PLANT ENZYMES: (PAPAIN AND BROMELAIN)

PAPAIN:

Source: It is the dried, purified latex obtained from the unripe fruit of Carica papaya
Family: Caricaceae.
Chemical nature: Papain is a mixture of enzyme, particularly proteolytic enzymes. It is also known as "Vegetable Pepsin" because it contains similar proteolytic enzymes of pepsin. But unlike pepsin, it can act in acid, neutral or alkaline media. Its maximum activity is between pH 5 & 6. It contains various enzymes they are:
• Various proteolytic enzymes like peptidase-I, which has the ability to convert protein into di and polypeptide.
• An amylolytic enzyme.
• An enzyme like pectase.
• Enzyme which as an action on fats
• A rennin like coagulation enzyme, which acts on casein (milk protein).
• Chymopapain and papaya proteinase.


METHOD OF PREPERATION:
COLLECTION OF LATEX AND PURIFICATION OF PAPAIN:
The matured unripe fruits are incised on four sides, because of which latex flows freely, which is collected in aluminium trays. It solidifies quickly. Repeated incisions are made at weekly intervals until exudation of latex ceases. The collected solidified latex is treated with an antioxidant like potassium bisulphate. It is then sieved to remove any extraneous matter. Drying is done in sun or by artificial means that is in a spray drier or in a shelf drier at 55oC to 60oC. Artificial drying is preferred because it gives better quality product.
It is purified by general purification and separation technique that is alternative precipitation and dissolution. Crude papain is dissolved in water and then precipitated with alcohol. It also can purified by affinity chromatography or by HPLC methods.

Separation by HPLC:
Substrate used: water soluble N-benzyl-L- arginine ethyl ester. The separation of substrate from reaction product benzoyl arginine was carried by reverse phase HPLC.

Column- µ Bonda pak CN column.
Mobile Phase- 0.05M ammonium acetate: methanol (85:15)
Eluate observed- 254nm


USES:
Medicinal, Pharmaceutical and Cosmetic:
• Used in treating a wide variety of illnesses or conditions, including infected wounds,sores,ulcers,chronic diarrhea, tumors, hay fever, esophageal obstruction due to meat impact, catarrh and psoriasis.
• Currently used as digestive aids and in preparations to control edema and inflammation associated with surgical or accidental trauma, infections, or allergies.
• It is also used in certain face creams, cleansers, face lift formulations, and dentifrices and as an active ingredient in enzyme cleaners for soft lenses.
• Chymopapain is used in the treatment of degenerative intervertabral disc disorders (low back pain, sciatica) due to herniated lumbar disc. When no other treatment is possible, then it is injected into intervertebral disc. It is administered by highly trained person because of the risk of anaphylactic shock.
• It is also used to relieve the symptoms of episiotomy.
• It is used for the tenderization of meat in meat packing industry, due to its proteolytic activity. (Hydrolysis of meat fibers and connective tissues).
• Papain is extensively used in stabilizing and chill proofing beer, hydrolyzing proteins that normally are soluble at room temperature but likely to precipitate from the beer when chilled. It is also in preparation of protein hydrosylates.
• Other potential uses include modifying dough in baking, clarifying fruit juices, in fish oil extraction, in manufacture of collagen heat-sealable films and manufacture of sausage casings.
• Papain is used as a digestive aid ingredient in capsules, tablets as it has pepsin like activity.



BROMELAIN:
Source: It is obtained form the juice of pineapple, Ananus cosmosus. It is also obtained
from the stem of the plant.
Family: Bromeliaceae.
Activity: It is a protein digesting and milk clotting enzyme.
Chemical constituent: Peptidase, anain, cosmosain etc. fruit is rich in soluble mono and disaccharides, inorganic acids and vitamins.


I. EXTRACTION OF BROMELAIN FROM FRUIT AND SKIN:

1. Fruits were cut into small pieces, weighed, macerated and juice was obtained.
2. Juice was pressed and filtered through cheese cloth.
3. pH of the juice was adjusted to 6.
4. Ammonium sulphate was added until saturation to precipitate the enzyme.
5. Partial purification was done by redissolving crude enzyme in NaCN and repeatedly precipitating it, firstly with 0.6% ammonium sulphate and then with acetone.
6. The precipitate is thoroughly washed with acetone and ether and dried in vacuum oven at low temperature. (Yield 0.06%-0.08%).


II.EXTRACTION OF ENZYME FROM STEM:

1. Matured pine apple stem was collected after final fruit harvest and freed from leaves and sucker stems.
2. Stems were weighed and placed in scrapping machine.
3. Juice was pressed and filtered through cheese cloth.Acetone was added to stem juice in two stages.
4. The first step was addition of two volume of acetone to two volumes of stem juice. The precipitate formed was discarded as it has low enzyme activity, poor color and stability.
5. Addition of another volume of acetone precipitated the main enzyme fraction.
6. This was collected by centrifugation and washed with acetone and diethyl ether and dried in vacuum oven.
7. Acetone was recovered from supernatant solution by distillation. (0.1%-0.6%)

(Bromelain activity is evaluated by milk clotting property. Maximum amount of enzyme of enzyme which clots milk in test tube under controlled conditions pH 3.8 in one minute)

Activity: It is protein digesting and milk clotting enzyme.

USES:
MEDICINAL, PHARMACUETICAL AND COSMETIC.
• Used primarily in preparations to treat inflammation and edema associated with surgical or accidental trauma, infections or allergies.
• Used in certain cosmetics such facial cleansers and bath preparations.
• To accelerate the tissue repair of post episiotomy (due to depolymerisation and permeability modification).
• Major uses are in meat tenderizing, manufacturing precooked cereals, modifying dough (bread, wafers, pizza etc.) and in chill-proofing beer.
• Other potential uses include preparation of protein hydrolysates, liquefying fish protein to facilitate fish oil extraction, clarifying fruit juices and manufacture of sausage casings as well as their removal from sausages.
• Used in some vitamin and herbal formulations, mainly as a digestive aid.
• Used in bating hide and desizing fabrics.

Reference: Text book of Pharmacognosy by Jean and Brunton
HPLC in enzymatic analysis by Rossamando.
Text book of Pharmacognosy by Brady and Tylor.

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