木瓜酵素.Papain Enzyme


Papain is a protease present in papaya which is useful in tenderizing meat and other proteins. Its utility is in breaking down the tough meat fibers and has been utilized for thousands of years in its native South America. It is sold as a component in powdered meat tenderizer available in most supermarkets. It is also sold as a dietary supplement to remedy indigestion.


ribbon diagram]An enzyme (in Greek ''en'' = in and ''zyme'' = leaven) is a protein, or protein complex, that catalyzes a chemical reaction and also controls the 3D orientation of the catalyzed substrates. Like any catalyst, enzymes work by lowering the activation energy of a reaction, thus allowing the reaction to proceed to its steady state or completion much faster than it otherwise would; the enzyme (again, as with any catalyst) remains unaltered by the completed reaction and can therefore continue catalysis. It is important to note that, as with all catalysts, all reactions catalyzed by enzymes must be 'spontaneous' (containing a net negative Gibbs free energy), ''i.e.'' with the enzyme, they run in the same direction as they would without the enzyme, just more quickly; the concept is similar to the likelihood of a ball rolling down a hill versus the likelihood of it rolling up the hill. Catalysis by an enzyme is analogous to removing a pebble that is stopping the ball from rolling down the hill; the reaction goes to completion more quickly, but the final product is identical. Given a particular starting set of conditions, the end products of a particular reaction (including net energy), once steady state is reached, must always be identical, independent of the specific individual pathway taken from beginning point to end point. This is required by the Law of Conservation of Energy, which would be violated by the possibility of a cycle of moving down a pathway releasing less net energy and back up a different pathway with higher net energy, or vice versa. An enzyme can, however, run a normally non-spontaneous reaction 'backwards' by coupling it to a spontaneous one, as long as the net free energy from the total of both reactions is negative. Enzymes are necessary within biological cells because most chemical reactions required by the cell would occur too slowly to sustain life; oxidation of organic food compounds to provide energy is one example.



A practical method is used to study chemical reactions in solution and in enzymes. In this method, referred to as the "Classical Trajectory Mapping" method, the trajectories are calculated on a classical potential surface and the free energy profile of the chemical reaction is calculated by using this classical surface as a reference state for the actual ground state potential described by the semiempirical AM1 method. The speed of our method is shown to be faster than that of the conventional approach because our method does not require the time-consuming quantum mechanical calculations at each iteration. This method was applied to proton-transfer reactions both in aqueous solution and in papain. The encouraging results indicate that our approach should be useful in studying chemical reactions in solution and and in enzymes.proteolytic” is a catch-all term referring to enzymes that digest protein. Supplemental forms can incorporate any of a wide variety of enzymes including Trypsin, Chymotrypsin, Pancreatin, Bromelain, Papain, and a range of fungal proteases. In the body, proteolytic digestive enzymes are produced in the pancreas, but supplemental forms of enzymes may come from fungal or bacterial sources, extraction from the pancreas o livestock animals (trypsin/chymotrypsin) or extraction from plants (such as Papain from the papaya and Bromelain from pineapples). The primary uses of proteolytic enzymes in dietary supplements are as digestive enzymes, anti-inflammatory agents and pain relievers.

The proton-transfer in papain:

As a second example, we studied the proton-transfer reaction in papain. This enzyme, which catalyzes the hydrolysis of polypeptide substrates, is a cysteine protease composed of 212 amino acid residues that include a cysteine (Cys-25) and a histidine (His-159) in the active site. Although in solution the normal pKa's for histidine and cysteine are around 6 and 10, respectively, experimental evidence16 indicates that in protein these two residues form an ion pair, i.e., a proton is transferred from the SH group of Cys-25 to Nd1 of His-159. The proton transfer reaction is described by the equation

where the notations, SH and Im, denote cysteine and histidine, respectively. The VB structures and the diabatic potential functions were similar to Eq.  8 and 9. Extra terms such as the torsional interactions have to be included in the classical force field to account for the imidazole ring, and the `s appearing in Eq. 9 are understood to denotes both water and the enzyme, which solvate the quantum mechanical atoms. Following Warshel's procedure10-12, we calibrated the van der Waals parameters of cysteine and histidine by using their corresponding pKa values in solution, and then we used the refined parameter set in the simulation of the protein papain. In our calculations, the imidazole ring of histidine and the side-chain group, i.e., CH3SH, of cysteine are treated quantum mechanically, while the other atoms of the systems are described by classical force fields.

In the simulations of papain, unlike in the first example, there are now hybrid bonds that connect the quantum mechanical and classical atoms. To handle this problem, we introduce a dummy atom along the hybrid bond and treat the dummy atom as a quantum mechanical hydrogen atom which does not interact with the rest of the classical atoms. This approach was used by several groups3,16-18 and was shown to be quite useful.

 [Animal & Fish Feed]

l          Splits chains of protein, producing small peptides and free fatty acids, and helps in protein digestion.

l          Hydrolyses starch molecules into smaller oligosaccharides, glucose and maltose.

l          Ensures availability of adequate nutrient supply for growth and performance.

l          Responsible for the absorption of ingested fats and mobilization of storage fats in animals.

l          Helps in degrading cellulose and various plant fibrous material.

l          Digests high molecular weight Arabinoxylans in animal feeds.

l          Degrades phytin phosphorus and converts it into available form, thereby reducing the quantity of supplemented phosphorus. A cocktail enzyme preparation, tailor-made to suit the customer's requirements, is also available.

l          The major use of Papain is in the drug heparin. vaccines and digestive enzyme. It is also as an excellent enzyme for breaking down proteins.

l          Is widely used for replacement therapy in pathological conditions in which the concentration of bile acids in upper intestine is low, such as biliary fistula, disease of the ileum, hepatic or extra-hepatic cholestasis.

l          Is an excellent fibrinolytic and caseinolytic enzyme which in combination with amylase and lipase proves to be formidable digestive aid. It may be administered in acid solution to increase the digestive power of gastric juices, particularly where there is a deficiency of pepsin secretions.


Topically, papain has been used for skin conditions such as psoriasis. It¨s ability to break down proteins is used to remove dead tissue from burns, to help skin injuries heal, to remove warts, and to treat ringworm. Cold sores caused by Herpes zoster virus have been treated successfully with both oral and topical papain-containing products. In one small study of individuals with Herpes zoster, an oral papain product was as effective as a prescription antiviral medication in resolving pain.  

Enzymes accelerate reactions within body cells. In the human body, the pancreas usually produces enzymes that break down foods into nutrients that the body can use for energy and other functions. Papain is an enzyme produced by the tropical fruit, papaya. Papain is proteolytic, which means that it digests proteins. Enzyme deficiencies are rare, but individuals who have cystic fibrosis or diseases of the pancreas may not produce enough natural enzymes to digest foods properly. Frequently, papain is included in prescription combinations of digestive enzymes to replace what individuals with these conditions cannot produce naturally. Because it improves digestion in general, papain has been used orally to treat less serious digestion disorders such as bloating and chronic indigestion. Since parasitic organisms are largely proteins, papain has sometimes been taken internally to eliminate intestinal worms.

Optimum pH: 6.0 - 7.0.

Extinction coefficient:  = 25.0

Isoelectric point: pH 9.6

Activators: Papain is activated by cysteine, sulfide, sulfite, etc. It is enhanced when heavy metal binding agents such as EDTA are also present. Kirschenbaum indicated that N-bromosuccinimide enhances the activity. Hall et al. report on the affects of acridine dyes. 

Inhibitors: Substances which react with sulfhydryl groups including heavy metals, carbonyl reagents. Westerik and Wolfenden have studied aldehydes as papain inhibitors and Sluyterman and have studied aldehydes as papain report on benzoylamidoacetonitrile as an inhibitor. See Shapira and Arnon on antibody inhibitors. Papain may be inactivated by H2O2 generated by [[gamma]]-irradiation of H2O- the active SH group being oxidized to sulfenic acid. See also Allison and Swain. 

Stability: Papain as a crystalline suspension is stable at 5°C for 6-12 months. Stabilizing agents are EDTA, cysteine and dimercaptopropanol.

To enhance stability as well as solubility it may be advantageous to convert crystalline papain to its mercury derivative (Brubacher and Bender 1966).


Fig. 1. Structures of S-nitroso compounds used in this study. SNO, S-nitroso compound; Pen-SNO, S-nitrosopenicillamine; Dansyl-SNO, S-nitrosodansyl.


Scheme 3. RSNO, S-nitrosothiol; SNO, S-nitroso compound; TNB, 5-thio-2-nitrobenzoate anion.



A titrimetric determinatin of the acid produced during the hydrolysis of benzoyl-L-arginine ethyl ester (BAEE). One unit will hydrolyze one micromole of benzoyl-L-arginine ethyl ester per minute at 25°C and pH 6.2 under the specified conditions.


  • Enzyme diluent (Activation buffer): Prepare fresh daily by mixing the following:

0.01 M EDTA

10 ml

0.06 M Mercaptoethanol

0.1 ml

0.05 M CysteineHCl

10 ml

Reagent grade water

70 ml

  • Substrate solution: Prepare fresh daily by mixing the following:

0.058 M BAEE

15.0 ml

0.01 M EDTA

0.8 ml

0.05 M CysteineHCl

0.8 ml


l          Adjust pH to 6.2 and dilute to a final volume of 21 ml with reagent grade water.

l          Titrant: 0.01-0.02 N NaOH, standardized


Activate enzyme by dissolving in enzyme diluent to a concentration of 0.05-0.1 mg/ml. Under these conditions activation is complete within 30 minutes.

Determination of protein concentration


The reaction can be measured with either an automatic titrator or a laboratory pH meter. The titration vessel should be maintained at 25°C.

Pipette the following into the titration vessel at 25°C:

Substrate solution

5.0 ml

3.0 M NaCl

5.0 ml

Reagent grade water

5.0 ml

At zero time add 0.1 ml of appropriately diluted enzyme and adjust the pH to 6.2. Record the amount of standardized NaOH added per minute to maintain the pH at 6.2 after a constant rate is achieved.



Mercuripapain must be activated before use. Mercury is removed from the enzyme in activation buffer. After 30 minutes in this solution, the enzyme is completely activated and the mercury has been chelated. The mercuripapain suspension contains no free mercury. The product has been extensively dialyzed prior to packaging.

      Papain has been employed to treat ulcers, dissolve membranes in diphtheria, and reduce swelling, fever and adhesions after surgery. With considerable risk, it has been applied on meat impacted in the gullet. Chemopapain is sometimes injected in cases of slipped spinal discs or pinched nerves. Precautions should be taken because some individuals are allergic to papain in any form and even to meat tenderized with papain.

 In tropical folk medicine, the fresh latex is smeared on boils, warts and freckles and given as a vermifuge. In India, it is applied on the uterus as an irritant to cause abortion. The unripe fruit is sometimes hazardously ingested to achieve abortion. Seeds, too, may bring on abortion. They are often taken as an emmenagogue and given as a vermifuge. The root is ground to a paste with salt, diluted with water and given as an enema to induce abortion. A root decoction is claimed to expel roundworms. Roots are also used to make salt.

 Crushed leaves wrapped around tough meat will tenderize it overnight. The leaf also functions as a vermifuge and as a primitive soap substitute in laundering. Dried leaves have been smoked to relieve asthma or as a tobacco substitute. Packages of dried, pulverized leaves are sold by "health food" stores for making tea, despite the fact that the leaf decoction is administered as a purgative for horses in Ghana and in the Ivory Coast it is a treatment for genito-urinary ailments. The dried leaf infusion is taken for stomach troubles in Ghana and they say it is purgative and may cause abortion.

 Studies at the University of Nigeria have revealed that extracts of ripe and unripe papaya fruits and of the seeds are active against gram-positive bacteria. Strong doses are effective against gram-negative bacteria. The substance has protein-like properties. The fresh crushed seeds yield the aglycone of glucotropaeolin benzyl isothiocyanate (BITC) which is bacteriostatic, bactericidal and fungicidal. A single effective does is 4-5 g seeds (25-30 mg BITC).