Anti-biofilm and wound-healing activity of chitosan-immobilized Ficin.

Biofouling is among the key factors slowing down healing of acute and chronic wounds. Here we report both anti-biofilm and wound-healing properties of the chitosan-immobilized Ficin. The proposed chitosan-adsorption approach allowed preserving ~90% of the initial total activity of the enzyme (when using azocasein as a substrate) with stabilization factor of 4.9, and ~70% of its specific enzymatic activity. In vitro, the chitosan-immobilized Ficin degraded staphylococcal biofilms, this way increasing the efficacy of antimicrobials against biofilm-embedded bacteria. In vivo, in the presence of Ficin (either soluble or immobilized), the S.aureus-infected skin wound areas in rats reduced twofold after 4 instead of 6 days treatment. Moreover, topical application of the immobilized enzyme resulted in a 3-log reduction of S. aureus cell count on the wound surfaces in 6 days, compared to more than 10 days required to achieve the same effect in control. Additional advantages include smoother reepithelisation, and new tissue formation exhibiting collagen structure characteristics closely reminiscent of those observed in the native tissue. Taken together, our data suggest that both soluble and immobilized Ficin appear beneficial for the treatment of biofilm-associated infections, as well as speeding up wound healing and microbial decontamination.

Influence of UV radiation on molecular structure and catalytic activity of free and immobilized bromelain, ficin and papain.

Our research has shown that the degree of photosensitivity of the cysteine proteases can be arranged in the following order: bromelain → ficin → papain. After the UV irradiation with 151 J·m-2 intensity of a bromelain solution, the enzyme activity has increased. No decrease in the catalytic capacity and the change in the size of the molecule was recorded in the 151-6040 J·m-2 range of irradiation intensities. A decrease in the catalytic capacity of ficin and the increase of its globule size occurred after exposure to a radiation of 3020 J·m-2 intensity. The decrease in papain activity was observed at the UV irradiation intensity of 453 J·m-2, and an increase of the papain globule size was detected at 755 J·m-2. Immobilization on chitosan matrix leads to the increase in the stability of heterogeneous biocatalysts with respect to UV irradiation in comparison with free enzymes. The changes in IR spectra of immobilized cysteine proteases practically do not affect the bands due to the protein component of the system: amide I, amide II, amide III. Therefore, it can be postulated that the chitosan matrix acts as photoprotector for immobilized ficin, bromelain and papain. The obtained results can be helpful for development of drugs based on chitosan and cysteine proteases in combination with phototherapy, as well as for choosing their sterilization conditions.