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Immobilization/stabilization of ficin extract on glutaraldehyde-activated agarose beads. Variables that control the final stability and activity in protein hydrolyses

Authors:Roberto Fernandez-Lafuente
Groups of research:Optimization of biocatalysts and bioprocesses
El-Hocine Siar*,1,2, Sara Arana-Peña*1, Oveimar Barbosa3, Mohammed Nasreddine Zidoune2, Roberto Fernandez-Lafuente1,*
1 Departamento de Biocatálisis.  Instituto de Catálisis-CSIC, Campus UAM-CSIC Madrid, Spain.
2 Equipe TEPA, Laboratoire LNTA, INATAA, Université des Frères Mentouri Constantine1, 25000, Constantine, Algeria
3 Departamento de Química, Facultad de Ciencias. Universidad del Tolima, Ibagué, Colombia.

Abstract: Ficin extract has been immobilized on different 4% aminated-agarose beads. Using just ion exchange, immobilization yield was poor and expressed activity did not surpass 10% of the offered enzyme, with no significant effects on enzyme stability. The treatment with glutaraldehyde of this ionically exchanged enzyme produced an almost full enzyme inactivation. Using aminated supports activated with glutaraldehyde, immobilization was optimal at pH 7 (at pH 5 immobilization yield was 80%, while at pH 9, the immobilized enzyme became inactivated). At pH 7, full immobilization was accomplished maintaining 40% activity versus a small synthetic substrate and 30% versus casein. Ficin stabilization upon immobilization could be observed, but it depended on the inactivation pH and the substrate employed, suggesting the complexity of the mechanism of inactivation of the immobilized enzyme. The maximum enzyme loading on the support was determined to be around 70 mg/g. The loading has no significant effect on the enzyme stability or enzyme activity using the synthetic substrate, but it had a significant effect on the activity using casein; the biocatalysts activity greatly decreased using more than 30 mg/g, suggesting that the near presence of other immobilized enzyme molecules may generate some steric hindrances for the casein hydrolysis.

Keywords:immobilization using glutaraldehyde, versatility of glutaraldehyde, steric problems in enzyme activity, effect of loading on enzyme activity.
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