Immobilization of Alcalase on Silica Supports Modified with Carbosilane and PAMAM Dendrimers

Enzyme immobilization is a powerful strategy for enzyme stabilization and recyclability. Materials covered with multipoint molecules are very attractive for this goal, since the number of active moieties to attach the enzyme increases with respect to monofunctional linkers. This work evaluates different dendrimers supported on silica to immobilize a protease enzyme, Alcalase. Five different dendrimers were employed: two carbosilane (CBS) dendrimers of different generations (SiO₂-G₀Si-NH₂ and SiO₂-G₁Si-NH₂), a CBS dendrimer with a polyphenoxo core (SiO₂-G₁O₃-NH₂), and two commercial polyamidoamine (PAMAM) dendrimers of different generations (SiO₂-G₀PAMAM-NH₂ and SiO₂-G₁PAMAM-NH₂). The results were compared with a silica support modified with a monofunctional molecule (2-aminoethanethiol). The effect of the dendrimer generation, the immobilization conditions (immobilization time, Alcalase/SiO₂ ratio, and presence of Ca²⁺ ions), and the digestion conditions (temperature, time, amount of support, and stirring speed) on Alcalase activity has been evaluated. Enzyme immobilization and its activity were highly affected by the kind of dendrimer and its generation, observing the most favorable behavior with SiO₂-G₀PAMAM-NH₂. The enzyme immobilized on this support was used in two consecutive digestions and, unlike CBS supports, it did not retain peptides released in the digestion.