Chitosan, the product of chitin deacetylation, is an excellent candidate for enzyme immobilization purposes. Here we demonstrate that papain, an endolytic cysteine protease (EC: 3.4.22.2) from Carica papaya latex immobilized on the matrixes of medium molecular (200 kDa) and high molecular (350 kDa) weight chitosans exhibits anti-biofilm activity and increases the antimicrobials efficiency against biofilm-embedded bacteria. Immobilization in glycine buffer (pH 9.0) allowed adsorption up to 30% of the total protein (mg g chitosan⁻¹) and specific activity (U mg protein⁻¹), leading to the preservation of more than 90% of the initial total activity (U mL⁻¹). While optimal pH and temperature of the immobilized papain did not change, the immobilized enzyme exhibited elevated thermal stability and 6–7-fold longer half-life time in comparison with the soluble papain. While one-half of the total enzyme dissociates from both carriers in 24 h, this property could be used for wound-dressing materials design with dosed release of the enzyme to overcome the relatively high cytotoxicity of soluble papain. Our results indicate that both soluble and immobilized papain efficiently destroy biofilms formed by Staphylococcus aureus and Staphylococcus epidermidis. As a consequence, papain, both soluble and immobilized on medium molecular weight chitosan, is capable of potentiating the efficacy of antimicrobials against biofilm-embedded Staphylococci. Thus, papain immobilized on medium molecular weight chitosan appears a presumably beneficial agent for outer wound treatment for biofilms destruction, increasing antimicrobial treatment effectiveness.

Plant enzymes such as ficin (EC 3.4.22.3), papain (EC 3.4.22.2) and bromelain (EC 3.4.22.4) are obtained from tropical plants. These biocatalysts belong to thiol proteases, in the active center of which cysteine is contained. Ficin, papain and bromelain have a wide substrate specificity, which provides a demand for their use in various industries. Enzymes in the free state are less commonly used; immobilized biocatalysts are the preferred form. The aim of this work was to determine the optimal concentration of a crosslinking agent in the covalent immobilization of ficin, papain and bromelain on a chitosan matrix. Ficin, papain and bromelain (Sigma) were chosen as objects of study. An acid-soluble chitosan (350 kDa, Bioprogress CJSC) was used as an immobilization carrier. The concentration range of glutaraldehyde (crosslinking agent) ranged from 1 to 25%. Suitable concentrations of glutaraldehyde for covalent immobilization were identified by the optimal ratio of protein content (mg per g of carrier), total activity (in units per ml of solution) and specific activity (in units per mg of protein). It was shown that for covalent immobilization of ficin and bromelain on a chitosan matrix, it is most promising to use 10% glutaraldehyde. For immobilization of papain on chitosan by covalent means, the concentration of glutaraldehyde equal to 20% is optimal.

Immobilized enzymes are the most sought-after preparations in the global market. They are used in medicine, veterinary medicine, the food industry, winemaking and brewing. The simplest method for immobilizing biocatalysts on insoluble carriers is the simple adsorption method. Its advantage is that it preserves the natural conformation of the enzyme, which slightly reduces its catalytic ability compared to the native form. In our study, we carried out the selection of optimal conditions for adsorption immobilization of acid-soluble chitosan (Mr = 350 kDa) enzymes of plant origin (ficin, papain and bromelain) on a matrix. Ficin (EC 3.4.22.3), papain (EC 3.4.22.2) and bromelain (EC 3.4.22.4) (Sigma) were chosen as the objects of study, azocasein (Sigma) was used as a substrate for hydrolysis and an acid-soluble high-molecular-weight chitosan (350 kDa) was used as an immobilization matrix, synthesized by Bioprogress CJSC. Suitable buffer systems for immobilization were identified by the optimal ratio of protein content and total and specific activity. Ficin is immobilized on a chitosan matrix using glycine buffer with a pH of 8.6. Glycine buffer with a pH of 8.6–10.5 is an optimal medium for sorption of papain on chitosan. Bromelain is immobilized on a chitosan matrix under Tris-glycine buffer with pH 8.5 conditions.

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.

Thermotoga maritima beta-fructosidases are enzymes that release beta-D-fructose from sucrose, raffinose, and fructan polymers such as inulin. The surfaces of beta-fructosidases 1UYP and 1W2T from Thermotoga maritima were studied in this work. It was showed that amino acids are not distributed equally on the surfaces of the enzymes. Several clusters of charged and hydrophobic residues were detected at pH 7.0. Such clusters were detected by calculation of the distances between them. It was determined that on surfaces of beta-fructosidases PDB ID: 1UYP and PDB ID: 1W2T, 96% and 95% of charged amino acids and also 50% and 42% of hydrophobic amino acids form clusters, respectively. Six clusters of charged amino acids on the surface of beta-fructosidase 1UYP and five clusters on the surface of beta-fructosidase 1W2T were detected. The composition of such clusters is presented. Both types of beta-fructosidase have three clusters of hydrophobic amino acids on their surface. These facts should be considered when choosing immobilization conditions. It was shown that a charged matrix is more promising for the immobilization of beta-fructosidases 1UYP and 1W2T from Thermotoga maritima due to the possibility of binding without any significant loss of activity due to their overlapping active center. Hydrophobic carriers are less promising due to the probable active site overlap. Such binding may have a loss of enzyme activity as a result.

Biopolymer-based hydrogels are promising platforms for hemostatic applications, yet most chitosan-based materials rely on passive plug formation rather than activation of the natural coagulation cascade. This study aimed to develop hemostatic hydrogels based on quaternized chitosan that activate the coagulation cascade through immobilized ficin. N-(2-Hydroxypropyl)-3-trimethylammonium chitosan (HTCC) with varying degrees of quaternization was synthesized and crosslinked with 1,4-butanediol diglycidyl ether to obtain Cross-HTCC hydrogels. The hydrogels were characterized by FTIR spectroscopy, ζ-potential measurements, and swelling tests. Ficin was immobilized by adsorption, and the effects of pH, contact time, and protein content on immobilization efficiency and proteolytic activity were examined. Ficin release and whole-blood coagulation were also assessed. The hydrogels exhibited ζ-potential values above 30 mV and swelling ratios of up to 28.9-fold. Ficin immobilization was optimal at pH 7.4, with maximum sorption reached after ~180 min. Ficin interacted with heterogeneous sorption sites through hydrogen bonding, electrostatic, and hydrophobic interactions, as confirmed by isotherm fitting, FTIR analysis, and molecular dynamics simulations. The immobilized enzyme was released within 24 h and significantly accelerated blood clotting compared with ficin-free hydrogels. These findings demonstrate that ficin-loaded Cross-HTCC hydrogels combine efficient enzyme loading with enhanced hemostatic performance and hold promise as multifunctional hemostatic materials.