The investigation of structure–function relationships in enzyme polysaccharide complexes provides a theoretical foundation for modulating enzyme properties and expanding their industrial applications. In this study, the interaction of cysteine proteases—bromelain, ficin, and papain—with a grafted chitosan–poly(N-vinylpyrrolidone) copolymers, Cs-g-PVP, was examined, and its effect on the catalytic and stability properties of the enzymes was assessed. Molecular docking and Fourier-transform infrared spectroscopy were used to analyze the topology of the resulting complexes and identify macromolecular fragments involved in binding. Based on the obtained results, it was hypothesized that complex formation would lead to a slight reduction in the catalytic activity of cysteine proteases. In vitro studies of the complexes confirmed this hypothesis, showing that the enzymes retained more than 63% of their proteolytic activity while their half-inactivation time during storage increased by up to ~12-fold. The investigated Cs-g-PVP copolymers demonstrated high efficiency as supports for the studied enzymes, capable of retaining up to 100% of the added enzymes.

Bleeding remains a leading cause of death in trauma and surgery, which drives the search for effective local hemostatic materials. Chitosan is a promising candidate because of its biocompatibility, biodegradability, and intrinsic hemostatic activity, yet chitosan hydrogels remain poorly characterized in terms of hemocompatibility. This study assessed the hemocompatibility and hemostatic activity of chitosan hydrogels with varying degrees of crosslinking and their complexes with ficin. Hydrogels were synthesized using 1,4-butanediol diglycidyl ether and characterized by ζ-potential measurements. Hemocompatibility was evaluated through hemolysis, hemoglobin spectroscopy, erythrocyte cytoarchitecture, and lymphocyte viability assays. Ficin was immobilized by adsorption and covalent binding, and hemostatic properties were assessed via swelling and kinetic blood coagulation. The hydrogels showed a statistically significant inverse dose-dependent hemolytic effect, attributed to their low colloidal stability. In contrast, hemoglobin spectra, erythrocyte cytoarchitecture, and lymphocyte viability showed no statistically significant changes. Adsorption immobilization provided higher protein loading and proteolytic activity than covalent binding. Ficin-containing compositions significantly accelerated blood coagulation compared with pristine hydrogels. These findings confirm the potential of the developed hydrogels and their ficin complexes as hemocompatible hemostatic materials.

Cysteine proteases (bromelain, ficin, and papain) are widely used in biotechnology and medicine, but their application is limited by rapid autolysis and oxidative inactivation. This study aimed to develop effective supports for these enzymes based on graft copolymers of sodium alginate and poly(N-vinylpyrrolidone) (SA-g-PVP) and to elucidate the structure–property relationships governing immobilization efficiency, catalytic activity, and storage stability. Copolymers were synthesized via radical solution polymerization under optimized conditions. Enzymes were immobilized by physical adsorption, and the resulting complexes were characterized by Fourier-transform infrared (FTIR) spectroscopy, protein content assays, proteolytic and amidase activity measurements, and molecular docking. The graft copolymer with a smaller particle size in solution provided a larger accessible surface area, leading to higher bromelain and papain loading. Ficin showed the opposite trend due to its unique surface amino acid composition. Immobilization dramatically increased storage stability: half-life values for bromelain, ficin, and papain reached up to 20, 14, and 14 days, respectively, compared to 1–3 days for the free enzymes. Molecular docking revealed that the dense polymer shell stabilizes the enzyme tertiary structure by forming multiple contacts with internal cavities and tunnels, thereby preventing autolysis and conformational unfolding. Collectively, these findings demonstrate that SA-g-PVP copolymers are promising, non-toxic supports for cysteine proteases, with ficin showing up to 100% activity recovery, making them suitable for food, cosmetic, and biomedical applications.

Bromelain is a widely used as model for papain-like cysteine proteases and shares significant sequence homology with bacterial and viral counterparts, making it a suitable target for computational screening of potential modulators. In this study, we performed molecular docking and protein–ligand interaction profiling for eight synthetic derivatives containing 6-chloroquinolone (compounds 1–4), 1,3,5 triazine (compounds 5–7) or furopyrrole (8) scaffolds. In the quinoline series, the position of the methyl group on the piperidine ring appeared to influence efficacy (4 methyl > unsubstituted > 2 methyl). Molecular docking and PLIP interaction profiling revealed that the highest computed binding affinities (up to –12.4 kcal/mol for 5) did not directly correlate with experimental inhibition, but identified the residues predicted by docking and PLIP to contribute to ligand interactions: Lys18, Phe29, Ile163, Trp180, Tyr185, and Ile186. Ligands 6 and 7 formed H-bonds with catalytic residues His158 and Cys26, respectively. Sequence alignment revealed 42–49% identity between bromelain and bacterial cysteine proteases from Klebsiella pneumoniae and Acinetobacter baumannii, suggesting that bromelain may serve as a tentative model system for the preliminary study of pathogen cysteine proteases, although direct extrapolation would require experimental validation. To experimentally assess these predictions, we conducted a preliminary activity screen using an azocasein proteolytic assay. Compound 1 exhibited a moderate inhibitory effect, whereas compounds 2–8 produced only marginal changes, which lacked statistical significance after correction for multiple comparisons. These findings provide a structural basis for the rational design of new protease inhibitors and identify compound 1 as a preliminary screening hit and a possible starting point for further optimization.