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).

Ficin: A protease extract with relevance in biotechnology and biocatalysis.

Due to the problems raised by the use of animal or microbial recombinant proteases, the use of proteases from vegetable origin is becoming increasingly popular.. Among them, sulfidryl proteases have a special interest. Ficin is an outstanding example of this kind of proteases. This paper aims to be to make a comprehensive review of the recent uses of this enzyme, including for example protein hydrolysis, the production of bioactive peptides and antibodies fragments (researchers point that ficin results are more reproducible than using other proteases), meat tenderization, milk coagulations in cheese making or peptide synthesis. Efforts to get industrial immobilized biocatalysts of the enzyme will be also described. The review shows the huge potential and brilliant prospect that this enzyme can have in the near future.

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.

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.

Use of standardized protease enzymes for antibody screening of blood donor samples with the microplate system AutoAnalyzer.

Papain, bromelin and ficin can be standardized by the casein method for use in red blood cell antibody screening tests. The minimal and optimal enzyme activity for detecting blood group antibodies in donors, using the new Automated Pre-Transfusion Blood Testing System Olympus PK7200 by the two-stage method for all three enzymes, is one casein unit. One casein unit of proteinase activity changed the red blood cell surface charge to a low plateau value as measured by electrophoretic mobility and sialic acid content, and removed sterically inhibiting structures of surface protein as detected by SDS-PAGE.

A new enzymatic method to obtain high-yield F(ab)2 suitable for clinical use from mouse IgGl.

Four different proteases were screened for their capability of selectively digesting murine monoclonal IgGl to obtain active F(ab)2. For the screening, a series of five different mouse monoclonal antibodies (IgGl, k) was used, recognizing different tumor-associated antigens and currently used for radioimmunoimaging studies. The enzymes (pepsin, bromelain, ficin and elastase) showed different fragmentation capability and the fragments obtained showed different stability and immunoreactivity. No digestion was noticed using elastase. Pepsin gave discontinuous results, in that its activity ranged from reduction of IgG to small inactive fragments to an inability to digest the immunoglobulin. Pepsin activity was strongly pH-dependent and immunoreactivity of the obtained fragments was not always conserved. Bromelain and, in particular, ficin gave excellent results. Digestion was always rapid and stable, all five MAbs were reduced to F(ab)2 in a comparable time range and with high yields. Moreover, ficin-obtained F(ab)2 showed a highly conserved immunoreactivity. Therefore, ficin was selected as the murine monoclonal IgGl digestion enzyme to obtain active bivalent antibody fragments. The digestion procedure gave a uniform result for all five different MAbs and was easily scaled up to produce hundreds of milligrams of F(ab)2.

Differences in substrate specificities for cysteine proteinases used in blood group serology, and the use of bromelain in a two-phase inhibitor technique.

Because substrate specificities differ between proteolytic enzymes and because knowledge of the optimal enzyme activity levels is necessary in order to standardize procedures used in antibody screening, a study was made of the best common assay method for the routinely employed enzymes bromelain, papain and ficin. Casein degradation was found better suited to this purpose than azoalbumin. With standardization achieved, a useful two-phase bromelain inhibitor technique was devised using bromelain at 20 casein units of activity. This method improved upon the one-stage bromelain technique in terms of sensitivity, freedom from false positive reactions and it compared well with the two-phase papain inhibitor technique.

Structure-activity relationship of the ficin hydrolysis of phenyl hippurates. Comparison with papain, actinidin, and bromelain.

A study of the hydrolysis of 30 substituted-phenyl hippurates by the enzyme ficin has been made. From the results the following quantitative structure--activity relationship (QSAR) has been derived: log 1/Km = 0.79 pi'3 + 0.58 sigma + 0.28 MR4,5 + 3.70. In this expression Km is the Michaelis constant, pi'3 refers to the more hydrophobic of the two meta substituents, and MR4,5 is the molar refractivity of substituents in the 4- and 5-positions of the phenyl ring. This QSAR is compared with those from papain, actinidin, bromelain B, and bromelain D.

Rh0(D) activity of red blood cells and stroma treated with proteolytic enzymes.

Agglutinability of red blood cells against anti-D increased remarkably when the cells were treated with proteolytic enzymes, such as bromelain, chymotrypsin, ficin, papain, pronase and trypsin. When stroma prepared from normal red blood cells was treated with any of proteolytic enzymes, however, Rh-Hr blood type activities were completely abolished. The similar results were obtained from stroma solubilized with detergents which was treated with enzymes after being prepared. Of all the enzymes, ficin acted more slowly than the others did. Neuraminidase or phospholipase A2 had no effect on Rh-Hr activities at all. SDS-polyacrylamide gel electrophoretic pattern of stroma prepared from bromelain, ficin and pronase-treated red blood cells were quite different from that of normal stroma.

Benzyloxycarbonylphenylalanylcitrulline p-nitroanilide as a substrate for papain and other plant cysteine proteinases.

After preliminary assays, with papain, bromelain and ficin, on a range of citrulline p-nitroanilides, values of Km and kcat. for the papain-catalysed hydrolysis of three derivatives, N alpha- benzyloxycarbonylcitrulline p-nitroanilide, benzyloxycarbonylphenylalanylcitrulline p-nitroanilide and benzyloxycarbonylglycylphenylalanylcitrulline p-nitroanilide, were obtained. It is concluded that benzyloxycarbonylphenylalanylcitrulline p-nitroanilide is a highly selective substrate for the sensitive detection and assay of the plant cysteine proteinases.

Comparison of the substrate conformations in the active sites of papain, chymopapain, ficin and bromelain by resonance Raman spectroscopy.

The resonance Raman spectra of several enzyme-substrate intermediates of papain, chymopapain, ficin and bromelain are reported. The intermediates are dithioacyl enzymes formed during the catalyzed hydrolysis of N-acylglycine thionoester substrates. Interpretation of the resonance Raman spectra allows us to compare, for the first time, the substrate geometries in a series of functioning intermediates from different enzymes. The substrates assume essentially identical conformations for papain, chymopapain and ficin and a similar, but not identical, conformation in the active site of bromelain. Each dithioacyl enzyme population appears to be made up of a single homogeneous conformational state. This state has been characterised in earlier studies of dithioacyl papains. It is designated as conformer B and is characterized by an attractive contact between the substrate's glycinic N atom and the active site cysteine S atom. It is now apparent that conformer B is of general significance in the mechanism of cysteine proteases.

Benzofuroxan as a thiol-specific reactivity probe. Kinetics of its reactions with papain, ficin, bromelain and low-molecular-weight thiols.

1. The characteristics of benzofuroxan (benzofurazan 1-oxide, benzo-2-oxa-1,3-diazole N-oxide) that relate to its application as a reactivity probe for the study of environments of thiol groups are discussed. 2. To establish a kinetic and mechanistic basis for its use as a probe, a kinetic study of its reaction with 2-mercaptoethanol was carried out. 3. This reaction appears to proceed by a rate-determining attack of the thiolate ion on one of the electrophilic centres of benzofuroxan (possibly C-6) to provide a low steady-state concentration of an intermediate adduct; rapid reaction of this adduct with a second molecule of thiol gives the disulphide and o-benzoquinone dioxime. 4. The effects of the different types of environment that proteins can provide on the kinetic characteristics of reactions of thiol groups with benzofuroxan are delineated. 5. Benzofuroxan was used as a thiolspecific reactivity probe to investigate the active centres of papain (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4). The results support the concept that the active centres of all three enzymes either contain a nucleophilic thiolate ion whose formation is characterized by a pKa of 3-4 and whose reaction with an electrophile can be assisted by interaction of a site of high electron density in the electrophile with active-centre imidazolium ion of pKa 8-9, or can provide such ions by protonic redistribution in enzyme-reagent or enzyme-substrate complexes.

On the activation of bovine plasma factor XIII. Amino acid sequence of the peptide released by thrombin and the terminal residues of the subunit polypeptides.

A blood coagulation factor, Factor XIII, was highly purified from bovine fresh plasma by a method similar to those used for human plasma Factor XIII. The isolated Factor XIII consisted of two subunit polypeptides, a and b chains, with molecular weights of 79,000 +/- 2,000 and 75,000 +/- 2,000, respectively. In the conversion of Factor XIII to the active enzyme, Factor XIIIa, by bovine thrombin [EC 3.4.21.5], a peptide was liberated. This peptide, designated tentatively as "activation peptide," was isolated by gel-filtration on a Sephadex G-75 column. It contained a total of 37 amino acid residues with a masked N-terminal residue and C-terminal arginine. The whole amino acid sequence of "Activation peptide" was established by the dansyl-Edman method and standard enzymatic techniques, and the masked N-terminal residue was identified as N-acetylserine by using a rat liver acylamino acid-releasing enzyme. This enzyme specifically cleaved the N-acetylserylglutamyl peptide bond serine and the remaining peptide, which was now reactive to 1-dimethylamino-naphthalene-5-sulfonyl chloride. A comparison of the sequences of human and bovine "Activation peptide" revealed five amino acids replacements, Ser-3 to Thr; Gly-5 to Arg; Ile-14 to Val; Thr-18 to Asn, and Pro-26 to Leu. Another difference was the deletion of Leu-34 in the human peptide. Adsorption chromatography on a hydroxylapatite column in the presence of 0.1% sodium dodecyl sulfate was developed as a preparative procedure for the resolution of the two subunit polypeptides, a or a' chain and b chain, constituting the protein molecule of Factor XIII or Factor XIIIa. End group analyses on the isolated pure chains revealed that the structural change of Factor XIII during activation with thrombin occurs only in the N-terminal portion of the a chain, not in the N-terminal end of the b chain or in the C-terminal ends of the a and b chains. From these results, it was concluded that the activation of bovine plasma Factor XIII by thrombin must be accompanied by a limited proteolysis of the arginyl-glycyl bond located in the N-terminal region of the a chain, liberating the "Activation peptide." The possibility of activating Factor XII with other porteinases was examined using Factor Xa [EC 3.4.21.6], Factor XIIa, kallikreins [EC 3.4.21.8], urokinase [EC 3.4.99.26], trypsin [EC 3.4.21.4], ficin [EC 3.4.22.3], papain [EC 3.4.22.2], and bromelain [EC 3.4.22.4]. Among these enzymes, only bromelain and trypsin showed clear activating effects.