Chinese name: papain English name: Papain (Carica papaya) molecular weight: about 20700 (calculation by sedimentation-diffusion method); about 23000 (calculated according to amino acid composition). Enzyme reaction: Papain is a thiol protease. It breaks down more and more extensive protein substrates than pancreatic proteolytic enzymes. It also has lipase activity. Unit definition: The amount of enzyme which hydrolyzes 1 μmol of molecular benzoyl-L-arginine ethyl ester at 1 °C at 25 ° C, pH 6.2 is 1 unit. Physical and chemical properties: Papain is a single peptide chain with 211 amino acid residues folded into two parts to form a crack. The enzyme molecule has only one sulfhydryl group and is essential for enzyme activity. Activators are cysteine, sulfide, sulfite and EDTA. Inhibitors include sulfhydryl reagents including heavy metal and carboxyl reagent hydrogen peroxide. Preparation method 1. Preparation of crude extract Dried papaya emulsion (180 g) and diatomaceous earth (100 g), washed sand (150 g) were mixed at room temperature with 200-300 mL of 0.04 mol/L cysteine ​​(6.3 G-cysteine ​​hydrochloride is dissolved in 1000 mL of 0.0054 mol/L sodium hydroxide, and an excess of alkali is required to adjust the pH of the water extract to 5.7) and thoroughly ground in a mortar. It can also be extracted with a masher. After the suspension was placed, the supernatant was decanted. Repeated grinding and extraction can be reused with 300 mL of cysteine ​​solution; the mortar is washed with a cysteine ​​solution to adjust the volume of the extract to 1 L. The resulting suspension was filtered through a cold chamber using a 18 cm Buchner funnel (0.5 cm layer of kaolin and filtered with suction). The time required for filtration is related to the thickness of the dry latex used, which can be slow. The filtrate (Part 1) is greenish yellow opaque and the pH should be close to 5.7. The next few steps should be carried out in the cold room unless otherwise noted. 2. Removal of pH 9 Insolubles Slowly add about 110 mL of mol/L sodium hydroxide with stirring to adjust the pH to 9. The resulting gray precipitate was removed by filtration or centrifugation (2600 rpm, 1 hour). The supernatant (Part 2) should be clarified. The precipitate containing the denatured protein is discarded. 3. Ammonium sulfate fractionation The second part was added with ammonium sulfate to a saturation of 0.40. After 1-2 hours, the suspension was centrifuged (2500 rpm). The supernatant was discarded (or reserved for papain production) and the pellet (Part 3) was washed once with 400-500 mL of 0.40 saturated ammonium sulfate. 4. Sodium chloride fractionation precipitation Part 3 was dissolved in 600 mL of 0.02 mol/L cysteine ​​(pH 7-7.5), and 60 g of solid sodium chloride was slowly added to precipitate papain. After 1 hour, centrifugation (2500 rpm, 1 hour), the precipitate was collected (Part 4), and the supernatant was discarded. 5. Crystallization Part 4 was suspended in 400 mL of 0.002 mol/L cysteine ​​pH 6.5 at room temperature. The suspension was immediately reconditioned to 6.5. The suspension was allowed to stand at room temperature for 30 minutes, at which time a crystallization of crystals was produced and allowed to stand at 4 ° C overnight. The crystals were collected by centrifugation (25,000 rpm, 4-5 hours) in a cold place (Part 5), and the supernatant was discarded. 6. Recrystallization Part 5 was dissolved in a minimum amount of distilled water at room temperature (protein concentration was about 1%), and a saturated sodium hydride solution (10 mL / 300 mL of protein solution) was added very slowly with stirring. When about 75% of the solution is added, the papain will begin to crystallize at room temperature. The suspension was placed at 4 ° C overnight, and the crystals were collected as before. Repeating the crystallization again slightly increases its specific activity. Papain can also be recrystallized as follows: dissolved in 70% methanol, salted out with lithium sulfate or lithium chloride. Storage: stable at 4 ° C; about 20% decreased in 6 months.
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