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papain
Papain, commonly known as "papaya enzyme," is a proteolytic enzyme derived from the latex of the Carica papaya fruit. Its English name is Papain, and it has a molecular weight estimated at approximately 20,700 Da using sedimentation-diffusion methods, while amino acid composition calculations suggest a value around 23,000 Da. As a thiol protease, papain exhibits broad substrate specificity, breaking down a wide range of proteins more effectively than many pancreatic proteases. In addition to its proteolytic activity, papain also possesses lipase activity.
The unit definition for papain is based on its ability to hydrolyze 1 μmol of benzoyl-L-arginine ethyl ester per minute at 25°C and pH 6.2. Structurally, papain consists of a single polypeptide chain with 211 amino acid residues that folds into two domains, creating a cleft where substrates can bind. The enzyme contains only one essential sulfhydryl group, which plays a critical role in its catalytic function. Activators such as cysteine, dithiothreitol (DTT), sodium sulfite, and EDTA enhance its activity, while inhibitors like heavy metals, sulfhydryl reagents, and hydrogen peroxide can suppress it.
To prepare papain, the process begins with the extraction of crude enzyme from dried papaya latex. A mixture of dried papaya emulsion, diatomaceous earth, and washed sand is ground with a cysteine solution to facilitate extraction. The suspension is filtered, and the filtrate is collected. Subsequent steps include adjusting the pH to 9 to remove insoluble impurities, followed by ammonium sulfate precipitation to concentrate the enzyme. Sodium chloride is then used to further precipitate papain, leading to crystallization. Crystals are obtained by suspending the precipitate in a low-concentration cysteine buffer and allowing it to crystallize over time. Recrystallization can be performed using methanol or lithium salts to increase purity and specific activity.
Papain is stable when stored at 4°C, though its activity may decrease by about 20% after six months. This enzyme is widely used in food processing, pharmaceuticals, and biotechnology due to its unique properties and high efficiency in protein digestion.