Immobilization of Pepsin on Different Nanomaterials and Its Application in Obtaining Bioactive Peptides

Enzyme immobilization is frequently used in catalytic processes as an alternative to improve enzyme activity and stability. Among the materials used as a support, nanomaterials have been successfully reported in the literature, mainly due to its high specific area. In this study, the nanomaterials graphene, reduced graphene oxide, boron nitride nanosheets, and gold nanoparticles were evaluated as a support for pepsin immobilization. The effectiveness of this process was evaluated by the content of immobilized protein and activity of immobilized pepsin, determined through casein hydrolysis, as well as circular dichroism to verify possible alterations in enzyme structure. The antioxidant properties of hydrolysates were assessed by DPPH, ABTS, FRAP, and β-carotene. All the materials studied presented a high efficiency of immobilization, with over 80% of the pepsin content bound to the support, the use of graphene derivatives as support led to a reduction in proteolytic activity, with the immobilized pepsin retaining only 6% of its activity. The 45 nm gold nanoparticle led to an increase in proteolytic activity from 0.050 to 0.104 U. No effect of amino group functionalization of boron nitride nanosheet was observed. The variations determined in the secondary structure, especially in the β-sheet content, corroborate the variations in activity after immobilization. The change in casein hydrolysates using pepsin immobilized in nanomaterials can be verified by reversed-phase chromatography. This change in the enzyme conformation affects the antioxidant properties of the casein hydrolysates. The results found in the present study contribute to the development of new biocatalysts based on the immobilization of enzymes in nanomaterials.