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RevistaINTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
Año2019
Volumen133
Páginas412-419
Internacional

AMINATION OF FICIN EXTRACT TO IMPROVE ITS IMMOBILIZATION ON GLYOXYL-AGAROSE: IMPROVED STABILITY AND ACTIVITY VERSUS CASEIN

Autores:Roberto Fernandez-Lafuente
Grupos de investigación:Optimización de biocatalizadores y bioprocesos enzimáticos
El-HocineSiar1,2, +, Roberto Morellon-Sterling1,+,  Mohammed Nasreddine Zidoune2, Roberto Fernandez-Lafuente1
1  Departamento de Biocatálisis, Instituto de Catálisis-CSIC, Campus UAM-CSIC, 28049 Madrid, Spain
2           Equipe TEPA, LaboratoireLNTA, INATAA, Université des Frères Mentouri Constantine 1, 25000 Constantine, Algeria
 
 
Ficin extract has been aminated using ethylenediamine and carbodiimide to transform all exposed carboxylic groups into amino groups, retaining around 80% of activity versus benzoyl-D,L-arginine p-nitroanilide hydrochloride (BANA) and 90% versus casein. This aminated enzyme was then immobilized on glyoxyl agarose beads. After optimization of the immobilization protocol (immobilization at pH 10 for just 1 h), the new biocatalyst was compared to that obtained using the non-aminated enzyme. Activity versus BANA was lower, but was higher versus casein. The new biocatalyst was more stable than the reference mainly at pH 7. The new biocatalyst permitted to have a more linear course and a higher hydrolysis yield of casein at 75ºC. Moreover, the activity of the new preparations was significantly higher than the reference or the free enzyme in 8 M urea, at pH 7 and 55ºC. The enzyme in an overloaded biocatalyst exhibited a much higher specific activity versus casein (75% of the low loaded biocatalysts) than the non-aminated enzyme (only 30%), suggesting a more appropriate enzyme orientation that decreased steric hindrances. Finally, the enzyme was reused for 5 cycles of casein hydrolysis at 40ºC and pH 7 without any decrease in enzyme activity.
 
Palabras clave:multipoint covalent attachment, enzyme orientation, : enzyme amination, casein hydrolysis, steric hindrances
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