Papain Preparation Research Articles

Preparation and Characterization of Papain Loaded Phosphatidyl Choline-PLGA Hybrid Nanoparticles as Novel Drug Delivery Systems

Preparation and Characterization of Papain Loaded Phosphatidyl Choline-PLGA Hybrid Nanoparticles as Novel Drug Delivery Systems

Determination of vitamin B12 in commercial pepsin preparations using immunoaffinity chromatography and LC‐MS/MS

AbstractVitamin B12 is a critical micronutrient in a vegan diet and thus dietary sources are limited and often low in the vitamin. Analytical determination of vitamin B12 requires laborious sample preparations including the proteolytic breakdown of the (food) matrix with proteases and the enrichment of the vitamin via immunoaffinity methods. Proteases like pepsin have an animal origin and thus might contain significant amounts of vitamin B12. We hypothesize that pepsin preparations potentially transfer residual vitamin B12 into the sample and falsify results. Hence the aim of our study was to determine trace amounts of cyano‐cobalamin by LC‐MS/MS in commercial protease enzyme preparations. The use of a stable 15N‐labeled cyano‐cobalamin as internal standard enabled us to detect vitamin B12 with a LOQ as low as 0.7 µg/l. Significant amounts of the vitamin were found in pepsin whereas the vitamin B12 content of papain preparations was below the detection limit. We conclude that sample preparation with pepsin bears the risk of false positive results, especially for samples with low vitamin B12 content, and we therefore recommend papain as an alternative protease for food sample preparation.

Preparation and Properties of Papain Precipitated from Fresh Latex of Papaya Fruits (Carica papaya)

Papain was precipitated from cysteine hydrochloride extract of fresh latex of papaya fruits planted in AlexandriaGovernorate, Egypt. Three precipitants; namely, ethyl alcohol, acetone and sodium chloride resulted in proteolyticactivity recovery 84.8 (of three fractions), 85.2 and 76.7 %, respectively. Acetone was found to be the most promisingrapid and simple conditions for preparing acetone dry powder of partially purified crude papain. Papain propertiesindicated that the maximum activity was attained at pH 7.5–8.0, and temperature of 75 -85°C. The activity was notaffected by Na+ or Ca++ ions. Various degrees of inhibition were exerted by Ni+,+, Co++, Pb ++,and Hg++., while Cu++,caused complete inhibition .On the other hand, the proteolytic activity increased to various levels, in the presenceof Sn++, cyanide, thioglycolic acid, and cysteine hydrochloride. The catalytic activation (49%) of Sn++ was found todepend to some extent on the concentration. A mixed stored papain-oryzae protease preparation ,was found to retain71, 63, and 54 % of its proteolytic activity upon storage at 25°C for 1, 2 ,and 3 months ,respectively. The retainedactivity was more pronounced, being 92, 80,and 69 %, respectively upon storage at –5°C for the aformentioned durations,accordingly, storage at –5°C for up to 2 months, was recommended. The storage stability of papain powder andsolutions were studied. A papain-oryzae protease preparation was formulated for further use in meat tenderization.

Optimization of the Process of Compound Enzymatic Hydrolysis of Soluble Protease Preparation

soluble protease preparations, including trypsin, papain and other preparations, are widely used in various fields, such as medicine, agriculture, industry and so on. Based on this, this paper takes the soluble protease as the main research object, and uses the experimental method of compound enzymolysis to observe and analyze the utilization of egg membrane protein, so as to improve the utilization rate of egg membrane protein.

Physical, Microbial, and Chemical Qualities of Dangke Produced by Different Temperatures and Papain Concentrations

Dangke, a dairy product of cow or buffalo, is a traditional food of Enrekang, South Sulawesi Province. Addition of papain in dangke preparation is responsible for the formation of solid texture of dangke. This study was aimed to find optimum conditions (temperature and concentration of papain enzyme) and their effects on physical, chemical, microbiological, and hedonic qualities of dangke. This study consisted of two stages: preparation of papain and dangke production with heating temperatures (70, 80, and 90 °C) and papain treatments (0.2%, 0.3%, and 0.4%). The experiment was conducted in a completely randomized design with a 3 x 3 factorial arrangement with three replicates. The first factor was the processing temperature consisted of 3 levels i.e., 70, 80, and 90 °C. The second factor was the papain concentration consisted of 3 levels i.e., 0.2%, 0.3%, and 0.4%. The obtained data were evaluated using analysis of variance (ANOVA), followed by Duncan’s Multiple Range Test to observe the significances among treatments. Papain and amino acids were characterized using descriptive methods and organoleptic study was performed by non-parametric test (Kruskal-Wallis). The highest protein concentration was found in commercial papain (Merck, 360.63 mg/100 g), while the protein content of papain used in this study was of 323.21 mg/100g. However, these enzymes had similar molecular weight of 19.17 kDa. The optimum condition of dangke preparation was found at heating temperature of 80 °C and 0.3% of papain concentration, resulting in the most desirable characteristics of dangke in terms of chemical, physical, and microbiological properties as well as hedonic evaluation.

Preparation and characterization of papain embedded in magnetic cellulose hydrogels prepared from tea residue

Papain was embedded and immobilized in hydrogels prepared from tea cellulose by dissolving in ionic liquid of 1-allyl-3-methylimidazolium chloride and coating with magnetic Fe3O4 via reaction of FeCl3/FeCl2 solution with ammonium hydroxide. The prepared magnetic-cellulose-hydrogel-embedded papain was characterized via vibrating sample magnetometer, scanning electron microscope, X-ray diffraction, Fourier transform infrared, thermogravimetry analysis and differential scanning calorimetry. Thermal stability, optimal pH, optimal temperature, Michaelis constant and maximum reaction rate were compared between free papain and magnetic-cellulose-hydrogel-embedded papain. The magnetic-cellulose-hydrogel-embedded papain was sensitive to magnetic field and showed paramagnetic behavior, higher thermal stability and lowed substrate affinity. The optimal pH and optimal temperature of the magnetic-cellulose-hydrogel-embedded papain were shifted to 8.0 and 90°C respectively.

Synthesis, functionalization, characterization, and in vitro evaluation of robust pH-sensitive CFNs–PA–CaCO3

The preparation, characterization, and application of Papain (PA) conjugated CaCO3-coated cobalt ferrite nanoparticles (CFNs–PA–CaCO3) is reported.

The Extraction of Peanut Protein Peptone Hydrolysate from Cold Pressed Peanut Meal by Papain

This paper studied the extraction of peanut protein peptone hydrolysate from cold pressed peanut meal by papain to provide a theoretical basis on peanut meal special microorganism culture makings products for further research. The study use orthogonal test method to optimize the method of papain preparation of peanut peptone, the best preparation conditions were: enzymolysis temperature 55 °C, pH7.5, substrate concentration 3%, the amount of enzyme 6%, enzymolysis time 6 hours. The Protein yield was 52.02% and the content of amino nitrogen was 0.772 mg %by this perparation condition.

The anthelmintic efficacy of natural plant cysteine proteinases against two rodent cestodes Hymenolepis diminuta and Hymenolepis microstoma in vitro

Little is known about the efficacy of cysteine proteinases (CP) as anthelmintics for cestode infections. We examined the effects of CPs on two rodent cestodes, Hymenolepis diminuta and H. microstoma in vitro. Our data showed that naturally occurring mixtures of CPs, such as those found in papaya latex, and relatively pure preparations of fruit bromelain, papain and stem bromelain, were active in vitro against both juvenile, artificially excysted scoleces, as well as against adult worms of both rodent cestodes. Significant dose-dependent reduction in motility, ultimately leading to death of the worms, was observed with both species, and against both freshly excysted scoleces and 14-day old pre-adult worms. The most effective was fruit bromelain (after 30min of incubation of juvenile H. diminuta and H. microstoma IC50=63 and 74μM, respectively, and for pre-adult worms=199 and 260μM, respectively). The least effective was stem bromelain (after 30min of incubation of juvenile H. diminuta and H. microstoma IC50=2855 and 2772μM, respectively, and for pre-adult worms=1374 and 1332μM, respectively) and the efficacies of papaya latex supernatant and papain were between these extremes. In all cases these values are higher than those reported previously for efficacy of CPs against intestinal nematodes, and in contrast to nematodes, all CPs were effective against cestodes in the absence of exogenous cysteine in incubation media. The CPs appeared to attack the tegument resulting in generalised erosion mainly on the strobila. The scolex was more resistant to CP attack but nevertheless some damage to the tegument on the scolex was detected.

The use of papain inhibitor immobilized onto polyaniline for bioaffinity chromatography of cysteine proteases

In this work, a papain inhibitor extracted from seeds of Adenanthera pavonina was covalently immobilized onto glutaraldehyde modified polyaniline (PANIG). The immobilization was very efficient, presenting 54.24% inhibitor retention in the following conditions: immobilization time of 30min, at 4°C, pH 7.0, inhibitor concentration of 20mg and 5mg of PANIG. The resultant material (PANIG-I) was tested as stationary phase for the cysteine proteases papain, ficin and bromelain purification through bioaffinity chromatography. A commercial preparation of papain was efficiently purified, resulting in a single band with 25kDa. SDS–PAGE of purified bromelain showed a characteristic band around 28kDa and this procedure resulted in 0.89-fold purification and 32.4% yield. The purification process was more effective for ficin reaching 2.6-fold purification and a single band of 25kDa in the SDS–PAGE. These results showed that the stationary phase containing A. pavonina inhibitors immobilized onto PANIG is a very promising material for a single step purification of cysteine proteases through bioaffinity chromatography.

Nanostructural modification of a model homogalacturonan with a novel pectin methylesterase: Effects of pH on nanostructure, enzyme mode of action and substrate functionality

A Carica papaya pectin methylesterase (CpL-PME) present in a commercial papain preparation was used to modify the amount and distribution of anionic charge in a population of a model pectic homogalacturonan (HG) at pH 4.5 and pH 7.5. Introduced negatively charged demethylesterified blocks (DMB) were excised as oligomers with limited endo-polygalacturonase (EPG) digestion, and then separated and quantified by high performance anion exchange chromatography. Exhaustive EPG digestion was also performed and the concentrations of mono-, di- and tri-galacturonic acid (GalA) produced were estimated. The CpL-PME mode of action was modeled while the HG population's nanostructure and the resulting functionality of the modified pectin were characterized. The results indicated that average DMB size and number of these “average” blocks per molecule increased as the overall degree of methylesterification (DM) decreased by CpL-PME treatment (P < 0.05). Average DMB size differed depending on the reaction pH at each DM. Absolute degree of blockiness (DBabs) increased as DM decreased and demonstrated a linear relationship with the DM at both reaction pH values. Significant positive correlations between DBabs and average DMB size were observed for both pH series (P < 0.05). The enzyme mode of action, modeled in silico, demonstrates a processive multiple attack mechanism. Functionally, the storage modulus of ionotropic gels formed from the processed substrates increased as DM decreased, and as DBabs and average DMB size increased with highly significant correlations (P < 0.001). These results indicate it is feasible to engineer pectin nanostructure and functionality with CpL-PME.

Purification and characterization of a papaya (Carica papaya L.) pectin methylesterase isolated from a commercial papain preparation

We purified a Carica papaya pectin methylesterase (CpL-PME; EC 3.1.1.11) from a commercial papain preparation. This CpL-PME was separated from the abundant cysteine endopeptidases activities using sequential hydrophobic interaction and cation-exchange chromatographies and then purified by affinity chromatography using Sepharose-immobilized kiwi PME inhibitor protein to obtain a single electrophoretically homogeneous protein. The enzyme was purified 92-fold with 38% yield, providing a specific activity of 1200 U/mg. The molecular weight was determined to be 35,135 by MALDI-TOF-MS in linear mode. MALDI-TOF-MS peptide mass fingerprinting following trypsin digestion indicated CpL-PME represents a novel Carica PME isoform. The CpL-PME required salt for activity, and it showed a broad activity range (pH 6–9) and moderate thermostability (optimum ca. 70°C). A calcium-insensitive methylated lime pectin treated with CpL-PME to reduce degree of methylesterification by 6% converted the substrate to high calcium sensitivity, indicating a processive mode of action. These properties support further research to apply CpL-PME to tailor pectin nanostructure.

Optimizing Jerky Drying Time with Minimal Product Impact

Jerky products are a popular snack in today's society. The process of making jerky products is very time con- suming -especially the dehydration process. If the process is sped up the producer will save time and money. This study focuses mainly on speeding up dehydration time with minimal potential impact on the final product. Beef jerky was our test product and was made from eye of round sliced to 5 mm, vacuum packaged and frozen to -10C. The product was marinated in a jerky seasoning for 1 hour. A two level factorial array experiment was designed to identify the optimum levels of four control factors that could affect dehydration time: pH, freeze/thaw, product orientation, and tenderization. Half of the packages were thawed at 5C and then refrozen to account for two levels of freeze-thaw cycles. The two pH levels were unadjusted and adjusted. White vinegar was added to the marinade to adjust the pH to within 5 to 5.25. Half of the jerky samples were tenderized by adding 0.5 mg of a commercial liquid papain preparation to the marinade. The meat was pasteurized under a wet bulb temperature of 60C for 12 min and dried for 5 hours at 65C dry bulb. Treat- ments were repeated in triplicate. Tenderizer had the greatest effect on reducing the dehydration time, followed by ad- justed pH and vertical orientation. Dehydration times for all experiments averaged 258 min and ranged from 146 to 386 min. A response optimization model predicted (95% confidence level) a dehydration time of 178 min.

Purification and characterization of a wound-inducible thaumatin-like protein from the latex of Carica papaya

A 22.137 kDa protein constituent of fresh latex was isolated both from the latex of regularly damaged papaya trees and from a commercially available papain preparation. The protein was purified up to apparent homogeneity and was shown to be absent in the latex of papaya trees that had never been previously mechanically injured. This suggests that the protein belongs to pathogenesis-related protein family, as expected for several other protein constituents of papaya latex. The protein was identified as a thaumatin-like protein (class 5 of the pathogenesis-related proteins) on the basis of its partial amino acid sequence. By sequence analysis of the Carica genome, three different forms of thaumatin-like protein were identified, where the latex constituent belongs to a well-known form, allowing the molecular modeling of its spatial structure. The papaya latex thaumatin-like protein was further characterized. The protein appears to be stable in the pH interval from 2 to 10 and resistant to chemical denaturation by guanidium chloride, with a Δ G water 0 of 15.2 kcal/mol and to proteolysis by the four papaya cysteine proteinases. The physiological role of this protein is discussed.

Activity of immobilized papain dehydrated by n-propanol in low-water media.

A propanol-rinsed enzyme preparations (PREP) of papain showed an activity of 59 nmol min(-1) (mg powder)(-1) in tert-butanol at the optimal water activity of 0.2. The immobilized papain was stable in aqueous media for 3 d at 4 degrees C. Solid-state buffers (bases and their HCl salts) suspended in the organic medium decreased the initial rate in all cases tested. The operational stability during Z-Gly-Phe-NH2 synthesis was improved when solid cysteine was added, doubling the yield after 24 h.

Plant lipases: biocatalyst aqueous environment in relation to optimal catalytic activity in lipase-catalyzed synthesis reactions.

Adsorption and desorption isotherms of two commercial enzyme preparations of papain and bromelain were determined with a Dynamic Vapor System. The Guggenheim-Anderson-deBoer (GAB) modeling of the obtained sorption isotherms allowed the definition of different levels of hydration of those samples. Afterward, these enzyme preparations were used as biocatalysts in water and solvent-free esterification and alcoholysis reactions. The evolution of the obtained fatty acid ester level as a function of the initial hydration level of the biocatalyst, i.e., thermodynamic water activity (a(w)) and water content, was studied. The results show an important correlation between the initial hydration level of the biocatalyst and its catalytic activity during the lipase-catalyzed synthesis reactions. Thus, the Carica papaya lipase (crude papain preparation) catalytic activity is highly dependent on the biocatalyst hydration state. The optimized synthesis reaction yield is obtained when the a(w) value of the enzyme preparation is stabilized at 0.22, which corresponds to 2% water content. This optimal level of hydration occurs on the linear part of the biocatalyst's sorption isotherm, where the water molecules can form a mono- or multiple layer with the protein network. The synthesis reaction yield decreases when the a(w) of the preparation is higher than 0.22, because the excess water molecules modify the system equilibrium leading to the reverse and competitive reaction, i.e., hydrolysis. These results show also that an optimal storage condition for the highly hydrophilic crude papain preparation is a relative humidity strictly lower than 70% to avoid an irreversible structural transition leading to a useless biocatalyst. Concerning the bromelain preparation, no effect of the hydration level on the catalytic activity during esterification reactions was observed. This biocatalyst has too weak a catalytic activity which makes it difficult to observe any differences. Furthermore, the bromelain preparation is far more hydrophobic as it adsorbs only 18 g of water per 100 g of dry material at a(w) around 0.90. No deliquescence of this enzymatic preparation is observed at this a(w) value.

Polyclonal-antibody-mediated insolubilization and stabilization of papain

Antisera raised in rabbits against native iodoacetamide- and iodoacetic acid-modified papain contained precipitating and non-inhibitory antibodies. Papain could be insolubilized as enzyme-antibody adducts with the gamma-globulin fraction derived from the antiserum. Two insolubilized papain preparations, designated A and B, with low and high antibody/enzyme ratios, respectively, exhibited high enzyme activity and markedly enhanced thermal and denaturant stabilities. However, papain antibody adduct B was superior in activity and stability over preparation A. The usefulness of immobilized papain in biopharmaceutical and other industries is also discussed.

Investigation of crude latex from various Carica papaya varietices for lipid bioconversions

AbstractThe protein contents in crude latices from various varieties of papaya (Carica papaya) and their catalytic activities in proteolysis, lipolysis, and interesterification reactions were studied with regard to the variety, the geographic location of cultures, and the frequency of fruit tapping. Biocatalytic activities of these raw materials were compared to several commercially available crude and purified preparations of papain. These investigations were carried out in order to have a better physicochemical characterization of these raw materials, to select the adequate papaya latex for protein or lipid bioconversions, and to valorize them on an industrial scale. For the purified preparations of papain, only proteolytic activity was obtained. All crude papaya latices exhibit proteolytic, lipolytic, and interesterification activities, and no relationship between the proteolytic and lipolytic activities was observed. The high multiple correlation coefficient (R) on the order of R=0.93–0.99, obtained from the regression analysis for the lipolytic and interesterification activities for all crude papaya latices investigated suggested that there was a correlation between these enzyme activities. However, for the same lipase preparation, the interesterification activity differed substantially depending on the type of interesterification reaction.

Lipase activity and fatty acid typoselectivities of plant extracts in hydrolysis and interesterification

AbstractLipase fatty acid typoselectivities of Euphorbia characias latex and commercially available crude preparation of bromelain were determined in the hydrolysis of homogeneous triacylglycerols (TAG) and natural TAG mixtures. Their activities were compared to a commercially available crude preparation of papain. Under optimal lipolysis conditions at pH 8.0 and 10 min of incubation time, maximal activities were observed at 45, 55, and 50°C, respectively, for E. characias latex, crude bromelain, and crude papain. Commercially available crude preparations of bromelain exhibited very poor hydrolysis activity. Latex from E. characias, which contained 340 mg of dried material per milliliter of fresh latex, exhibited a high lipase activity and a short‐chain fatty acid preference in the hydrolysis of homogeneous TAG. For all substrates, it showed a better activity than crude papain. Lipase fatty acid typoselectivities of crude bromelain and crude papain also were studied in interesterification reactions of tributyrin with a series of homogeneous TAG. Experiments showed that crude bromelain [water activity (Aw)∶ 0.21] had no activity in interesterification. Regarding reactions with crude papain (Aw∶ 0.55), yields of newly formed TAG decreased with increasing chain length of TAG, except for the reaction with trimargarin. For interesterification of tributyrin with unsaturated TAG, triolein reacted faster than polyunsaturated TAG. During these interesterification reactions, the proportion of new TAG with two butyroyl residues was higher than new TAG with only one butyroyl residue. This phenomenon was more pronounced for reactions with long‐chain TAG.

Association between sensitization to natural rubber latex and papain

Several studies have documented clinical and immunologic cross-reactivity between natural rubber latex (NRL) and a variety of fruits, mainly banana, avocado, and kiwi?, 2 We fortuitously observed that a nurse with NRL allergy also showed skin reactivity to papain, although she had had no known exposure to this enzyme, which is extracted from the latex of the papaya tree (Carica papaya): Papain is widely used in the food industry (meat tenderizer, beer clearing agent), as well as in pharmaceuticals (local treatment of necrotic ulcers, digestive aid, cleaning agent for contact lenses, chemonucleolysis) and cosmetic products? -5 The latex of C. papaya and commerCial preparations of papain contain a number of characterized enzymes, including cysteine proteinases (papain itself, chymopapain, caricain, and endoproteinase Gly-C; molecular weight, 23 to 24 kd) and chitinase (molecular weight, 28 kd). 6 Papain has been documented as causing IgE-mediated allergic reactions after domestic and occupational exposure. 34 Quarre et al. 7 recently reported an association between allergy to NRL and papain in a subject with occupational exposure to both allergens. The purpose of this study was to assess the prevalence of sensitization to papain among health care workers with and without NRL allergy. METHODS Subjects