Activity Of Papain Research Articles

A novel cystatin in Psoroptes ovis var. cuniculi: molecular characterization, serodiagnostic potential, and its anti-inflammatory property on rabbit peripheral blood mononuclear cells

BackgroundThe ectoparasite Psoroptes ovis var. cuniculi causes substantial economic losses to the global rabbit industry. Currently, microscopy for identifying Psoroptes mite in skin scrapings, as the “diagnosis gold standard,” remains a challenge owing to its poor sensitivity in detecting low-level and/or early stage mite infestations. Additionally, Psoroptes infestations rapidly trigger cutaneous inflammation, thus the mites might produce some molecules to deal with the harmful effects of inflammation for their long-time survival on the host skin, but these molecules are still mostly unknown.MethodsTo seek a sensitive diagnostic method and illuminate the new antiinflammatory molecules, we characterized a novel cystatin of P. ovis var. cuniculi (PsoCys) using bioinformatics and molecular biology methods.ResultsThe results showed that PsoCys comprised the classical features of the type II cystatin superfamily including an N-terminal glycine residue, a central QXVXG motif, and a C-terminal LW motif. In mixed stages of mites, the transcription level of PsoCys was significantly higher in “fed” mites than in “starved” mites (P < 0.001), and among the different life-cycle stages of “fed” mites, the expression of PsoCys was higher in adult males than in larva, nymph, and adult females (P < 0.001). The established indirect ELISA based on recombinant PsoCys (rPsoCys-iELISA) presented 95.4% sensitivity and 95.7% specificity. The area under the receiver operating characteristic curve (AUC) for this method was 0.991, indicating its excellent diagnostic performance. Moreover, rPsoCys-iELISA had advantages over microscopy for detecting low-level and/or early stage mite infestations (90% versus 40% in artificial infestation cases at 3 weeks post-infestation; 61.9% versus 22.6% in clinical cases). In addition, rPsoCys could inhibit the activity of papain and cathepsin B in vitro, and significantly suppressed mRNA levels of toll-like receptors (TLR 1, 2, 4, and 6) and downstream molecules (NF-κB, p38, MyD88, IL-10, and IFN-γ) in LPS-stimulated rabbit PBMCs, indicating its anti-inflammatory property.ConclusionsOur findings indicated that PsoCys was a novel type II cystatin of Psoroptes mites, and it served as a potential serological diagnostic antigen for detecting low-level and/or early stage mite infestations, as well as a novel anti-inflammatory molecule of Psoroptes mites.Graphical abstract

Green Nanotechnology Through Papain Nanoparticles: Preclinical in vitro and in vivo Evaluation of Imaging Triple-Negative Breast Tumors.

Recent advancements in nanomedicine and nanotechnology have expanded the scope of multifunctional nanostructures, offering innovative solutions for targeted drug delivery and diagnostic agents in oncology and nuclear medicine. Nanoparticles, particularly those derived from natural sources, hold immense potential in overcoming biological barriers to enhance therapeutic efficacy and diagnostic accuracy. Papain, a natural plant protease derived from Carica papaya, emerges as a promising candidate for green nanotechnology-based applications due to its diverse medicinal properties, including anticancer properties. This study presents a novel approach in nanomedicine and oncology, exploring the potential of green nanotechnology by developing and evaluating technetium-99m radiolabeled papain nanoparticles (99mTc-P-NPs) for imaging breast tumors. The study aimed to investigate the efficacy and specificity of these nanoparticles in breast cancer models through preclinical in vitro and in vivo assessments. Papain nanoparticles (P-NPs) were synthesized using a radiation-driven method and underwent thorough characterization, including size, surface morphology, surface charge, and cytotoxicity assessment. Subsequently, P-NPs were radiolabeled with technetium-99m (99mTc), and in vitro and in vivo studies were conducted to evaluate cellular uptake at tumor sites, along with biodistribution, SPECT/CT imaging, autoradiography, and immunohistochemistry assays, using breast cancer models. The synthesized P-NPs exhibited a size mean diameter of 9.3 ± 1.9 nm and a spherical shape. The in vitro cytotoxic activity of native papain and P-NPs showed low cytotoxicity in HUVEC, MDA-MB231, and 4T1 cells. The achieved radiochemical yield was 94.2 ± 3.1% that were sufficiently stable (≥90%) for 6 h. The tumor uptake achieved in the 4T1 model was 2.49 ± 0.32% IA/g at 2h and 1.51 ± 0.20% IA/g at 6h. In the spontaneous breast cancer model, 1.19 ± 0.20% IA/g at 2h and 0.86 ± 0.31% IA/g at 6h. SPECT/CT imaging has shown substantial tumor uptake of the new nanoradiopharmaceutical and clear tumor visualization. 99mTc-P-NPs exhibited a high affinity to tumoral cells confirmed by ex vivo autoradiography and immunohistochemistry assays. The findings underscore the potential of green nanotechnology-driven papain nanoparticles as promising agents for molecular imaging of breast and other tumors through SPECT/CT imaging. The results represent a substantial step forward in the application of papain nanoparticles as carriers of diagnostic and therapeutic radionuclides to deliver diagnostic/therapeutic payloads site-specifically to tumor sites for the development of a new generation of nanoradiopharmaceuticals.

Evaluation of the Effects of Papain on Schistosoma mansoni: Miracidial Infection Capacity, Infection Prevalence, Cercarial Shedding and Molecular Changes in Biomphalaria alexandrina.

The aim of the present study is to assess the molluscicidal, larvicidal and genotoxicological activities of papain and how it can affect the host-parasite interactions. Toxicity of papain on snails by making series of concentrations to calculate LC50, and then study its larvicide effect on the free larval stages of S. mansoni and infection rate of snails. Papain has a molluscicidal activity on adult snails of Biomphalaria alexandrina with a lethal concentration LC50 equals to 43.1mg/L. In addition, it has activity on miracidia with half Lethal time (LT50) of 16.11min., and on cercariae with 12.1min. compared to control ones. The sub lethal concentration LC10 and LC25 (6.9 or 24.1mg/L, respectively) decreased the survival rate of snails at the first cercarial shedding, the rate of infection, the average total number of cercariae per snail, the shedding period and the life span of snails, while the prepatent period was significantly increased than the control ones. The morphological alterations in cercariae after exposure to papain were occurred where the cercariae lacked motility and some had a dark tail with complete detachment of head and tail. Compared to the control group, the levels of cytochrome oxidase subunit I (COI) and (ND1) genes significantly decreased in snails after exposure to papain. Papain could be used as a potential molluscicide for elimination of schistosomiasis and decrease its transmission and deterioration of host-parasite interaction.

Thermal Inactivation, Denaturation and Aggregation Processes of Papain-Like Proteases.

The investigation into the behavior of ficin, bromelain, papain under thermal conditions holds both theoretical and practical significance. The production processes of medicines and cosmetics often involve exposure to high temperatures, particularly during the final product sterilization phase. Hence, it's crucial to identify the "critical" temperatures for each component within the mixture for effective technological regulation. In light of this, the objective of this study was to examine the thermal inactivation, aggregation, and denaturation processes of three papain-like proteases: ficin, bromelain, papain. To achieve this goal, the following experiments were conducted: (1) determination of the quantity of inactivated proteases using enzyme kinetics with BAPNA as a substrate; (2) differential scanning calorimetry (DSC); (3) assessment of protein aggregation using dynamic light scattering (DLS) and spectrophotometric analysis at 280 nm. Our findings suggest that the inactivation of ficin and papain exhibits single decay step which characterized by a rapid decline, then preservation of the same residual activity by enzyme stabilization. Only bromelain shows two steps with different kinetics. The molecular sizes of the active and inactive forms are similar across ficin, bromelain, and papain. Furthermore, the denaturation of these forms occurs at approximately the same rate and is accompanied by protein aggregation.

Fabrication of functional diatomaceous earth with enhanced papain enzyme adsorption

Diatomaceous earth (DE) is a sedimentary rock comprising fossilized shells of diatoms, which are a type of algae. The shells of the diatoms are made of silicon dioxide, which is also the main component of DE. DE has excellent moisture absorption and desorption properties and is widely used as an adsorbent, abrasive, insulator, catalyst, and carrier in the stationary phase. It has also been widely studied for its application as a new silicon material in emerging growth fields, such as biosensors. However, because organic matter is adsorbed on the surface of DE, the organic matter must be removed to exploit the properties of mesoporous silica. In this study, the organic matter on the DE surface was removed by alkali treatment to expose the pores and increase the adsorption capacity. Additionally, papain was physisorbed onto DE using a rotator, and l-cysteine was added for papain activation. Under these optimised conditions, the activation of the papain enzyme was effective, resulting in improved DE performance. The adsorption power of DE was correlated with the amount of papain adsorbed, which, in turn, was correlated with its absorbance. Trichloroacetic acid was added to stop the reaction, and the absorbance was measured. The prepared samples were designated as papain/DE (P/DE) complexes. The final concentration of the P/DE was 1 mg/mL. In addition, cross-linked and non-cross-linked P/DE samples were prepared using N-hydroxysuccinimide and water-soluble carbodiimide in a 5:3 ratio and compared. The results showed that surface treatment and functionalisation of DE improved its functionality (enzyme activity), indicating its potential as a new DE material.

The ACE-inhibitory activity of alcalase, papain and pepsin lupin protein hydrolysates

Recently, Lupinus angustifolius proteins revealed their high potential to liberate bioactive peptides after hydrolysis. In our study, we examined the release of angiotensin I-converting enzyme inhibitory peptides from extracted lupin proteins after digestion by alcalase, papain, and pepsin. Three enzyme-to-substrate ratios were evaluated with three hydrolysis duration times. First, the degree of hydrolysis was determined using the ortho-phthalaldehyde aldehyde (OPA) method and then, the HPLC-DAD method was applied to measure the ACE-inhibitory activity. According to our results, all hydrolysates possessed ACE inhibitory properties. The calculated IC50 concentrations varied from 0.26 ± 0.59 to 2.19 ± 0.23 μg.ml-1. The peptides with the lowest IC50 values were produced by pepsin (ΔIC50 = 0.33 ± 0.40 μg.ml-1), followed by papain (ΔIC50 = 0.67 ± 0.61 μg.ml-1), and alcalase (ΔIC50 = 1.78 ± 0.92 μg.ml-1). Further research on bioavailability is required to demonstrate the beneficial effects of bioactive peptides on human health, since they have to resist digestive processing pass the intestinal barrier, and reach the bloodstream and intended organs.

Nematicidal and Insecticidal Activity of Proteases from Carica papaya and Ananas comosus

Plant proteases are well known for their various industrial applications. Papain, present in papaya latex (Carica papaya) and pineapple bromelain (Ananas comosus), is undoubtedly the most studied and widely used vegetable protease in the food and pharmaceutical industry worldwide. However, its potential as a biopesticide has been little explored. The objective of this study was to evaluate the activity of proteases from Carica papaya latex and peel and crown of Ananas comosus fruits on agricultural pests. To evaluate proteolytic activity on nematodes, extracts, and approximately 50 juveniles of Panagrellus sp. were placed in microtubes. To evaluate the insecticidal effect, larvae and pupae of Tenebrio molitor L. were submerged in active and denatured extracts. Additionally, larvae of T. molitor were fed an artificial diet at doses of 0, 100, 250, and 500 mg/g of wheat bran. The weight and number of dead larvae were recorded, and feeding behavior was evaluated. The proteases of papaya latex and papain caused reduction (p &lt; 0.05) on Panagrellus sp. The extracts showed a toxic effect (p &lt; 0.05) against the larvae of T. molitor. Active papain resulted in the absence of wings in 53.3% of adults from the pupae, and no malformation caused by denatured papain was observed. No mortality was observed in larvae fed an artificial diet. However, there was a strong feed reduction, reduction in the relative rate of consumption, reduction in growth and feed conversion efficiency caused by papaya latex. The results of this study show that plant proteases have the potential for the development of sustainable alternatives for the control of arthropod pests and parasitic nematodes.

Synergistic precipitant powered self-assembly of papain for cross-linked enzyme crystals preparation

In this study, we prepared cross-linked enzyme crystals (CLECs) of papain to further broaden the application of the enzyme with high activity in extreme environments. Initially, papain crystals were successfully obtained based on the micro-batch, batch, and expanded batch crystallization experiments. Specifically, ammonium sulfate and polyethylene glycol 6000 (PEG6000) were synergistically used as the precipitants, while l-cysteine was applied to enhance the activity of papain. Furthermore, the interaction between l-cysteine and papain was modeled by molecular docking technique. It was found that l-cysteine could form a hydrogen bond with aspartic acid residue (Asp) at site 158, and the electrostatic attraction with lysine residue (Lys) at site 156 was also quite obvious. Then the enzyme crystals were cross-linked by glutaraldehyde at optimized conditions. The papain CLECs were identified by various methods, and it was found that the thermal stability and enzymatic activity both increased compared to the raw enzyme. More importantly, it could be applied at more rigorous conditions, for example, pH of 4.

Starch- and carboxymethyl cellulose-based films as active beauty masks with papain incorporation

This work aimed to evaluate the effects of the incorporation of papain in biopolymeric-based beauty face masks with exfoliation activity of the skin. The masks were produced by casting with starch and carboxymethyl cellulose blend (50:50 weight percentages). The macro and microstructure, protein distribution, thickness, moisture content, water contact angle, solubility matter, and mechanical properties were evaluated. Moreover, the in vitro proteolytic and exfoliation activity and storage stability were also evaluated. The films with papain had a more concise matrix which provided higher mechanical properties and lower water solubility when compared to the control film (without papain). Films with 1, 2, and 5 % of papain had enzymatic activity for casein and porcine skin substrates. The micrographs of porcine skin treated with 2 and 5 % of papain showed more difference when compared to the control sample, indicating the enzymatic exfoliation. Differently from the solution of papain, the enzyme that was immobilized in the films maintained its activity for up 90 days during the storage stability assay. Based on the physicochemical properties and proteolytic activities, the films preserved the exfoliation activity of papain and have interesting characteristics to act as beauty masks in the cosmetic field.

Evaluating different levels of papain as texture modifying agent in bovine meat loaf containing transglutaminase

In this study, bovine meat loaves were produced with different levels of papain (0.00125%, 0.0025%, 0.00375%, and 0.005%) combined with transglutaminase (1%). The effect of this reformulation on pH, instrumental color, water activity, proximate composition, texture, yield, and scanning electron microscopy (SEM) of meat loaves was investigated. In addition, the enzymatic activity of papain was also analyzed. The papain addition increased the pH and the yield of the samples. The hardness was progressively reduced with the increase of papain level. Such changes could be seen through the images recorded by SEM, where an extremely fragmented structure was observed in treatments with higher papain concentration. Papain showed an optimum temperature of 80 °C. This study allowed to observe an intense proteolytic effect in all treatments, despite the papain concentration. Therefore, lower levels should be applied so that the product does not alter its sensory characteristics, such as soft and crumbly texture.

A new application of papain: As a peroxidase-like catalyst for fluorometric detection of uric acid

Papain, as a classical cysteine protease, has been widely used in the food, pharmaceutical, chemical, and cosmetic fields. However, there are few information about the peroxidase-like activity of papain catalyzed substrate to produce fluorescence. In this study, we found that papain can catalyze H2O2 to convert o-phenylenediamine (OPD), and generate fluorescence emission at 550 nm under 430 nm excitation. Based on this foundation, we report a papain/OPD/H2O2 system for fluorescence detection of uric acid. The method exhibits a wide linear range of 10–1000 μM with a limit-of-detection of 4.6 μM, and has been successfully used to detect uric acid in human serum. This study paves the way for the application of papain as catalyst for fluorescence detection of different target biomolecules, such as cholesterol, glucose, lactate, for which H2O2 is a product of oxidoreductase enzymes.

The peroxidase-like activity of papain colorimetrically detects H2O2 and glucose with high sensitivity

To date, several glucometer methods are available for patient-guided glycemic control, which diminishes the onset and severity of diabetic complications. In a dipstick urine glucose test, horseradish peroxidase (HRP) oxidizes tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2) to quantitatively produce a colorimetric signal and predict glucose concentration. Papain, a sulfhydryl protease secreted by the latex of Carica papaya, displays HRP-like activity with high selectivity. However, the current papain-based glucometer method lacks sensitivity. We hypothesized that catalyzing the oxidation of papain under varying buffer conditions (e.g., papain, TMB, and acetic acid concentrations) would improve glucose detection sensitivity. To test this, we performed glucose oxidase (GOx)-mediated glucose oxidation and papain-mediated TMB oxidation under varying sample conditions in vitro and spontaneously coupled them. The aims of this study were to validate the coupling of GOx-mediated glucose oxidation and papain-mediated TMB oxidation and identify the optimized conditions that maximize catalytic activity. We confirmed that glucose oxidase quantitatively produces H2O2 (in a glucose concentration-dependent manner), the presence of which catalyzes papain-mediated production of a colorimetric signal. When two oxidation reactions were coupled, we identified that the sensitivity of papain’s glucose detection increased by ten-fold compared to previous studies with a markedly high correlation (R2 = 0.99). In particular, substituting sodium acetate-acetic acid (HAc-NaAc) buffer with acetic acid and increasing the sample incubation time and papain concentration markedly increased the sensitivity of colorimetric signals. The optimized protocols can be integrated into emerging hydrogel technology to develop a non-invasive glucometer patch predicting blood glucose from the patient’s produced sweat.

Biosynthesis of amidated pectins with ultra-high viscosity and low gelation restriction through ultra-low temperature enzymatic method

In traditional industry, amidated pectin was usually prepared by adding ammonia gas under the alkaline condition or ethanol system which degrades the galacturonic acid chain of pectin and weakens the advantage of amidated pectin in gel. In this study, amidated pectin with ultra-high viscosity and low gelation restriction were prepared by grafting with the neutral aliphatic amino acids through enzymatic method at ultra-low temperature to extend the application of the pectin in food, daily use chemical, medicine and other fields. The amino acid grafting ratio of alanine amidated pectin (V-PE), valine amidated pectin (A-PE) and leucine amidated pectin (L-PE) were 14.12%, 24.36% and 18.94%, respectively. The results of dynamic viscosity showed that the dynamic viscosity of high methoxy pectin (HMP) increased from 186 to 39799 (V-PE), 20396 (A-PE) and 23781 mPa.s (L-PE), respectively. Through ultra-low temperature enzymatic, the V-PE with the highest amino acid grafting ratio had the highest solution viscosity and gel hardness. Furthermore, the hydrogel has better thermal stability, which can maintain the three-dimensional network structure of the gel at higher temperatures without collapse. Amidated pectin with ultra-high viscosity and low gelation restriction can provide valuable information in applications such as food and medicine. • Pectin was amidated by neutral aliphatic amino acids through enzymatic method. • In the two-phase system, the amide synthase activity of papain was dominant. • The amidated pectin can form hydrogels under low gelation restriction. • Compared with high methoxy pectin, amidated pectin has high dynamic viscosity.

Antimicrobial activity, Cytotoxicity and Anti inflammatory activity of Papain and Bromelain based caries removal gel decorated with Silver Nanoparticles against oral microbes

Antimicrobial activity, Cytotoxicity and Anti inflammatory activity of Papain and Bromelain based caries removal gel decorated with Silver Nanoparticles against oral microbes

In vitro effect on the proteolytic activity of papain with proteins of the skin as substrate.

This work aims to evaluate the effects of enzyme concentration, pH, temperature and time course degree of hydrolysis (DH) of papain regarding further development of pharmaceutical and cosmetic formulations. The hydrolysis of casein, collagen, keratin and porcine skin at pH and temperature ranges of the human skin was evaluated. Also, low contact times of enzyme-substrate were studied. The incorporation of 3mM of cysteine improved the caseinolytic (PU), collagenolytic (CU) and keratinolytic (KU) activities of papain. In general, the increase from 0.1 to 1.0 or 2.0mgmL-1 of papain improved PU, CU and KU. When 2.0mgmL-1 of papain was used, the highest DH of casein, collagen and keratin was obtained at 240min (14%, 35% and 6% respectively). The decrease in pH and temperature reduced all proteolytic activities, but papain maintained at least 50% and 40% of its activity at 26°C and pH 4.5 respectively. Scanning electron micrographs of the surface of the skin showed that papain application had exfoliating activity. This preformulation study demonstrated that papain concentration, time of application and pH of the product should be evaluated when developing a product to promote the hydrolysis of the proteins of the skin.

Peptide conformational imprints enhanced the catalytic activity of papain for esterification.

Peptide conformational imprints (PCIs) offer a promising perspective to directly generate binding sites for preserving enzymes with high catalytic activity and stability. In this study, we synthesized a new chiral cross-linker cost-effectively for controlling the matrix morphology of PCIs on magnetic particles (PCIMPs) to stabilize their recognition capability. Meanwhile, based on the flank part of the sequences on papain (PAP), three epitope peptides were selected and synthesized. Molecularly imprinted polymers (MIPs) were then fabricated in the presence of the epitope peptide using our new cross-linker on magnetic particles (MPs) to generate PCIMPs. PCIMPs were formed with helical cavities that complement the PAP structure to adsorb specifically at the targeted position of PAP. PCIMPs65–79 were found to have the best binding parameters to the PAP with K d = 0.087 μM and B max = 4.56 μM. Upon esterification of N-Boc-His-OH, proton nuclear magnetic resonance (1H-NMR) was used to monitor the yield of the reaction and evaluate the activity of PAP/PCIMPs. The kinetic parameters of PAP/PCIMPs65–79 were calculated as V max = 3.0 μM s−1, K m = 5 × 10−2 M, k cat = 1.1 × 10–1 s−1, and k cat/K m = 2.2 M−1 s−1. In addition, PAP is bound tightly to PCIMPs to sustain its activity after four consecutive cycles.

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

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

Determination of chlorothalonil levels through inhibitory effect on papain activity at protein-decorated liquid crystal interfaces.

A liquid crystal (LC)-based assay was developed to detect chlorothalonil (CHL). The detection principle is based on (i) the electrostatic interaction between the positively charged protein protamine (PRO) with the negatively charged phospholipid dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG) and (ii) the CHL-mediated inhibition of papain (PAP) activity. The aqueous/LC interface was decorated with a monolayer of DOPG and PRO that self-assembled via electrostatic interactions. PAP can hydrolyze PRO, resulting in the realignment of an LC by DOPG, inducing a shift in the LC response from bright to dark. The addition of CHL can inhibit the activity of PAP, leading to the attraction of PRO to DOPG and the consequent disruption of the LC orientation. The orientation change of the LC in the presence or absence of CHL can be observed from the changes in its optical appearance using a polarized light microscope. Under optimal conditions, the developed assay achieved a detection limit of 0.196 pg mL−1 within a range of determination of 0.65–200 pg mL−1. The selectivity of the assay was verified in the presence of carbendazim and imidacloprid. The practical application of the proposed assay was demonstrated by its use to determine the levels of CHL in food extracts and environmental samples, which yielded recoveries and relative standard deviations (RSD) in the ranges of 87.39–99.663% and 1.03–6.32%, respectively.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00604-022-05396-1.

Preferential and competitive role of hydrophilic/hydrophobic interactions quantifying amino acid-based ILs for papain stabilization

A plethora of amino acid based ionic liquid (AAIL) has been synthesized recently in order to thermally and structurally stabilize the proteins, however, critical impact of AAIL cations and anions hydrophilicity has been significantly ignored and subsided. Herein, the pivotal role of cations competitive hydrophilicity has been envisaged by investigating the proteolytic activity, structural stability, and alteration in microenvironment of papain in the presence of varying concentration of cholinium tryptophan ionic liquids [CHO][Trp]IL and tetraethylammonium tryptophan ionic liquids [TEA][Trp]IL. The proteolytic activity of papain in increasing concentration of [CHO][Trp]IL and [TEA][Trp]IL shows that at lower concentration of [CHO][Trp]IL more hydrophilic cholinium cation enhanced the papain activity from 100 to 131 %, however detrimental effects on the activity have been observed at lower concentration of [TEA][Trp]IL due to less hydrophilic nature of tetraethylammonium anions. Moreover, the highly interactive nature of [TEA][Trp]IL can be concluded by Fourier transmission infrared spectroscopy (FTIR). The UV–vis, steady state fluorescence and circular dichroism results also concluded the prowess of AAILs hydrophilic cation for stabilization nature. However, at higher concentration detrimental effects have been observed even for AAILs which are having hydrophilic cations. Thus, the study highlighted that [CHO][Trp]IL can enhance the activity and structure stability of papain more significantly while maintaining its surrounding microenvironment due to more hydrophilic nature of cholinium cation in contrast to [TEA][Trp]IL which consists of less hydrophilic tetraethylammonium cation. Moreover, with increase in the concentration of both AAILs, detrimental effects on the proteolytic activity and structural stability of papain have been observed. To differentiate further between the polar and non-polar interactions, protein-ligand molecular docking studies of papain and cations of both AAILs, have been performed using AutoDock Vina and AutoDock tools 1.5.7. The evaluated results corroborated the data obtained by FTIR, spectroscopic studies and enzymatic activity. From docking, activity and spectroscopic studies, it can be observed that at cholinium cation vehemently prefers polar interaction by forming hydrogen bonding with Arg-64 and Asp-158, however, tetraethylammonium cation longs for the polar interactions by forming van der Waals interactions with Ala-162, Val-133 and Val-207. Moreover, at the higher concentration of AAILs no extraordinary distinction between hydrophilic/hydrophobic natures has been observed and both AAILs denatured the papain at higher concentration irrespective of hydrophilic/hydrophobic nature of cation.

Identification of Proteins Responsible for High Activity of Cysteine Proteinase Inhibitor in the Blood of Nile Tilapia Oreochromis niloticus

Cysteine proteinase inhibitors (CPIs) protect tissues and organs against cysteine proteinases in animal blood and have attracted much attention for use in food processing and medical sciences for humans and animals. Several CPI proteins, which include stefins, cystatins, kininogens, histidine-rich glycoproteins (HRG) and fetuins, have been identified and characterized in mammals. Fish blood also contains high CPI activity, but the identity of the major protein responsible for this activity has not been clarified. This study was conducted to screen CPI activity by examining papain inhibitory activity from various different tissues in Nile tilapia Oreochromis niloticus and to identify major proteins for the activity in the blood. CPI activity was highest in the serum among the tissues screened in this study (at least fourfold higher than in other tissues)(P &lt; 0.05). Major proteins for CPI activity in serum were purified using a CNBr-activated sepharose 4B column, gel filtration and an ion exchange FPLC column. From these purifications, two proteins with strong CPI activity were isolated and partially sequenced. Based on their molecular weights and partial amino sequences, the two major proteins with CPI activity from the blood in this species were found to be fetuin B (60 kDa) and kininogen (54 kDa).