Complexation of Bromelain, Ficin, and Papain with the Graft Copolymer of Carboxymethyl Cellulose Sodium Salt and N-Vinylimidazole Enhances Enzyme Proteolytic Activity.

This study investigates the features of interactions between cysteine proteases (bromelain, ficin, and papain) and a graft copolymer of carboxymethyl cellulose sodium salt with N-vinylimidazole. The objective is to understand the influence of this interactions on the proteolytic activity and stability of the enzymes. The enzymes were immobilized through complexation with the carrier. The interaction mechanism was examined using Fourier-transform infrared spectroscopy and flexible molecular docking simulations. The findings reveal that the enzymes interact with the functional groups of the carrier via amino acid residues, resulting in the formation of secondary structure elements and enzyme's active sites. These interactions induce modulation of active site of the enzymes, leading to an enhancement in their proteolytic activity. Furthermore, the immobilized enzymes demonstrate superior stability compared to their native counterparts. Notably, during a 21-day incubation period, no protein release from the conjugates was observed. These results suggest that the complexation of the enzymes with the graft copolymer has the potential to improve their performance as biocatalysts, with applications in various fields such as biomedicine, pharmaceutics, and biotechnology.

Novel Biocatalysts Based on Bromelain Immobilized on Functionalized Chitosans and Research on Their Structural Features.

Enzyme immobilization on various carriers represents an effective approach to improve their stability, reusability, and even change their catalytic properties. Here, we show the mechanism of interaction of cysteine protease bromelain with the water-soluble derivatives of chitosan-carboxymethylchitosan, N-(2-hydroxypropyl)-3-trimethylammonium chitosan, chitosan sulfate, and chitosan acetate-during immobilization and characterize the structural features and catalytic properties of obtained complexes. Chitosan sulfate and carboxymethylchitosan form the highest number of hydrogen bonds with bromelain in comparison with chitosan acetate and N-(2-hydroxypropyl)-3-trimethylammonium chitosan, leading to a higher yield of protein immobilization on chitosan sulfate and carboxymethylchitosan (up to 58 and 65%, respectively). In addition, all derivatives of chitosan studied in this work form hydrogen bonds with His158 located in the active site of bromelain (except N-(2-hydroxypropyl)-3-trimethylammonium chitosan), apparently explaining a significant decrease in the activity of biocatalysts. The N-(2-hydroxypropyl)-3-trimethylammonium chitosan displays only physical interactions with His158, thus possibly modulating the structure of the bromelain active site and leading to the hyperactivation of the enzyme, up to 208% of the total activity and 158% of the specific activity. The FTIR analysis revealed that interaction between N-(2-hydroxypropyl)-3-trimethylammonium chitosan and bromelain did not significantly change the enzyme structure. Perhaps this is due to the slowing down of aggregation and the autolysis processes during the complex formation of bromelain with a carrier, with a minimal modification of enzyme structure and its active site orientation.

Novel Immobilized Biocatalysts Based on Cysteine Proteases Bound to 2-(4-Acetamido-2-sulfanilamide) Chitosan and Research on Their Structural Features.

Briefly, 2-(4-Acetamido-2-sulfanilamide) chitosan, which is a chitosan water-soluble derivative, with molecular weights of 200, 350, and 600 kDa, was successfully synthesized. The immobilization of ficin, papain, and bromelain was carried out by complexation with these polymers. The interaction mechanism of 2-(4-acetamido-2-sulfanilamide) chitosan with bromelain, ficin, and papain was studied using FTIR spectroscopy. It was found that the hydroxy, thionyl, and amino groups of 2-(4-acetamido-2-sulfanilamide) chitosan were involved in the complexation process. Molecular docking research showed that all amino acid residues of the active site of papain formed hydrogen bonds with the immobilization matrix, while only two catalytically valuable amino acid residues took part in the H-bond formation for bromelain and ficin. The spectral and in silico data were in good agreement with the catalytic activity evaluation data. Immobilized papain was more active compared to the other immobilized proteases. Moreover, the total and specific proteolytic activity of papain immobilized on the carrier with a molecular weight of 350 kDa were higher compared to the native one due to the hyperactivation. The optimal ratio of protein content (mg × g -1 of carrier), total activity (U × mL-1 of solution), and specific activity (U × mg-1 of protein) was determined for the enzymes immobilized on 2-(4-acetamido-2-sulfanilamide) chitosan with a molecular weight of 350 kDa.

Chitosan Graft Copolymers with N-Vinylimidazole as Promising Matrices for Immobilization of Bromelain, Ficin, and Papain.

This work aims to synthesize graft copolymers of chitosan and N-vinylimidazole (VI) with different compositions to be used as matrices for the immobilization of cysteine proteases-bromelain, ficin, and papain. The copolymers are synthesized by free radical solution copolymerization with a potassium persulfate-sodium metabisulfite blend initiator. The copolymers have a relatively high frequency of grafting and yields. All the synthesized graft copolymers are water-soluble, and their solutions are characterized by DLS and laser Doppler microelectrophoresis. The copolymers are self-assembled in aqueous solutions, and they have a cationic nature and pH-sensitivity correlating to the VI content. The FTIR data demonstrate that synthesized graft copolymers conjugate cysteine proteases. The synthesized copolymer adsorbs more enzyme macromolecules compared to non-modified chitosan with the same molecular weight. The proteolytic activity of the immobilized enzymes is increased up to 100% compared to native ones. The immobilized ficin retains up to 97% of the initial activity after a one-day incubation, the immobilized bromelain retains 69% of activity after a 3-day incubation, and the immobilized papain retains 57% of the initial activity after a 7-day incubation. Therefore, the synthesized copolymers can be used as matrices for the immobilization of bromelain, ficin, and papain.

Second derivative analysis of synthesized spectra for resolution and identification of overlapped absorption bands of amino acid residues in proteins: Bromelain and ficin spectra in the 240-320 nm range.

We establish the origin and formation of peaks in UV absorption spectra of proteins by applying the second derivative analysis to (i) spectra of the native protein, (ii) to its model spectra "synthesized" as a sum of partial free amino acid spectra and (iii) to absorption spectra of the free amino acids. We show that the bromelain peaks at 248.2, 253.2, 258.4 and 264.2 nm are due to phenylalanine maxima; the predictable peak at 279.6 nm (which is almost coincident with the extremum of the zero-order spectrum at 279.4 nm) is mainly due to tyrosine maximum, while the peaks at 274.6 and 290.6 nm are due to tryptophan maximum; 268.0 nm peak to the superposition of tyrosine and phenylalanine maxima, and 283.4 nm peak to the superposition of tyrosine and tryptophan maxima. Similar results are obtained for ficin: the peaks at 248.4, 253.0 and 258.8 nm are formed by the phenylalanine maxima, the predictable peak at 264.4 nm accords with the corresponding bromelain 264.2 nm peak; the 279.4 nm peak almost coincides with the zero order spectrum peak (279.6 nm), but it is expressed stronger than that of bromelain due to a different ratio of tyrosine to tryptophan side groups. The peaks at 273.4 and 290.6 nm are associated with tryptophan, the 268.0 nm peak being mainly due to tyrosine (and fractionally to phenylalanine); and the 283.8 nm peak belongs to tyrosine and, to a greater extent, to tryptophan. We demonstrate that the amino acid residues of tryptophan, tyrosine and phenylalanine undergo correspondingly the largest, intermediate and the lowest positive (red) wavelength shift in the zero-order protein absorption spectrum with respect to the model (synthesized) spectrum. The difference appearing in the positions of the bromelain and ficin absorption band peaks is determined by superposition of relative contributions from amino acid residues. This superposition is resulted from (i) linear combination of amino acid residues spectra and (ii) their different (non-uniform) wavelength shifts as functions of microenvironment of these residues' chromophores. The proposed approach to the analysis of the protein absorption spectra with the help of "synthesized" spectra can be transferred to other objects studied in analytical and organic chemistry of high molecular compounds containing monomer units with various chromophores.