Imobilização de alcalase® em carcopol 996 incorporado com extrato de romã

Abstract

Alcalase® is a serine protease-type proteolytic enzyme capable of degrading adhesins and biofilms produced by some gram-negative bacteria. In addition, it has hydrolytic activity on proteins, being efficient in breaking peptide bonds. Although it has remarkable catalytic activity, its application in soluble form faces challenges, such as low operational stability and susceptibility to denaturation in environments with variations in pH or in the presence of solvents. These limitations compromise its economic viability in industrial processes. In this context, enzyme immobilization emerges as an effective strategy to improve enzyme activity and stability in commercial applications. Polyacrylic acid, also known as Carbopol, is a biodegradable, skin-biocompatible, non-toxic and stable polymer that can be used in the formulation of pharmaceutical products and as a support for enzyme immobilization. Other molecules of interest for immobilization include phenols, tannins, flavonoids and terpenes, present in the commercial extract of Punica granatum L., also known as pomegranate. Polyphenols and tannins have antimicrobial activity, while flavonoids and terpenes have anti-inflammatory action. Antimicrobial activity contributes to the treatment of infected wounds and can accelerate the healing process. Given the increasing resistance to synthetic antibiotics, this work proposes the immobilization of the enzyme Alcalase® in Carbopol 996, incorporating the commercial pomegranate extract into the produced Carbopol 996 gel. Currently, enzymatic treatment of wounds and the use of medicinal plant extracts are used separately in the healing process. Thus, the combination of enzyme immobilization with the incorporation of pomegranate extract represents an innovative strategy. The present study performed enzymatic activity assays, electrophoresis, quantification of total phenols, Fourier transform infrared spectroscopy (FTIR), determination of zeta potential and viscosity analysis. Despite the presence of protease inhibitors in the pomegranate extract, the immobilized bioproduct maintained its enzymatic activity 0.030 ± 0.002 U/mL in the precipitate and 0.003 ± 0.001 U/mL in the supernatant, indicating that the immobilization process was successful. In addition, the pomegranate extract preserved its phenolic activity after being incorporated into the immobilization gel containing the enzyme. FTIR analysis demonstrated that there was no loss of the main functional groups of the substances used in the immobilization process. The rheological characterization indicated that the gel presents non-Newtonian behavior of the pseudoplastic type. Therefore, the study achieved its objectives by performing enzyme immobilization and incorporation of commercial pomegranate extract into Carbopol 996 gel, preserving the essential characteristics of both components. The results suggest that the bioproduct obtained has potential as an alternative in the treatment of wounds, although additional tests are needed to evaluate the release of the active components and their healing efficacy.