Abstract
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•Ficin extract has been immobilized on glyoxyl agarose.•Carbodiimide mediated amination of the immobilized enzyme altered stability and activity.•Glutaraldehyde modification of immobilized ficin increased stability and activity versus casein.•The glutaraldehyde modified enzyme can be used at higher temperature in hydrolysis of casein.
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 66 °C 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.