Papain

Papain is a low-specific proteolytic enzyme contained in carieapapaya. It is widely present in the roots, stems, leaves and fruits of carieapapaya, with the most abundant content in immature latex. Papain belongs to sulfhydryl protease, of which the active site contains cysteine. It has the characteristics of high enzyme activity, good thermal stability and safety. Hence it is widely used in food, medicine, feed, chemical, cosmetic, leather and textile industries.

Structure

Papain consists of a single peptide chain with 212 amino acid residues. There are at least three amino acid residues present in the active site of the papain, including Cys25, His159 and Asp158. The enzyme activity will be inhibited when Cys25 is oxidized or bound to a metal ion, the reducing agent cysteine (or sulfite) or EDTA can restore the enzyme activity. The other six cysteine residues form three pairs of disulfide bonds, both of which were not at the active site.

Chemical Properties

Papain is a protease that hydrolyze proteins in acidic, neutral, and alkaline environments. It is white to light yellow powder with slightly hygroscopicity. Papain is soluble in water and glycerol, aqueous solution is colorless or pale yellow, sometimes milky white. It is almost insoluble in ethanol, chloroform, ether, and other organic solvents. Papain is a sulfhydryl-containing (-SH) endopeptidase with protease and esterase activity. It has a wide range of specificities and has strong ability to hydrolyze animal and plant proteins, peptides, esters and amides, but it hardly hydrolyzes peptone. The most suitable pH value of papain is 6~7, isoelectric point (pI) is 8.75. The most suitable temperature is 55~65℃, but it has strong heat resistance and will not be completely inactivated even at 90℃; Papain is inhibited by oxidants and activated by reductants.

Catalytic Mechanisms

Papain hydrolyzes the carboxyl terminal of arginine and lysine in proteins or polypeptides and preferentially hydrolyzes those peptide bonds that have amino acids with two carboxyl groups at the N-terminus or aromatic L-amino acids. Mechanisms of papain cutting peptide bonds include deprotonation of Cys-25 under the action of His-159. Then Cys-25 nucleophilic attack the carbonyl carbon of peptide chains and is covalently linked with it to form an acyl-enzyme intermediate. Papain then interacts with a water molecule to deacylate and release the carbonyl terminal of the peptide.

Production Methods

Direct drying: The production method is simple and fast, but the finished product is only crude enzyme with impurities, poor color, exceeded microorganism, and low enzyme activity. This method is mostly used by individual factories and cannot meet the quality of food health and safety.

Spray drying: Spray drying is conducted after remove of some impurities by centrifugation. Enzyme activity of finished products is relatively high with less impurities. However, the products are easy to adhere to the wall when spray leading to great loss of enzyme activity and poor water-soluble. Meanwhile the stability of enzyme activity is also relatively poor.

Membrane separation method: The production method not only avoids the disadvantages of the above two methods, but also has the advantages of safety, little loss of enzyme activity, good enzyme activity stability, high purity, white color, and low microbial count.

Application

Food industry: Papain is mainly used for cold hardiness of beer (hydrolyze proteins in beer to avoid turbid after cold storage), meat softening (hydrolyze muscle proteins and collagen), preparation of cereal pre-cooking, and other food industries. The application of cold hardiness of beer and meat softening is far more extensive than other proteases.

Scientific research: Papain can be used in cell culture experiments and used to separate cells. Papain’s treatment of small tissue blocks for 10 minutes can break the extracellular matrix to interrupt the connections between cells. Protease inhibitors are then used to stop the reaction and prevent the papain from further lysing cells. In immunology, papain can be used to separate Fc fragments of immunoglobulins from Fab fragments.