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 biotechnological formulations of cysteine proteases, immobilized on chitosan. Structure, stability and activity.

Bromelain, papain, and ficin are studied the most for meat tenderization, but have limited application due to their short lifetime. The aim of this work is to identify the adsorption mechanisms of these cysteine proteases on chitosan to improve the enzymes' stability. It is known that immobilization can lead to a significant loss of enzyme activity, which we observed during the sorption of bromelain (protease activity compared to soluble enzyme is 49% for medium and 64% for high molecular weight chitosan), papain (34 and 28% respectively) and ficin (69 and 70% respectively). Immobilization on the chitosan matrix leads to a partial destruction of protein helical structure (from 5 to 19%). Using computer modelling, we have shown that the sorption of cysteine proteases on chitosan is carried out by molecule regions located on the border of domains L and R, including active cites of the enzymes, which explains the decrease in their catalytic activity upon immobilization. The immobilization on chitosan does not shift the optimal range of pH (7.5) and temperature values (60 °C for bromelain and papain, 37-60 °C for ficin), but significantly increases the stability of biocatalysts (from 5.8 times for bromelain to 7.6 times for papain).

Investigating the calcium binding characteristics of black bean protein hydrolysate.

The black bean protein has been widely utilized to prepare hydrolysates with different bioactive properties. Herein, we hydrolyzed the black bean protein to prepare hydrolysate with calcium binding activity and characterized its behavior. Our results showed that ficin was superior in obtaining hydrolysate with calcium binding capacity in comparison with trypsin, alcalase and bromelain. In particular, the optimal capacity of ficin hydrolysate reached 77.54 ± 1.61 µg mg-1, where the optimal hydrolysis conditions of ficin were a temperature of 70 °C, a pH value of 6.2, an enzyme concentration of 1.61% and a time of 3 h. This might be due to high proportions of aspartic acid and glutamic acid (35.59%). Further spectral analysis evidenced the formation of hydrolysate-calcium complexes, demonstrating that the interaction between hydrolysate and calcium ions primarily occur on carboxyl oxygen atoms and amino nitrogen atoms. These findings provide a possible utilization of black bean hydrolysate to serve as a calcium supplement nutraceutical to enhance the absorption and bioavailability.

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.

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.

Assessment of the Inhibitory Effects of Ficin-hydrolyzed Gelatin Derived from Squid (Uroteuthis duvauceli) on Breast Cancer Cell Lines and Animal Model.

Marine novel natural products have been applied for cancer therapy. Enzyme-digested gelatin hydrolysates have proven to serve as promising sources of potent biologically active peptides. Potential anti-breast cancer properties of the extracted Ficin-digesterd gelatin hydrolysate from Indian squid (Uroteuthis duvauceli) was extensively characterized by cellular and animal models. Gelatin was extracted from squid skin, hydrolyzed by Ficin, and characterized by standard physico-chemical methods. Ficin-digested gelatin hydrolysate was used at various doses of 0-0.1 mg/mL for assessment of MCF-7 and MDA-MB-231 breast cancer cells versus HUVEC normal cells. Cytotoxicity, phase-contrast morphological examination, apoptosis/necrosis, clonal-growth, cell-migration, Matrix-metalloproteinases (MMPs) zymography, and Western blotting were used for cellular assessments. For animal studies, breast tumor-induced BALB/c mice received hydrolyzed gelatin regimen, followed by tumor size/growth and immune-histochemical analyses. Significant inhibition of MCF-7 and MDA-MB-231 with no cytotoxicity on HUVEC cells were detected. Apoptosis was increased in cancer cells, as revealed by elevated ratio of cleaved caspase-3 and PARP. MMP-2 and MMP-9 activities in both cancer cells were diminished. In mice, gelatin hydrolysate prevented weight loss, decreased tumor size, induced p53, and down-regulated Ki67 levels. These findings suggest that Ficin-digested gelatin hydrolysate could be a beneficial candidate for novel breast cancer therapies.

Biochemical, immunological and kinetic characterization and partial sequence analysis of a thiol proteinase inhibitor from Bubalus bubalis kidney: An attempt targeting kidney disorders.

In the present study a thiol proteinase inhibitor was isolated from buffalo kidney making use of ammonium sulphate precipitation and gel filtration chromatography on Sephacryl S-100HR column. Purified inhibitor is homogeneous as it displayed a single band in gel electrophoresis both under reducing and non-reducing environment and is of 65KDa as revealed by gel filtration and SDS PAGE. Kinetic studies revealed the presence of reversible accompanied with competitive mode of inhibition; showing maximum efficacy against papain (Ki=2.90×10-4). It was maximally active at pH 8.0 and was stable for a period of 30, 60 and 90 days at 37, 4 and -20°C respectively. Immunological studies confirmed its purity of epitopes as a single precipitin line is obtained in immunodiffusion. N-terminal analysis revealed that it shared a good homology with mouse kidney cystatin as well as with Human Cys C and Cys E thereby advocating its use as a model for various human oriented studies which targets how the kidney cystatin level varies in accordance with various drugs that are currently being used as a target for variety of diseases.

Structural and functional studies on a variant of cystatin purified from brain of Capra hircus.

Cystatins, known for their ubiquitous presence in mammalian system are thiol protease inhibitors serving important physiological functions. Here, we present a variant of cystatin isolated from brain of Capra hircus (goat) which is glycosylated but lacks disulphide bonds. Caprine brain cystatin (CBC) was isolated using alkaline treatment, ammonium sulphate fractionation (40-60%) and gel filtration chromatography on Sephacryl S-100HR column with an overall yield of 26.29% and 322-fold purification. The inhibitor gave a molecular mass of ~44 kDa as determined by SDS-PAGE and gel filtration behaviour. The Stokes radius and diffusion coefficient of CBC were 27.14 Å and 8.18 × 10-7 cm2 s-1, respectively. Kinetic data revealed that CBC inhibited thiol proteases reversibly and competitively, with the highest inhibition towards papain (Ki = 4.10 nM) followed by ficin and bromelain. CBC possessed 34.7% α-helical content as observed by CD spectroscopy. UV, fluorescence, CD and FTIR spectroscopy revealed significant conformational change upon CBC-papain complex formation. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic parameters - ΔH, ΔS, ΔG along with N (binding stoichiometry) for CBC-papain complex formation. Binding stoichiometry (N = .97 ± .07 sites) for the CBC-papain complex indicates that cystatin is surrounded by nearly one papain molecule. Negative ΔH (-5.78 kcal mol-1) and positive ΔS (11.01 cal mol-1 deg-1) values suggest that the interaction between CBC and papain is enthalpically as well as entropically favoured process. The overall negative ΔG (-9.19 kcal mol-1) value implies a spontaneous CBC-papain interaction.

Surface Plasmon Resonance Imaging biosensor for cystatin determination based on the application of bromelain, ficin and chymopapain.

A Surface Plasmon Resonance Imaging (SPRI) sensor based on bromelain or chymopapain or ficin has been developed for specific cystatin determination. Cystatin was captured from a solution by immobilized bromelain or chymopapain or ficin due to the formation of an enzyme-inhibitor complex on the biosensor surface. The influence of bromelain, chymopapain or ficin concentration, as well as the pH of the interaction on the SPRI signal, was investigated and optimized. Sensor dynamic response range is between 0-0.6 µg/ml and the detection limit is equal to 0.1 µg/ml. In order to demonstrate the sensor potential, cystatin was determined in blood plasma, urine and saliva, showing good agreement with the data reported in the literature.