Amination of ficin extract to improve its immobilization on glyoxyl-agarose: Improved stability and activity versus casein

Abstract

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 75 °C. 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 55 °C. 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 40 °C and pH 7 without any decrease in enzyme activity.