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RevistaPROCESS BIOCHEMISTRY
Año2018
Volumen73
Páginas109-116
Internacional

Solid phase chemical modification of agarose glyoxyl-ficin: Improving activity and stability properties by amination and modification with glutaraldehyde

Autores:Roberto Fernandez-Lafuente
Grupos de investigación:Optimización de biocatalizadores y bioprocesos enzimáticos
1           Departamento de Biocatálisis, Instituto de Catálisis-CSIC, Campus UAM-CSIC, 28049 Madrid, Spain
2           Equipe TEPA, Laboratoire LNTA, INATAA, Université des Frères Mentouri Constantine 1, 25000 Constantine, Algeria; zidounem@yahoo.fr
3           Departamento de Química, Facultad de Ciencias, Universidad del Tolima, 730006299 Ibagué, Colombia; oveimar@gmail.com
           Both authors have evenly contributed to this paper.



Ficin extract immobilized on glyoxyl-agarose was chemically modified using carbodiimide and ethylenediamine (full or partial amination of the carboxylic groups of the enzyme) or glutaraldehyde (modifying primary amino groups) under different conditions. Aminated enzyme altered its activity versus casein and benzoyl-arginine-p-nitroanilide (BANA), e.g., the activity versus casein even slightly increased while versus BANA it decreased, being this more significant at pH 9 (the fully aminated biocatalyst increased the activity versus casein by 10% but it decreased more than 5 folds versus BANA), greatly altering the enzyme specificity. The amination improved the enzyme stability at pH 5 while stability was impoverished at pH 9. Glutaraldehyde treatment usually decreased the activity versus BANA while it improved the activity versus casein (by around a factor of 2 when using 1% glutaraldehyde). Enzyme thermostability was enhanced, mainly at pH 7. This permitted to have more linear casein hydrolysis courses at pH 7 and 66ºC compared to the unmodified enzyme. Amination followed by glutaraldehyde treatment drove to the almost full enzyme inactivation. Thus, these treatments may be considered interesting for preparing an improved biocatalyst of ficin, mainly in proteolytic applications, and may be considered (mainly the amination) a possibility to further improve enzyme immobilization.

Palabras clave:enzyme crosslinking, Enzyme stabilization, enzyme modulation, Chemical immobilization of immobilized enzymes, enzyme amination, hydrolysis of proteins.
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