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Papain and its application
What is papain?
Papain is an enzyme with a protein hydrolytic function and cysteine protease activity. The enzyme belongs to a superfamily that consists of a single polypeptide chain containing three disulfide bonds and a sulfhydryl group essential for enzymatic activity and is derived from the green papaya (Figure 1), where the papain activity depends on the green colour of the fruit. Papain (product number:P128675,etc.) has an important role in many important functions in all living organisms32. For example, it has a wide range of proteolytic functions for short-chain peptides, proteins, esterified amino acids and amide bonds, and has a wide range of functions in pharmaceutical and food ingredients. It specifically cleaves positively charged amino acid peptide chains, mainly lysine (product no.D139442,etc.), arginine (product no.A118651,etc.) and phenylalanine (product no.Z341221,etc.) residues.
Molecular weight: 23406 Da (amino acid sequence)16, the activity is the most suitable pH:6.0-7.0; The optimum temperature of activity is 65℃22, and the spectral characteristic is λmax:278 nm19；Extinction coefficient：E1% = 2519，EmM = 57.6 (280 nm)20.
Inhibitors: Emur64 (product number:E109034,etc.), cystamine dihydrochloride(productnumber:C153647,etc.), chymotrypsin inhibitor (product number:C113165, ebse(product number:E425045,etc.), leucopeptin trifluoroacetate (product number:L332045).
Activators: cysteine (product number:D304972,etc.), heavy metal chelating agents such as EDTA, N-bromosuccinimide (product number:B105057).
Unit definition: hydrolysis of 1.0μ mol en-α-benzoyl-L-arginine ethyl ester per minute at 25 °C, pH6.2
Figure 1.?Isolated from green papaya
Specificity
Papain can digest most of the protein substrates and has a wider range than trypsin (product number:T274333). Papain has a wide range of specificity and can break the peptide bonds of basic amino acids, leucine or glycine. It can also hydrolyze lipids and amides. Papain is more inclined to amino acids with large hydrophobic side chains at P2 position, and does not accept valine at P1' position.
Solubility and solution stability
Papain is soluble in water with 10mg/ml. Before use, dilute it in a buffer containing 5mM L-cysteine.?Its activation/stabilizers include EDTA, cysteine and dimercaptopropanol21. Although papain solution has good temperature stability, its stability depends on pH.?Under acidic conditions, papain solution is not stable, for example, when pH is lower than 2.8, its activity decreases significantly.?In the dissolved state, the activity of papain loses about 1-2% per day, which may be caused by self-decomposition and/or oxidation. The common inactive form of papain obtained during separation is a mixed disulfide formed between sulfhydryl and free cysteine at the active site of the protein23. The papain solution also remained stable in several denaturants, for example, remained intact after recrystallization in 70% methanol and 8m urea solution. However, the activity of papain decreased significantly after being treated with 10% trichloroacetyl or 6m guanidine hydrochloride (product number:G108673,etc.).
A wide range of applications
Life science industry
1.?Papain is widely used in cell separation, and it has been proved to be more efficient and less destructive than other proteases in some tissues. For example, papain can be used to isolate active, morphologically intact cortical neurons of postnatal rats2. Papain can isolate a large number of active smooth muscle cells in the process of isolating smooth muscle cells, and does not affect the sensitivity of cells to stimulants5.
2.?The partial hydrolysis of papain can be used to study the structure of enzymes and other proteins.
3.?Papain can be used in erythrocyte serology. It enhances or destroys the reactivity of many red blood cell antigens by modifying the surface of red blood cells. It can be used as an auxiliary means of classification, antibody screening and antibody identification. Papain is also used in platelet serology9.
4.?Papain can also be used in the enzymatic synthesis of amino acids, peptide chains and other molecules10-13.
5.?Fab and F (ab') 2 antibody fragments can be used in experimental systems where FC domain cannot be found. In this case, it is more appropriate to use only antigen binding fragments (Fab).?Papain can be used to prepare Fab fragments of immunoglobulin G routinely.?Immunoglobulin M can also be digested by papain to produce a large number of homologous Fab fragments15.?Papain cleaves the antibody into two Fab fragments whose variable regions specifically recognize antigens, and a Fc fragment14.?Its cutting position is above the hinge region of the disulfide bond between the heavy chain and the light chain, and below the disulfide bond site between the heavy chain and the light chain, thus forming two separate Fab fragment univalents (each containing a single antibody binding site) and a complete Fc fragment.?Each fragment can be purified by gel filtration, ion exchange or affinity chromatography. In lymph nodes, spleen and other tissues, or in peripheral blood preparations, cells containing Fc receptors (such as macrophages, monocytes, B lymphocytes, natural killer cells, etc.) can bind to the FC domain of complete antibodies, resulting in background staining in areas without target antigens.?The application of Fab fragment ensures that the antibody only binds to the antigen rather than the Fc receptor.?These fragments can also be used for cell staining in the presence of plasma because they cannot bind to the complement that can dissolve the cell.?The application of Fab fragments makes the localization of target antigens more accurate, such as in tissue staining under electron microscopy (figure 2).
Figure 2. Pepsin and papain cleavage
Health care industry
Papain supports the immune system that treats tumors because it controls and alters white blood cells in the immune response. Swelling and redness of joints and prostate are reduced24. Papain from raw papaya showed 90% activity. In 2012, Hitesch et al studied the role of this enzyme by caking formation and absorbance determination. The enzymatic degradation compounds of levetiracetam and granules which have dangerous effects on the cell system were obtained.?The results of spectrophotometry and TLC analysis showed that the effect was good25. Papain also has antifungal properties. In 2010, Chukwuemka and Anthony reported that papaya can reduce the fungal infection that causes papaya to rot, investigated different fungi obtained from Rhizopus, Aspergillus and mucus, found inhibition bands to reduce hyphae, and statistically tested that the use of papaya can extend the shelf life of papaya26.?Papain can reduce the allergic reaction of exudative dyspepsia syndrome, reduce hydrochloric acid and reduce the tolerance to gelatin. It has been shown to have significant analgesic and anti-inflammatory effects on head and tooth pain allergies without side effects27. It plays a vital role in skin and wound care, and Hawaii and Tahiti have proven successful in using concentrated papain extracted from papaya to treat skin wounds, burns, bed sores, irritation and rashes.?In 2012, Detrich used papain to treat sports injuries and sports feet, and 3-8 days was the recovery time after using papain28.
Food industry
In the world, papain is a widely studied and most commonly used enzyme in the food industry. Papain is used to tenderize meat proteins, especially myofibril and connective tissue.?5% or 3% of hen leg slices were treated with different concentrations of papain (0.25%, 0.05%, 0.075%, 0.1%) to improve meat tenderness.?The concentration of 0.25% papain showed significantly higher tenderness at the 3% level than the 0% control group.?The injection multiple and bifurcation plus soaking technique were used to cut the meat. In this study, 0.25% papain combined with bifurcation and soaking was used to tenderize the waste chicken to improve its functional characteristics in order to achieve the purpose of effective utilization29.?Papain plays an important role as a clarifier in food industry. Because it breaks down proteins, it can be digested and plays an important role in controlling indigestion and other digestive and gastrointestinal diseases30.?The results showed that papain improved the melting and extensibility of Nabursi cheese in different pastries, pizza and Kunafa cheese by cross-linking casein. Nabursi cheese treated with papain has good fiber structure, good effect and good storage performance in the use of pizza and pastry, which leads to melting or elongation caused by high salt, which is an effective method. Papain is used as a softener in chewable gum31.
Summary
In this paper, the basic structure, mechanism, production and industrial application of papain were reviewed. Papain is one of the most widely used hydrolases in the market and has a good market prospect. Through recombinant DNA and immobilization technology, papain is expected to become an ideal material for industrial application. Researchers can explore more effective applications by understanding the detailed mechanism and function of papain. Then, there will be new papain functions to meet people's huge demand for improving their lives.
References
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