo Torres 210, Universidad Industrial de Santander, Bucaramanga, Colombia.
d: Universidade Federal de Alfenas, 37130-000, Alfenas, MG, Brazil
 
Lecitase Ultra (a quimeric fosfolipase commercialized by Novozymes) has been immobilized via two different strategies: mild covalent attachment on cyanogen bromide agarose beads and interfacial activation on octyl-agarose beads. Both immobilized preparations have been submitted to different individual or cascade chemical modifications (amination, glutaraldehyde or 2,4,6-trinitrobenzensulfonic acid (TNBS)  modification) in order to check the effect of these modifications on the catalytic features of the immobilized enzymes (including stability and substrate specificity under different conditions). The first point to be remarked is that the immobilization strongly affects the enzyme catalytic features: octyl-Lecitase was more active versus p-nitrophenylbutyrate but less active versus methyl phenylacetate than the covalent preparations. Moreover, the effects of the chemical modifications strongly depend on the immobilization strategy used. For example, using one immobilization protocol a modification improves activity, while for the other immobilzed enzyme is even negative. Most of the modifications presented a positive effect on some enzyme properties under certain conditions, although in certain cases that modification presented a negative effect under other conditions. For example, glutaraldehyde modification of immobilized or modified and aminated enzyme permitted to improve enzyme stability of both immobilized enzymes at pH 7 and 9 (around a 10-fold), but only the aminated enzyme improved the enzyme stability at pH 5 by glutaraldehyde treatment. This occurred even though some intermolecular crosslinking could be detected via SDS-PAGE. Amination improved the stability of octyl-Lecitase, but even has a slightly negative effect using the covalent preparation. Modification with TNBS only improved enzyme stability of the covalent preparation at pH 9 (by a 10 fold factor).
Keywords:Enzyme stabilization, Enzyme immobilization, glutaraldehyde, enzyme chemical modification, enzyme hyperactivation, 2, 4, 6-trinitrobenzensulfonic acid .
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