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.

Thermostable ficin from jelly fig (Ficus pumila var. awkeotsang) latex: purification, identification and characterization.

BACKGROUND: The achenes/seeds of endemic jelly fig (Ficus pumila var. awkeotsang) fruit have been applied to prepare a traditional beverage in Taiwan. Upon fruit harvest, jelly fig latex exuded from stalks was discarded. Protease activity was monitored in its latex. Proteases capable of hydrolyzing proteins have many application aspects based on diverse characteristics. Commercial plant proteases are frequently from latex. RESULTS: The latex protease of jelly fig, termed FaFicin, was purified to homogeneity with a molecular mass of ~32 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. According to liquid chromatographic-tandem mass spectrometric analyses, the expected protein band of protease was matched to ficin A, ficin B or chymopapain from common fig or papaya. Iodoacetamide, an inhibitor of cysteine protease, inhibited its protease activity completely. Hence FaFicin was identified as a papain-like cysteine protease (PLCP), exhibiting more than 80% and 70% activity as assayed at pH 5-8 and 40-70 °C, respectively. It maintained ~89% of initial activity after 120 min at 55 °C and pH 7. Moreover, FaFicin could degrade the myosin and actin of meat, and clot milk. CONCLUSION: The ficin FaFicin was obtained, purified and identified as a PLCP member from agricultural waste: jelly fig latex. It possessed activity under a wide range of pH values and temperature, and exhibited excellent thermostability. Based on its initial evaluation as a meat tenderizer and milk clotting reagent, the application of FaFicin was possible, which may extend utilization of jelly fig. © 2022 Society of Chemical Industry.

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).

Enhancing the peroxidase activity and decreasing the protease activity of ficin with rational modification and its application to one-step colorimetric detection of glucose.

Ficin has dual enzyme activity, i.e., protease and peroxidase-like activity. In some respects, its application is limited by the protease activity of ficin. Herein, we used tris (2-carboxyethyl) phosphine hydrochloride (TCEP) to break the three pairs of disulfide bonds of ficin, and then blocked the free thiol groups with N-ethylmaleimide (NEM) to synthesize ficin-TN. The results showed that ficin-TN had increased peroxidase-like activity and reduced protease activity. According to this phenomenon, we have exploited a colorimetric method with high sensitivity and selectivity for the one-step detection of glucose. Comparing with ficin, ficin-TN has wider detection range (0.1-300 µM) and lower detection limit (88 nM), and our method is simpler and more timesaving than other two-step methods. Furthermore, the actual appliances of ficin-TN for glucose detection in human serum have been illustrated with satisfied result, suggesting that its promising utilization in various fields.

Broad range of substrate specificities in papain and fig latex enzymes preparations improve enumeration of Listeria monocytogenes.

Numerous applications of proteolytic enzymes include dissociation of fermented meat products for the enumeration of `foodborne pathogenic bacteria. The use of trypsin for this cause is abandoned due to the high concentration of the enzyme affecting released bacteria. Papain, as a suggested replacement, and fig latex preparation with high extent of papain-like enzymes have the potential to be applied for bacteria enumeration. Both enzymatic preparations, originating from papaya and fig, showed a broader range of substrate specificities including gelatinolytic activity, especially prominent in the case of ficin and attributed to both, cysteine protease ficin and serine protease by the analysis of 2D zymography with specific inhibitors. The activity towards native collagen, mild in the case of papain, and extensive in the case of fig latex was proved by structural analysis of digested collagen by infrared spectroscopy. Further exploration of their potential for dissociation of fermented meat products showed that both papain and fig latex enzymes are stable in the presence of detergents Tween 20 and Triton X-100 and effective in the enumeration of Listeria monocytogenes. Gelatenolytic activity, and at least partial collagenolytic activity and stability in procedure conditions make papaya and fig latex proteases potent for this application in significantly lower concentrations than previously used enzymes. As a mixture of proteolytic enzymes with divergent characteristics, fig latex preparation shows higher efficiency in Listeria monocytogenes release than papain, conserved even in the presence of stronger non-ionic detergent Triton X-100.

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.

Synthesis of ficin-protected AuNCs in a droplet-based microreactor for sensing serum ferric ions.

A droplet-based microfluidic synthesis approach for preparation of ficin capped gold nano clusters (AuNCs) was developed. Well dispersed AuNCs could be procured within 8 min. Upon excitation wavelength at 340 nm, the resultant AuNCs exhibited a strong blue fluorescence with the maximum emission at 450 nm. Due to the aggregation-induced "turn-off" fluorescence mechanism, the synthesized AuNCs as a fluorescent probe displayed high sensitivity and good selectivity for sensing ferric ions. The relative fluorescence intensity versus ferric ions concentration yielded a good linear calibration in the range of 10.0-1000.0 µM (R2 = 0.998) and the limit of detection was 4.1 µM. Moreover, the possible mechanism for abated fluorescence intensity of AuNCs by adding ferric ions was discussed briefly. Further, the as-prepared fluorescent AuNCs was successfully applied for the detection of serum ferric ions. The results indicated that the droplet-based microfluidic synthesis system could provide a new way for the rapid preparation of AuNCs with good polydispersity and have potential as the sensing probes for the analysis of ferric ions in real biological samples.

Amination of ficin extract to improve its immobilization on glyoxyl-agarose: Improved stability and activity versus casein.

Ficin extract has been aminated using ethylenediamine and carbodiimide to transform all exposed carboxylic groups into amino groups, retaining around 80% of activity versus benzoyl-d,l-arginine p-nitroanilide hydrochloride (BANA) and 90% versus casein. This aminated enzyme was then immobilized on glyoxyl agarose beads. After optimization of the immobilization protocol (immobilization at pH 10 for just 1 h), the new biocatalyst was compared to that obtained using the non-aminated enzyme. Activity versus BANA was lower, but was higher versus casein. The new biocatalyst was more stable than the reference mainly at pH 7. The new biocatalyst permitted to have a more linear course and a higher hydrolysis yield of casein at 75 °C. Moreover, the activity of the new preparations was significantly higher than the reference or the free enzyme in 8 M urea, at pH 7 and 55 °C. The enzyme in an overloaded biocatalyst exhibited a much higher specific activity versus casein (75% of the low loaded biocatalysts) than the non-aminated enzyme (only 30%), suggesting a more appropriate enzyme orientation that decreased steric hindrances. Finally, the enzyme was reused for 5 cycles of casein hydrolysis at 40 °C and pH 7 without any decrease in enzyme activity.

Competitive method for fluorescent dopamine detection in cerebrospinal fluid based on the peroxidase-like activity of ficin.

Dopamine (DA), a catecholamine neurotransmitter, is considered to be an important indicator for mental diseases detection in the clinic. In this study, a novel fluorescent sensing platform consisting of the ficin-H2O2-tyramine system for determining DA in cerebrospinal fluids (CSF) was established. The proposed method is based on the fact that ficin, a mimetic peroxidase, can catalyze H2O2 decomposition into OH radicals, which can oxidize non-fluorescent tyramine into fluorescent dityramine. When DA was introduced, DA can compete with tyramine for OH and resulting in the oxidation reaction of tyramine inhibited along with the fluorescence intensity of the system decreased, which provides a unique strategy for fluorescence detection of DA. Under optimal conditions, the fluorescence intensity decreased linearly with the DA level over a wide concentration range from 0.05 to 12.0 µM (R2 = 0.995) with a detection limit of 46 nM (3σ/k). More importantly, the proposed sensing approach exhibits high sensitivity, good selectivity and has been successfully applied to DA sensing in complex biological samples, which made it hold great potential for DA determination in chemical and biological analytical applications.

Encapsulation and immobilization of ficin extract in electrospun polymeric nanofibers.

In this work, ficin extract (3.4.22.3, >1.0 u/mg) was immobilized for the first time in poly (vinyl alcohol) (PVA) electrospun nanofibers in order to preserve its tridimensional molecular structure. The analysis of the morphology of the electrospun nanofibers was carried out using scanning electron microscopy (SEM) showing a diameter in the range of 124-194 nm. The interaction of the ficin extract with the nanofibers structure was analyzed by infrared spectroscopy (FTIR) analysis. The immobilization step was achieved through crosslinking involving the exposure to glutaraldehyde (GA) vapor. The enzyme catalytic behavior was followed by ultraviolet spectroscopy (UV) using the Earlanger method using Nα-benzoyl-l-arginine-4-nitroanilide hydrochloride (BAPA) as substrate. The maximum catalytic activity was reached with 1 h of crosslinking, 20% of enzyme loading and pH 8. The immobilized ficin extract showed 92% of the enzyme activity of the crude ficin extract. The enzymatic activity of the immobilized ficin extract was conserved after a total of nine reuse cycles and maintained after being stored for 25 days. Finally, both the glass transition (Tg) and heat of fusion (Hf) were affected by number of enzyme molecules inside the polymeric nanofibers matrix according to the study of the thermal properties by differential scanning calorimetry (DSC).

Enhancing the peroxidase-like activity of ficin via heme binding and colorimetric detection for uric acid.

Ficin, a classical sulfhydryl protease, was found to possess intrinsic peroxidase-like activity. In this paper, we have put forward a novel strategy to improving the peroxidase-like activity of ficin through binding heme. Heme-ficin complexes were successfully obtained by simple one-step syntheticism. The results demonstrated that the catalytic activity and efficiency of heme-ficin complexes were about 1.7 times and 3 times higher than those of native ficin, respectively. Taking advantages of the high peroxidase-like activity, the heme-ficin complexes were used for colorimetric determination of uric acid with a low detection limit of 0.25 µM. Based on the excellent selectivity and sensitivity, we detected the concentration of uric acid in human serum successfully. On the basis of these findings, the heme-ficin complexes are promising for wide applications in various fields. Thus we not only optimized the peroxidase-like activity of the ficin, but also established a new strategy for development of artificial enzyme mimics by mimicking the architecture of the active site in horseradish peroxidase.

Intrinsic Peroxidase-like Activity of Ficin.

Ficin is classified as a sulfhydryl protease isolated from the latex of fig trees. In most cases, a particular enzyme fits a few types of substrate and catalyzes one type of reaction. In this investigation, we found sufficient proofs for the intrinsic peroxidase-like activity of ficin and designed experiments to examine its effectiveness in a variety of scenarios. Ficin can transform peroxidase substrates to colored products in the existence of H2O2. Our results also indicate that the active sites of peroxidase-like activity of ficin are different from that of protease, which reveals that one enzyme may catalyze more than one kind of substrate to perform different types of reactions. On the basis of these findings, H2O2 releasing from MCF-7 cells was detected successfully. Our findings support a wider application of ficin in biochemistry and open up the possibility of utilizing ficin as enzymatic mimics in biotechnology and environmental monitoring.

Ficin-Treated Red Cells Help Identify Clinically Significant Alloantibodies Masked as Reactions of Undetermined Specificity in Gel Microtubes.

Non-specific antibodies or antibodies of undetermined significance (AUS) often pose problems for a blood bank technologist and physician. It is well known that antibodies can weaken and evanesce over time, thus eluding detection by routine blood bank techniques. Special enhancement techniques exist (eg, ficin treatment); however, they are often underutilized due to concerns over expense. Ficin is known to enhance reactivity caused by antibodies in the ABO, Rh, Kidd, Lewis, I, and P blood group systems, while destroying reactivity of antibodies in the Duffy, and MNS blood group systems. Herein, we discuss our protocol for using ficin treatment to determine the specificity of antibodies that would otherwise be classified as AUS. Of the 97 AUS specimens that were treated with ficin, we were able to identify 25 new alloantibodies that would have otherwise been missed without ficin treatment. Thus, we believe our protocol enhances transfusion safety, while minimizing additional workload and cost.

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.

Characterisation of general proteolytic, milk clotting and antifungal activity of Ficus carica latex during fruit ripening.

BACKGROUND: The physiological role of fig latex is to protect the plant from pathogens. Latex is a rich source of proteases, predominantly ficin. Fig latex also contains collagenolytic protease and chitinolytic enzymes. Our aim was to investigate changes in protein composition, enzyme and antifungal activities of fig latex during fruit ripening. RESULTS: Comparison of latex samples in different time periods showed a uniform increase of protein concentration in chronological order. The content of collagenolytic protease did not differ significantly in the latex samples, while the content of ficin decreased. Ficin-specific activity towards casein was the highest at the beginning of fruit development (about 80 U mg(-1)). Specific milk clotting activity increased as well as the abundance of casein band in the clots. Specific chitinolytic activity at the beginning of flowering was 6.5 times higher than the activity in the period when fruits are ripe. Antifungal activity is the most extensive in spring. CONCLUSION: Ficin forms with different casein specificities are present in different proportions during fruit ripening, which is of importance for applications in the dairy industry. The protection mechanism against insects and fungi, which relies on chitinolytic activity, is the most important in the early phases of flowering and is replaced with other strategies over time.

A novel form of ficin from Ficus carica latex: Purification and characterization.

A novel ficin form, named ficin E, was purified from fig tree latex by a combination of cation-exchange chromatography on SP-Sepharose Fast Flow, Thiopropyl Sepharose 4B and fplc-gel filtration chromatography. The new ficin appeared not to be sensitive to thiol derivatization by a polyethylene glycol derivative, allowing its purification. The protease is homogeneous according to PAGE, SDS-PAGE, mass spectrometry, N-terminal micro-sequencing analyses and E-64 active site titration. N-terminal sequencing of the first ten residues has shown high identity with the other known ficin (iso)forms. The molecular weight was found to be (24,294±10)Da by mass spectrometry, a lower value than the apparent molecular weight observed on SDS-PAGE, around 27 kDa. Far-UV CD data revealed a secondary structure content of 22% α-helix and 26% ß-sheet. The protein is not glycosylated as shown by carbohydrate analysis. pH and temperature measurements indicated maxima activity at pH 6.0 and 50 °C, respectively. Preliminary pH stability analyses have shown that the protease conserved its compact structure in slightly acidic, neutral and alkaline media but at acidic pH (<3), the formation of some relaxed or molten state was evidenced by 8-anilino-1-naphtalenesulfonic acid binding characteristics. Comparison with the known ficins A, B, C, D1 and D2 (iso)forms revealed that ficin E showed activity profile that looked like ficin A against two chromogenic substrates while it resembled ficins D1 and D2 against three fluorogenic substrates. Enzymatic activity of ficin E was not affected by Mg(2+), Ca(2+) and Mn(2+) at a concentration up to 10mM. However, the activity was completely suppressed by Zn(2+) at a concentration of 1mM. Inhibitory activity measurements clearly identified the enzyme as a cysteine protease, being unaffected by synthetic (Pefabloc SC, benzamidine) and by natural proteinaceous (aprotinin) serine proteases inhibitors, by aspartic proteases inhibitors (pepstatin A) and by metallo-proteases inhibitors (EDTA, EGTA). Surprisingly, it was well affected by the metallo-protease inhibitor o-phenanthroline. The enzymatic activity was however completely blocked by cysteine proteases inhibitors (E-64, iodoacetamide), by thiol-blocking compounds (HgCl2) and by cysteine/serine proteases inhibitors (TLCK and TPCK). This is a novel ficin form according to peptide mass fingerprint analysis, specific amidase activity, SDS-PAGE and PAGE electrophoretic mobility, N-terminal sequencing and unproneness to thiol pegylation.

Cationic heterooligopeptides by ficain-catalyzed co-oligomerization of lysine and methionine ethylesters.

Oligopeptides are of high importance for various industrial applications, e.g. cosmetical or medical. Homooligomerizations and co-oligomerizations with anionic amino acid esters are well described but a successful synthesis of cationic heterooligopeptides has been missing so far. The present study reports the ficain-catalyzed heterooligomerizations of LysOEt with MetOEt, leading to cationic heterooligopeptides with a yield up to 49.5% (w/w). MALDI-ToF/ToF-MS analyses proved successful syntheses of cationic heterooligopeptides with a DP between 7 and 10 amino acid residues, with the enzyme exhibiting a clear preference for methionine.

A major target for warm immunoglobulin G autoantibodies: the third external loop of Band 3.

BACKGROUND: Rh proteins and the Wr(b) antigen, which results from an interaction between Band 3 and glycophorin A, are the most common targets for warm immunoglobulin (Ig)G autoantibodies. Apart from autoanti-Di(b) , a scarce specificity, IgG warm autoantibodies specific for Band 3 have never been characterized by serologic methods. STUDY DESIGN AND METHODS: Blood samples from 120 patients with autoimmune hemolytic anemia (AIHA) and IgG-coated red blood cells (RBCs) were studied by serologic methods. Some autoantibodies were investigated by immunochemical methods. RESULTS: Autoantibodies against the third external loop of Band 3 have a distinctive pattern of reactivity in that they fail to react after RBC treatment with α-chymotrypsin and pronase, whereas papain, ficin, and trypsin have no effect. Eleven (9%) patients had pure anti-Band 3 autoantibodies. Autoanti-Band 3 antibodies were associated with other specificities in 66 (55%) patients. Immunoprecipitation and rare RBCs from a Wu+ homozygote, known to have an unusual pattern of reactivity after protease treatment, were used to confirm the Band 3 specificity. Treatment with sodium hypochlorite, believed to oxidize the Met residue at Position 559 in the third loop, showed that these autoantibodies were heterogeneous. Most antibodies reacted optimally at 37 °C, but two patients had incomplete cold IgG autoantibodies. Unlike autoantibodies to Rh proteins, warm autoanti-Band 3 activate complement and are almost totally bound to autologous RBCs. CONCLUSION: We describe the first cases of warm IgG autoantibodies specific for the third loop of Band 3. This external loop also appears as a major target in patients with warm antibody AIHA.

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.