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Bromelain

Catalog Number: 101142
Bromelain


CAS #: 9001-00-7

Source: Pineapple

E.C. 3.4.22.4

Activity: Approximately 1100 to 1500 GDU units/gm

Unit Definition (Gelatin Digestion Unit - GDU): One unit will hydrolyze 1.0 mg of amino nitrogen from gelatin in 20 minutes at pH 4.5 at 45°C.

Preferential Cleavage: Lys-, Ala-, Tyr-, Gly-

Description: A glycoprotein which is a highly active thiol proteinase (non-specific cysteine protease), which splits glycine esters but not lysine esters; it is also a protein-digesting and milk-clotting enzyme. It is found in the leaves and stems of the pineapple plant. Substrates susceptible to bromelain include all of the common protein materials such as gelatin, casein, gluten, collagen, elastin, globulins an muscle fiber protein.

Physical Description: Yellowish-white to tan powder

Solubility: Readily soluble in water, insoluble in most organic solvents such as acetone, ether, ethanol and methanol.

Optimal pH: The optimum pH value is influenced by the nature of the substrate, by the concentration and type of buffer, and by the presence of reducing agents. The most common range is within pH 5 to 7, but it can function also in the pH range of 3 to 9. Bromelain is stable at pH 3.0 to 6.5 and once it has combined with its substrate, the activity is no longer susceptible to the effect of the pH.

Optimum Temperature: A temperature range from 45-65°C is regarded favorable for the digestive process and 62°C as the optimum point. Its activity decreases at 70°C or higher.

Activators: Bromelain can be activated by cysteine, bisulfite salt, NaCN, H2S, Na2S and benzoate; however, bromelain is usually sufficiently active without the addition of activators. Generally, if bromelain loses an appreciable amount of its activity by undue storage or preparation conditions, the enzyme can be markedly reactivated by cysteine.

Inhibitors: Bromelain is inhibited by Hg++, Ag+, Cu++, a-1-antitrypsin, estatin A & B, Iodoacetate, TLCK, TPCK.

Molecular Weight: Approximately 33,000.

Gelatin Digestion Assay Procedure

Reagents:

1. Gelatin Substrate (5.0%): To 25.00 g of Gelatin (high-test-275 bloom) in an 800 ml beaker, add 400 ml of boiling deionized water. Add one drop of antifoam and stir. Maintain the temperature of the gelatin solution at 85oC to 86oC in a water bath with occasional stirring. Cool to 45oC and immediately adjust to pH 4.5 with 2N NaOH. Dilute to 500 ml with deionized water. This substrate should be made fresh daily.

2. Buffer Solution: Add 5.7 ml Glacial Acetic Acid and 150 g Sodium Chloride to 700 ml deionized water and adjust to pH 4.5 with 50% NaOH. Dilute to one liter.

3. Sodium Hydroxide, 0.05N: Dissolve 2 g of NaOH in deionized water. Transfer to a 1000 ml volumetric flask and dilute to volume with deionized water.

4. Assay Solution: Weigh exactly 100 mg of Bromelain in a 50 ml beaker and add 20 ml buffer solution. Stir until the bromelain is well dispersed. transfer to a 100 ml volumetric flask, wash down the beaker and make up to volume with deionized water. This solution must be tested within 30 minutes of the time the buffer solution is added.

Assay

1. Pipette 25 ml of substrate into a 100 ml beaker and hold in a water bath at 45oC until temperature equilibrium is established.
2. Add 1.0 ml of enzyme solution, start timing and stir briefly.
3. After exactly 20.0 minutes of digestion at 45oC, add 2 drops of 3% Hydrogen Peroxide solution and stir briefly.
4. Remove from water bath, adjust to pH 6.0 with NaOH and add 10 ml of 37% formaldehyde.
5. Titrate to pH 7.8 with 0.05N NaOH. This is the sample titer, T.
6. For the blank, pipette 25 ml of substrate into a 100 ml beaker as above, add 2 drops of 3% Hydrogen Peroxide, then 1.0 ml of enzyme solution and hold at 45oC for exactly 20.0 minutes.
7. Remove from water bath, adjust to pH 6.0 with NaOH and add 10 ml of 37% formaldehyde.
8. Titrate to pH 7.8 with 0.05N NaOH. This is the blank titer, B.


Casein Digesting Units

Reagents:

1. Casein Substrate: Dissolve 0.6 g Hammerstein casein in 80 ul 0.05M Na2HPO4, and add 0.1 N HCl or 0.1 NaOH to pH 7.0, dilute with water to 100 ul. Prepare fresh daily.

2. Protein Precipitant: Dissolve 179.7 g CCl3COOH, 180.5 g CH3COONa, 198.2 CH3COOH in water to make 1000 ml (stock solution). Before use, dilute one volume of the stock solution with water to ten volume.

3. Bromelain Solution: Dissolve about 80 mg of Bromelain weighed accurately in physiological saline to make 1,000 ml (30-80 u/ml)

Assay:

1. Pipet 1.0 ml Bromelain solution into a test tube, and place in a water bath at 37°C for about 3 minutes.
2. To the test tube, add 5.0 ml of casein substrate prewarmed at 37°C.
3. After exactly 10 minutes, add 5.0 ml of protein precipitant to the tube.
4. Place the tube in the water bath, for 30 minutes, allow to fully coagulate the precipitated protein.
5. Filter through filter paper, read the absorbance (Et), at 275 nm, of the filtrate.
6. Into another test tube, pipet 1.0 ml Bromelain solution, add 5.0 ml protein precipitant, and then 5.0 ml casein substrate.
7. After filtration, read the absorbance (Eb) of the filtrate at 275 nm.
8. Read the absorbance (Es) of L-tyrosine solution containing 50 ug per ml at 275 nm.

The potency of Bromelain is calculated by the following equation:




Unit Conversions:

600 Milk Clotting Units = 16,500 casein digesting units
500 gelatin digesting units
1 Bromelain-Tyrosine Units (BTU) = 1.1 gelatin digesting units

References:
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  10. Chandler, D.S., Mynott, T.L., "Bromelain protects piglets from diarrhoea caused by oral challange with K88 positive enterotoxigenic Escherichia coli." Gut, v. 43:2, 196-202 (1998).
  11. Hale, L.P., Haynes, B.F., "Bromelain treatment of human T cells removes CD44, CD45RA, E2/MIC2, CD6, CD7, CD8, and Leu 8/LAM1 surface molecules and markedly enhances CD2-mediated T cell activation." J. Immunol., v. 149:12, 3809-3816 (1992).
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  13. Bernkop-Schnurch, A., Giovanelli, R., Valenta, C., "Peroral Administration of Enzymes: Strategies to Improve the Galenic of Dosage Forms for Trypsin and Bromelain." Drug Development and Industrial Pharmacy, v. 26:2, 115-121 (2000).
  14. Mynott, T.L., Guandalini, S., Raimondi, F., Fasano, A., "Bromelain prevents secretion caused by Vibrio cholerae and Escherichia coli enterotoxins in rabbit ileum in vitro." Gastroenterology, v. 113:1, 175-184 (1997).
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