Investigation of the peptides with calcium chelating capacity in hydrolysate derived from spent hen meat.

Calcium peptide chelates are developed as efficient supplements for preventing calcium deficiency. Spent hen meat (SHM) contains a high percentage of proteins but is generally wasted due to the disadvantages such as hard texture. We chose the underutilized SHM to produce peptides to bind calcium by proteolysis and aimed to investigate chelation between calcium and peptides in hydrolysate for a sustainable purpose. The optimized proteolysis conditions calculated from the result of response surface methodology for two-step hydrolysis were 0.30% (wenzyme/wmeat) for papain with a hydrolysis time of 3.5 h and 0.18% (wenzyme/wmeat) for flavourzyme with a hydrolysis time of 2.8 h. The enzymatic hydrolysate (EH) showed a binding capacity of 63.8 ± 1.8 mg calcium/g protein. Ethanol separation for EH improved the capacity up to a higher value of 68.6 ± 0.6 mg calcium/g protein with a high association constant of 420 M-1 (25°C) indicating high stability. The separated fraction with a higher amount of Glu, Asp, Lys, and Arg had higher calcium-binding capacity, which was related to the number of ─COOH and ─NH2 groups in peptide side chains according to the result from amino acid analysis and Fourier transform infrared spectroscopy. Two-step enzymatic hydrolysis and ethanol separation were an efficient combination to produce peptide mixtures derived from SHM with high calcium-binding capacity. The high percentage of hydrophilic amino acids in the separated fraction was concluded to increase calcium-binding capacity. This work provides foundations for increasing spent hen utilization and developing calcium peptide chelates based on underutilized meat.

Comparison of prediction models for soy protein isolate hydrolysates bitterness built using sensory, spectrofluorometric and chromatographic data from varying enzymes and degree of hydrolysis.

The bitterness of soy protein isolate hydrolysates prepared using five proteases at varying degree of hydrolysis (DH) and its relation to physicochemical properties, i.e., surface hydrophobicity (H0), relative hydrophobicity (RH), and molecular weight (MW), were studied and developed for predictive modelling using machine learning. Bitter scores were collected from sensory analysis and assigned as the target, while the physicochemical properties were assigned as the features. The modelling involved data pre-processing with local outlier factor; model development with support vector machine, linear regression, adaptive boosting, and K-nearest neighbors algorithms; and performance evaluation by 10-fold stratified cross-validation. The results indicated that alcalase hydrolysates were the most bitter, followed by protamex, flavorzyme, papain, and bromelain. Distinctive correlation results were found among the physicochemical properties, influenced by the disparity of each protease. Among the features, the combination of RH-MW fitted various classification models and resulted in the best prediction performance.

Characterization of the structure, antioxidant activity and hypoglycemic activity of soy (Glycine max L.) protein hydrolysates.

This study aimed to hydrolyze soy isolate protein (SPI) using five enzymes (alcalase, pepsin, trypsin, papain, and bromelain) in order to obtain five enzymatic hydrolysates and to elucidate the effect of enzymes on structural and biological activities of the resulting hydrolysates. The antioxidant and hypoglycemic activities of the soy protein isolate hydrolysates (SPIEHs) were evaluated through in silico analysis, revealing that the alcalase hydrolysate exhibited the highest potential, followed by the papain and bromelain hydrolysates. Subsequently, the degree of hydrolysis (DH), molecular weight distribution (MWD), amino acid composition, structure, antioxidant activities, and hypoglycemic activity in vitro of SPIEHs were analyzed. After enzymatic treatment, the particle size, polymer dispersity index (PDI), ζ-potentials, ß-sheet content and α-helix content of SPIEHs was decreased, and the maximum emission wavelength of all SPIEHs exhibited red-shifted, which all suggesting the structure of SPIEHs was unfolded. More total amino acids (TAAs), aromatic amino acids (AAAs), and hydrophobic amino acids (HAAs) were found in alcalase hydrolysate. For 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, metal ion chelating activity, α-glucosidase inhibitory activity and α-amylase inhibitory activity, alcalase hydrolysate had the lowest IC50; alcalase hydrolysate and papain hydrolysate had the lowest IC50 for hydroxyl radical scavenging activity. Physiological activity of SPIEHs was evaluated thoroughly by 5-Axe cobweb charts, and the results revealed that alcalase hydrolysate exhibited the greatest biological activities.

Therapeutic potential of metal ions for COVID-19: insights from the papain-like protease of SARS-CoV-2.

Coronaviruses have been responsible for multiple challenging global pandemics, including coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Papain-like protease (PLpro), one of two cysteine proteases responsible for the maturation and infectivity of SARS-CoV-2, processes and liberates functional proteins from the viral polyproteins and cleaves ubiquitin and ISG15 modifications to inhibit innate immune sensing. Consequently, PLpro is an attractive target for developing COVID-19 therapies. PLpro contains a zinc-finger domain important for substrate binding and structural stability. However, the impact of metal ions on the activity and biophysical properties of SARS-CoV-2 PLpro has not been comprehensively studied. Here, we assessed the impacts of metal ions on the catalytic activity of PLpro. Zinc had the largest inhibitory effect on PLpro, followed by manganese. Calcium, magnesium, and iron had smaller or no effects on PLpro activity. EDTA at a concentration of 0.5 mM was essential for PLpro activity, likely by chelating trace metals that inhibit PLpro. IC50 values for ZnCl2, ZnSO4, and MnCl2 of 0.42 ± 0.02 mM, 0.35 ± 0.01 mM, and 2.6 ± 0.3 mM were obtained in the presence of 0.5 mM EDTA; in the absence of EDTA, the estimated IC50 of ZnCl2 was 14 µM. Tryptophan intrinsic fluorescence analysis confirmed the binding of zinc and manganese to PLpro, and differential scanning calorimetry revealed that zinc but not manganese reduced ΔHcal of PLpro. The results of this study provide a reference for further work targeting PLpro to prevent and treat COVID-19.

The effects of plant cysteine proteinases on the nematode cuticle.

BACKGROUND: Plant-derived cysteine proteinases of the papain family (CPs) attack nematodes by digesting the cuticle, leading to rupture and death of the worm. The nematode cuticle is composed of collagens and cuticlins, but the specific molecular target(s) for the proteinases have yet to be identified. METHODS: This study followed the course of nematode cuticle disruption using immunohistochemistry, scanning electron microscopy and proteomics, using a free-living nematode, Caenorhabditis elegans and the murine GI nematode Heligmosomoides bakeri (H. polygyrus) as target organisms. RESULTS: Immunohistochemistry indicated that DPY-7 collagen is a target for CPs on the cuticle of C. elegans. The time course of loss of DPY-7 from the cuticle allowed us to use it to visualise the process of cuticle disruption. There was a marked difference in the time course of damage to the cuticles of the two species of nematode, with H. bakeri being more rapidly hydrolysed. In general, the CPs' mode of attack on the nematode cuticle was by degrading the structural proteins, leading to loss of integrity of the cuticle, and finally death of the nematode. Proteomic analysis failed conclusively to identify structural targets for CPs, but preliminary data suggested that COL-87 and CUT-19 may be important targets for the CPs, the digestion of which may contribute to cuticle disruption and death of the worm. Cuticle globin was also identified as a cuticular target. The presence of more than one target protein may slow the development of resistance against this new class of anthelmintic. CONCLUSIONS: Scanning electron microscopy and immunohistochemistry allowed the process of disruption of the cuticle to be followed with time. Cuticle collagens and cuticlins are molecular targets for plant cysteine proteinases. However, the presence of tyrosine cross-links in nematode cuticle proteins seriously impeded protein identification by proteomic analyses. Multiple cuticle targets exist, probably making resistance to this new anthelmintic slow to develop.

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).

Extraction, anti-tyrosinase, and antioxidant activities of the collagen hydrolysate derived from Rhopilema hispidum.

The study was conducted to determine anti-tyrosinase and antioxidant activities of the extracted collagen hydrolysate (CH) derived from Malaysian jellyfish, Rhopilema hispidum. Collagen was extracted using 1:1 (w:v) 0.1 M NaOH solution at temperature 25 °C for 48 hr followed by treatment of 1:2 (w:v) distilled water for another 24 hr and freeze-dried. The extracted collagen was hydrolyzed using papain at optimum temperature, pH and enzyme/substrate ratio [E/S] of 60 °C, 7.0 and 1:50, respectively. CH was found to exhibit tyrosinase inhibitory activity, DPPH radical scavenging and metal ion-chelating assays up to 64, 28, and 83%, respectively, after 8 hr of hydrolysis process. The molecular weight of CH was found <10 kDa consisting of mainly Gly (19.219%), Glu (10.428%), and Arg (8.848%). The UV-visible spectrum analysis showed a major and minor peak at 218 and 276 nm, accordingly. The FTIR spectroscopy confirmed the amide groups in CH. The SEM images demonstrated spongy and porous structure of CH. In the cytotoxicity study, CH has no cytotoxicity against mouse embryonic 3T3 fibroblast cell line with IC50 value >500 µg/ml. Results revealed that the CH generated from this study has a potential to be developed as active ingredient in cosmeceutical application.

Production and Characterization of Antioxidative Hydrolysates and Peptides from Corn Gluten Meal Using Papain, Ficin, and Bromelain.

There has been a growing interest in developing natural antioxidants with high efficiency and low cost. Bioactive protein hydrolysates could be a potential source of natural and safer antioxidants. The objectives of this study were to hydrolyze corn gluten meal using three plant-derived proteases, namely papain, ficin, and bromelain, to produce antioxidative hydrolysates and peptides and to characterize the antioxidant performances using both chemical assays and a ground meat model. The optimum hydrolysis time for papain was 3 h, and for ficin and bromelain was 4 h. The hydrolysates were further separated by sequential ultrafiltration to 5 hydrolysate fractions named F1 to F5 from low molecular weight (MW) (<1 kDa) to high MW range (>10 kDa), which were further characterized for TPC, free radical scavenging capacity against DPPH and ABTS, and metal chelating activity. The fraction F4 produced by papain (CH-P4), F1 produced by ficin (CH-F1), and F3 produced by bromelain (CH-B3) showed the strongest antioxidant activity and yield, respectively. These three fractions were incorporated into ground pork to determine their inhibition effects on lipid oxidation during a 16-day storage period. The inhibition effect was enhanced with the addition of higher amount of hydrolysate (e.g., 1000 vs. 500 mg/kg). The CH-P4 reduced lipid oxidation in ground meat by as much as 30.45%, and CH-B3 reduced oxidation by 27.2% at the same level, but the inhibition was only 13.83% with 1000 mg/kg of CH-F1. The study demonstrated that CGM protein hydrolysates and peptides could be used as naturally derived antioxidant in retarding lipid oxidation and improving product storage stability.

Ternary nanoparticle complex of antibiotic, polyelectrolyte, and mucolytic enzyme as a potential antibiotic delivery system in bronchiectasis therapy.

Antibiotic-polyelectrolyte nanoparticle complex (or nanoplex in short) has been recently demonstrated as a superior antibiotic delivery system to the native antibiotic in bronchiectasis therapy owed to its ability to overcome the lung's mucus barrier and generate high localized antibiotic exposure in the infected sites. The present work aimed to further improve the mucus permeability, hence the antibacterial efficacy of the nanoplex, by incorporating mucolytic enzyme papain (PAP) at the nanoplex formation step to produce PAP-decorated antibiotic-polyelectrolyte nanoplex exhibiting built-in mucolytic capability. Ciprofloxacin (CIP) and dextran sulfate (DXT) were used as the models for antibiotics and polyelectrolyte, respectively. The results showed that the PAP inclusion had minimal effects on the physical characteristics, preparation efficiency, and dissolution of the CIP-DXT nanoplex. The optimal CIP-(DXT-PAP) nanoplex exhibited size and zeta potential of approximately 200 nm and -50 mV with CIP and PAP payloads of 60% and 32% (w/w), respectively. The nanoplex was prepared at high efficiency with larger than 80% CIP and PAP utilization rates. The CIP-(DXT-PAP) nanoplex exhibited tenfold improvement in the mucus permeability compared to its CIP-DXT nanoplex counterpart, resulting in the former's superior bactericidal activity against clinical Pseudomonas aeruginosa biofilm in the presence of mucus barrier. A trade-off, nevertheless, existed between antibacterial efficacy and cytotoxicity towards human lung epithelium cells upon the incorporation of PAP above a certain concentration threshold. Therefore, the optimal dosing of the CIP-(DXT-PAP) nanoplex must be carefully determined.

The mechanistic effect of bromelain and papain on tenderization in jumbo squid (Dosidicus gigas) muscle.

Jumbo squid (Dosidicus gigas) muscle is rather hard and tough, which directly affects consumer acceptance. In this study, the tenderization effect of bromelain and papain on squid muscle during enzymolysis is examined and compared with an untreated control and water-treated sample. Squid mantle were incubated with different solutions (water, bromelain, and papain solution) for 40 min in a 30 °C water bath. Then, the mantle samples were subjected to water holding capacity (WHC) analysis, texture evaluation, biochemical determination, and histological observations. The results revealed that bromelain and papain disadvantageously decrease the water holding capacity when compared to the control and water-treated samples. Furthermore, following tenderization with bromelain or papain, muscle hardness, shear force, myofibrillar protein content, and Ca2+ ATPase activity were all significantly decreased. Additionally, some essential amino acids were released following tenderization. When examining the myofibrillar fragmentation index (MFI), bromelain and papain were shown to cause high levels of hydrolysis in myofibrillar and sarcoplasmic proteins. Moreover, microstructural imaging indicated that the tenderization treatments disrupted myofibrils and generated a larger number of small fragments in the muscle tissues, subsequently decreasing microstructure stability and integrity. SDS-PAGE analysis confirmed that bromelain and papain have a high proteolytic activity, with some small peptides and/or short fragments detected post-tenderization. The results presented herein demonstrated that bromelain and papain improved squid muscle tenderness and can be utilized to ensure a more desirable squid product.

Peptidomic Investigation of the Interplay between Enzymatic Tenderization and the Digestibility of Beef Semimembranosus Proteins.

This work investigated the influence of enzymatic tenderization on digestibility changes of beef semimembranosus proteins using peptidomics methods. Hydrolysis by proteinase K and bromelain elevated the average bitterness index of identified peptides by generating high-Q values peptides (1714-1790 Cal/mol), including KDLFDPIIQ, LIDDHFLFDKPVSPL, and QLIDDHFLFDKPVSPLLL. Proteolysis during enzymatic tenderization acted as a "pre-digestion" step and significantly elevated the degree of hydrolysis of beef protein (by 4.5-17.3%) in subsequent simulated gastrointestinal digestion. Peptidomics analysis of digests revealed large variations in the peptide composition, which was positively correlated with the degree of proteolysis during enzymatic tenderization. Enzymatic tenderization with proteinase K- (for 0.5 h) or bromelain-treated samples largely increased the survival rate (by 65.5 or 82.8%) of peptides during simulated digestion, possibly because of the "secondary enzyme-substrate interaction" effect. This work could provide a new sight into the possible influence of enzymatic tenderization on meat nutrition.

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.

High angiotensin-I converting enzyme (ACE) inhibitory activity of Alcalase-digested green soybean (Glycine max) hydrolysates.

As a protein-rich, underutilized crop, green soybean could be exploited to produce hydrolysates containing angiotensin-I converting enzyme (ACE) inhibitory peptides. Defatted green soybean was hydrolyzed using four different food-grade proteases (Alcalase, Papain, Flavourzyme and Bromelain) and their ACE inhibitory activities were evaluated. The Alcalase-generated green soybean hydrolysate showed the highest ACE inhibitory activity (IC50: 0.14 mg/mL at 6 h hydrolysis time) followed by Papain (IC50: 0.20 mg/mL at 5 h hydrolysis time), Bromelain (IC50: 0.36 mg/mL at 6 h hydrolysis time) and Flavourzyme (IC50: 1.14 mg/mL at 6 h hydrolysis time) hydrolysates. The Alcalase-generated hydrolysate was profiled based on its hydrophobicity and isoelectric point using reversed phase high performance liquid chromatography (RP-HPLC) and isoelectric point focusing (IEF) fractionators. The Alcalase-generated green soybean hydrolysate comprising of peptides EAQRLLF, PSLRSYLAE, PDRSIHGRQLAE, FITAFR and RGQVLS, revealed the highest ACE inhibitory activity of 94.19%, 99.31%, 92.92%, 101.51% and 90.40%, respectively, while their IC50 values were 878 µM, 532 µM, 1552 µM, 1342 µM and 993 µM, respectively. It can be concluded that Alcalase-digested green soybean hydrolysates could be exploited as a source of peptides to be incorporated into functional foods with antihypertensive activity.

Mucus permeating self-emulsifying drug delivery systems (SEDDS): About the impact of mucolytic enzymes.

This study aimed to improve the mucus permeating properties of self-emulsifying drug delivery systems (SEDDS) by anchoring lipidized bromelain, papain and trypsin using palmitoyl chloride. SEDDS containing enzyme-palmitate conjugates were characterized regarding droplet size and zeta potential. Their mucus permeating properties were evaluated by Transwell diffusion and rotating tube method using fluorescein diacetate (FDA) as marker. Degree of substitution of modified enzymes was 35.3%, 47.8% and 38.5% for bromelain-palmitate, papain-palmitate and trypsin-palmitate, respectively. SEDDS as control and SEDDS containing enzyme-palmitate conjugates displayed a droplet size less than 50nm and 180-312nm as well as a zeta potential of -3 to -4 and -4 to -5mV, respectively. The highest percentage of permeation was achieved by introducing 5% papain-palmitate into SEDDS. It could enhance the mucus permeation of SEDDS in porcine intestinal mucus 4.6-fold and 2-fold as evaluated by Transwell diffusion and rotating tube method, respectively. It is concluded that mucus permeation of SEDDS can be strongly improved by incorporation of enzyme-palmitate conjugates.

Camel milk protein hydrolysates with improved technofunctional properties and enhanced antioxidant potential in in vitro and in food model systems.

Camel milk protein hydrolysates (CMPH) were generated using proteolytic enzymes, such as alcalase, bromelain, and papain, to explore the effect on the technofunctional properties and antioxidant potential under in vitro and in real food model systems. Characterization of the CMPH via degree of hydrolysis, sodium dodecyl sulfate-PAGE, and HPLC revealed that different proteins in camel milk underwent degradation at different degrees after enzymatic hydrolysis using 3 different enzymes for 2, 4, and 6 h, with papain displaying the highest degradation. Technofunctional properties, such as emulsifying activity index, surface hydrophobicity, and protein solubility, were higher in CMPH than unhydrolyzed camel milk proteins. However, the water and fat absorption capacity were lower in CMPH compared with unhydrolyzed camel milk proteins. Antioxidant properties as assessed by 2,2-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) and 2,2-diphenyl-1-picrylhydrazyl radical scavenging activities and metal-chelating activity were enhanced after hydrolysis, in contrast to ferric-reducing antioxidant power which showed a decrease after hydrolysis. The CMPH were also tested in real food model systems for their potential to inhibit lipid peroxidation in fish mince and grape seed oil-in-water emulsion, and we found that papain-produced hydrolysate displayed higher inhibition than alcalase- and bromelain-produced hydrolysates. Therefore, the CMPH demonstrated effective antioxidant potential in vitro as well as in real food systems and showed enhanced functional properties, which guarantees their potential applications in functional foods. The present study is one of few reports available on CMPH being explored in vitro as well as in real food model systems.

Fluorescence spectroscopic analysis of the interaction of papain and bromelain with l-ascorbic acid, α-tocopherol, ß-carotene and astaxanthin.

In this study, the interaction between four classic dietary antioxidants (including l-ascorbic acid, α-tocopherol, ß-carotene and astaxanthin) and papain/bromelain was investigated by fluorescence spectroscopy. The results show that the quenching mechanisms are all static quenching at lower concentrations of antioxidants, but at higher concentrations of antioxidants, predominantly by the "sphere of action" quenching mechanisms. The binding processes of the four antioxidants to papain/bromelain are all synergistically driven by enthalpy and entropy, and the major driving forces are electrostatic effect and hydrophobic interactions. The binding constants of papain/bromelain with the four antioxidants are in the following order as: astaxanthin-papain >ß-carotene-papain > astaxanthin-bromelain >l-ascorbic acid-papain >l-ascorbic acid-bromelain >ß-carotene-bromelain >α-tocopherol-papain >α-tocopherol-bromelain. Synchronous fluorescence spectroscopy shows the interaction between l-ascorbic acid/ß-carotene/astaxanthin and papain/bromelain decreases the hydrophobicity of the microenvironment of tryptophan (Trp) and tyrosine (Tyr) residues. The hydrophobicity of Trp is increased while the hydrophility of Tyr is increased in the presence of α-tocopherol.

Protease-functionalized mucus penetrating microparticles: In-vivo evidence for their potential.

The focus of the current study was to explore whether immobilization of proteases to microparticles could result in their enhanced penetration into mucus. The proteases papain (PAP) and bromelain (BROM) were covalently attached to a polyacrylate (PAA; Carbopol 971P) via amide bond formation based on carbodiimide reaction. Microparticles containing these conjugates were generated via ionic gelation with calcium chloride and were characterized regarding size, surface charge, enzymatic activity and fluorescein diacetate (FDA) loading efficiency. Furthermore, mucus penetration potential of these microparticles was evaluated in-vitro on freshly collected porcine intestinal mucus, on intact intestinal mucosa and in-vivo in Sprague-Dawley rats. Results showed mean diameter of microparticles ranging between 2-3µm and surface charge between -8 to -18mV. The addition of PAA-microparticles to porcine intestinal mucus led to a 1.39-fold increase in dynamic viscosity whereas a 3.10- and 2.12-fold decrease was observed in case of PAA-PAP and PAA-BROM microparticles, respectively. Mucus penetration studies showed a 4.27- and 2.21- fold higher permeation of FDA loaded PAA-PAP and PAA-BROM microparticles as compared to PAA microparticles, respectively. Extent of mucus diffusion determined via silicon tube assay illustrated 3.96- fold higher penetration for PAA-PAP microparticles and 1.99- fold for PAA-BROM microparticles. An in-vitro analysis on porcine intestinal mucosa described up to 16- and 7.35-fold higher degree of retention and furthermore, during in-vivo evaluation in Sprague-Dawley rats a 3.35- and 2.07-fold higher penetration behavior was observed in small intestine for PAA-PAP and PAA-BROM microparticles as compared to PAA microparticles, respectively. According to these results, evidence for microparticles decorated with proteases in order to overcome the mucus barrier and to reach the absorption lining has been provided that offers wide ranging applications in mucosal drug delivery.

Development of an Economical Method to Reduce the Extractable Latex Protein Levels in Finished Dipped Rubber Products.

Natural rubber latex (NRL) allergy is caused by the extractable latex proteins in dipped rubber products. It is a major concern for the consumers who are sensitive to the allergenic extractable proteins (EP) in products such as NRL gloves. Objective of this research was to develop an economical method to reduce the EP in finished dipped NRL products. In order to reduce the EP levels, two natural proteases, bromelain from pineapple and papain from papaya, were extracted and partially purified using (NH4)2SO4. According to the newly developed method, different glove samples were treated with a 5% solution of each partially purified enzyme, for 2 hours at 60°C. Residual amounts of in treated samples were quantified using the modified Lowry assay (ASTM D5712-10). Bromelain displayed a 54 (±11)% reduction of the EP from the dipped rubber products, whereas it was 58 (±8)% with papain. These results clearly indicate that the selected natural proteases, bromelain, and papain contribute significantly towards the reduction of the total EP in finished NRL products. Application of bromelain enzyme for the aforementioned purpose has not been reported up to date, whereas papain has been used to treat raw NRL towards reducing the EP.

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.

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.