What is the Papain?
Papain, also known as papain, is a proteolytic enzyme. Papain is a low-specificity proteolytic enzyme contained in Carieapaya. It is widely present in the roots, stems, leaves and fruits of Carieapaya, and is most abundant in immature milk. The active center of papain contains cysteine and is a thiol protease. It has the characteristics of high enzyme activity, good thermal stability, natural health and safety, etc., so it is widely used in food, medicine, feed, daily chemicals, leather and textile industries. application.
Papain structure and chemical properties
Immature fruits of papaya contain various proteolytic enzymes such as papain, Chymopapain A, Chym opapain B, and Papaya Peptidase B. And it is known that the primary structures of the four cysteine proteases have a high degree of homology. Among them, papain is a thiol protease that can hydrolyze the carboxyl terminus of arginine and lysine in proteins and peptides, and can preferentially hydrolyze those amino acids or aromatic L-amino acid peptide bonds that have two carboxyl groups at the N-terminal of the peptide bond.
Papain is a proteolytic enzyme with a molecular weight of 23,406 and consists of a single peptide chain containing 212 amino acid residues. There are at least three amino acid residues present in the active center of the enzyme. They are Cys25, His159 and Asp158. When Cys25 is oxidized by oxidants or combined with metal ions, the activity of the enzyme is inhibited, and the reducing agent cysteine (or Sulfites) or EDTA can restore enzyme activity. The other six cysteine residues formed three pairs of disulfide bonds, and none of them were in the active site. Pure papain products can contain: (1) papain, molecular weight 21,000, accounting for approximately 10% of soluble protein; (2) chymotrypsin, molecular weight 26,000, accounting for approximately 45% of soluble protein; (3) lysozyme, molecular weight 25,000, accounting for about 20% of soluble protein; and different enzymes such as cellulase.
Papain is a protease that can decompose proteins in acidic, neutral, and alkaline environments. Its appearance is white to light yellow powder, slightly hygroscopic; papain is soluble in water and glycerin, and the aqueous solution is colorless or light yellow, sometimes milky white; it is almost insoluble in organic solvents such as ethanol, chloroform, and ether. Papain is a sulfhydryl (-SH)-containing endopeptidase with protease and esterase activities, broad specificity, and strong hydrolysis ability for animal and plant proteins, polypeptides, esters, amides, etc. But it can hardly break down peptone.The most suitable pH value of papain is 6 to 7 (generally 3 to 9.5 is acceptable), and it also works in neutral or acidic conditions. The isoelectric point (pI) is 8.75; the most suitable temperature of papain is 55 to 65°C ( Generally, it can be anywhere from 10 to 85℃), it has strong heat resistance and will not be completely deactivated at 90℃; it is inhibited by oxidants and activated by reducing substances.
Papain production method
The crude product is obtained by extracting the emulsion from the immature fruit of papaya, coagulating, settling and drying it. In general industry, crude products are mainly used. There are three production methods for papain, namely direct hot air drying, spray drying and membrane separation freeze-drying.
1.Dry directly
The production method is relatively simple and fast, but the finished product is only a crude enzyme with many impurities, poor color, excessive microorganisms, and low enzyme activity, only 600,000 to 800,000 units/g. Mostly used by individual factories, it can no longer meet the requirements of food quality, hygiene and safety.
2.Spray drying
This method first removes some impurities through centrifugation and then spray-dries. The activity of the produced finished enzyme is relatively high, reaching about 1 million units/g, with relatively few impurities and a relatively white color. However, the product easily sticks to the wall when sprayed, causing a large loss of enzyme activity, relatively poor water solubility, and poor enzyme activity stability.
3.Membrane separation
This production method not only avoids the shortcomings of the above two methods, but also has a hygienic and safe production process and a high yield. The enzyme activity of the finished product produced is small, and the enzyme activity can generally reach 2.8 million to 3.5 million units/g, with a maximum of 4 million units/g, with good enzyme activity stability, high purity, white color, low bacterial count, and strict operation can reach pharmaceutical grade.
The extraction process conditions are ultrasonic power of 300W, ultrasonic treatment time of 200s, and pulp mass fraction of 30%. After ultrasonic enhanced extraction, the enzyme activity is increased to 1.71 times the original; the enzyme activity is below 40°C and the pH is 5.4 to 6.0. Relatively stable; EDTA, Cys and vitamin C have activating effects on enzyme activity, CuS04 and ZnCl2 have inhibitory effects, while KCl, NaCI, CaCl2 and MgS04 have little effect on enzyme activity.
The separation and purification process of papain: using preliminary collection treatment, 20% and 40% ammonium sulfate fractional precipitation, SP-sephadexC50 column chromatography, and hydroxyapatite column chromatography, papain is separated and purified from papaya milk. A pure enzyme with a specific activity of 1184U/mg can be obtained, with an activity recovery rate of 55.79% and a purity of 99.31%. It is a high-purity papain.
Papain application range
Food industry
In the food industry, papain can be used for meat tenderization, beer clarification, biscuit loosening and other purposes.
Beer clarifier
The main reason why beer becomes cloudy when refrigerated is that the protein in beer easily combines with polyphenols to form macromolecular complexes. The papain in the beer clarifier has broad specificity for proteins that form turbidity. It can degrade large molecular proteins into small molecules and improve the solubility of the protein and polyphenol complex. On the other hand, papain is A biologically active protein that can form a stable balance with the polyphenols that cause refrigeration turbidity in beer, thereby preventing refrigeration turbidity in beer.
2.Meat tenderizer
Papain can cleave the collagen and muscle fibers in meat to loosen the structure of the meat. Because papain is a cysteinyl protease, it can degrade collagen fibers and connective tissue proteins. It degrades actomyosin and collagen into small molecule peptides and even amino acids, causing muscle filaments and tendon filaments to break, making meat It becomes tender and smooth, and simplifies the protein structure to make it easier for the human body to digest and absorb after consumption.
3.Cookie softener
The enzymatic reaction of papain is used to degrade the protein of the dough into small molecule peptides or aminoases, which reduces the tensile resistance of the dough, making the dough softer, more plastic, less elastic, and easier to form. The dosage varies depending on the processing method of the biscuit factory and the protein content in the dough. Research shows that it is better to add 0.6-10,000 units/g per kilogram of dough.
Scientific research application
Papain can be used in cell culture experiments and is used to separate cells in the first step of preparing cell culture. After treating the small tissue pieces with enzymes for 10 minutes, the extracellular matrix connecting the cells can be broken; then use protease inhibitors to stop the reaction to prevent papain from further cleavage of the cells themselves; and finally use Pasteurpipette to break up the tissue pieces into individual pieces. cell suspension.
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