Data Sheet - Bromelain

Source:
Stem of pineapple (Ananas comosus (L.) MERR.)
Systematic name:
Peptidyl peptide hydrolase; EC 3.4.22.32
Other names:
Bromelin, Ananase.
CAS-No.
37189-34-7
E.C.-No.
 3.4.22.32
EG-Indexnummer
647-005-00-X

Occurrence

Bromelain is the collective name for proteolytic enzymes found in various members of the family Bromeliaceae. Bromelain from the pineapple (Ananas comosus) is the most studied. The highest enzyme concentration occurs in the lower portion of mature pineapple plant stems, appreciable quantities are also present in fruits and leaves.

Characteristics

Specificity: Two forms (A and B) of Bromelain with similar specificity have been isolated from pineapple stem (1). Bromelain hydrolyzes proteins, peptides, amides and esters of amino acids and peptides; preferential cleavage site is the carbonyl end of lysine, alanine, tyrosine and glycine (1,2). For relative activities on a number of amino acid substrates see Barman (3). The following side activities can be detected in Bromelain preparations: amylase, phosphatase, peroxidase, the latter being very labile upon storage.

Effectors: Bromelain is a thiol proteinase, i.e. it contains in the active centre a highly reactive cysteine which is essential for catalysis. Therefore, the enzyme can be activated by reducing compounds, e.g. cysteine, 2-mercaptoethanol, dithiothreitol, KCN (4). On the other hand, Bromelain is irreversibly inhibited by alkylating agents such as N-ethylmaleimide (NEM), iodoacetic acid and 1,3-dibromoacetone (4). Reversible inhibition is caused by inorganic Hg ion, organic Hg compounds and tetrathionate (4).

Catalytic optima: The optimum pH for catalytic activity depends on the nature of substrate, type and concentration of buffer and the presence or absence of a reducing agent. The optimum pH range is about 4.5-7.5. The optimum temperature is 35-45 °C (5), the maximum operating temperature for industrial application (reaction time ≤4h) is 50 °C (2).

Stability: Bromelain is stable at pH 3-6 and at temperatures up to 60 °C (5). Bromelain powder can be stored originally packed and tightly closed up to two years at < 8 °C without loss of activity. Upon opening the activity gradually decreases, unless the enzyme is immediately mixed with diluent (lactose) which prevents the protein from self-inactivation. Activity losses caused by unproper storage or preparation conditions can be abolished to an appreciable extent by the addition of cysteine.

Solubility: Bromelain powder is partly soluble in water, insoluble in most organic solvents.

Molecular weight: 33,200 daltons.
Composition: Bromelain is a glycoprotein constituted of one single polypeptide chain with 1 glycan per molecule. The number of amino acids per molecule is not yet unequivocally established. The NH2-terminal amino acid is valine, the COOH-terminal is glycine.

Isoelectric point: 9.55

Spectral data: E280 (1%, 1cm) = 20.1.

Assay

The method described here is the one given by Lauwers and Scharpé (4).

Principle
Bromelain hydrolyzes casein into small peptides, which cannot be precipitated with a specific reagent. Thus, after incubation (10 min at 35 °C) undigested casein is removed and the amount of peptides remaining in solution is determined spectrophotometrically at 275 nm.

Unit definition
1 FIP unit of Bromelain is the amount of enzyme that hydrolyzes casein under the standard conditions into not acid-precipitable peptides at an initial rate such that there is liberated per minute an amount of peptides which gives the same absorbance at 275 nm as 1 °mole tyrosine.

Reagents

  1. Tris-buffer: 0.05 M tris/HCl pH 7.15. Dissolve 6.06 g tris (= tris-(hydroxymethyl)aminomethane) in 600 ml distilled water. Adjust with 1 N HCl at 25 °C to pH 7.15 and add water to a final volume of 1000 ml. At 35 °C the pH must be 7.00.
  2. Substrate activation solution: 80 mM cysteine, 10 mM EDTA in Tris-buffer (A). Dissolve 242 mg cysteine and 93 mg EDTA x 2H2O in Tris-buffer (A), adjust to pH 7.15 and fill up to 25 ml with Tris-buffer (A). Prepare fresh daily.
  3. Substrate solution: Add 2.5 g casein (e.g. casein for biochemistry, Merck 2242, Merck, Darmstadt, F.R.G.; FIP controlled) to 10 ml distilled water, mix, add 15 ml 0.1 N NaOH and mix thoroughly with a magnetic stirrer for 30 min at 35 °C until all casein is dissolved. Then add 50 ml of Tris-buffer (A) and 10 ml of substrate activation solution (B), adjust pH to 7.15 at 25 °C and make up to 100 ml with Tris-buffer (A). Prepare fresh daily.
  4. Enzyme activation solution: 5 mM cysteine, 1 mM EDTA, 10 mM KCl in tris buffer (A). Dissolve 60.5 mg cysteine, 37.2 mg EDTA x 2H2O and 74.6 mg KCl in Tris-buffer (A), adjust to pH 7.15 and make up to 100 ml with Tris-buffer (A). Prepare fresh daily.
  5. Enzyme solution: Dissolve Bromelain in enzyme activation solution (D). The solution should contain 0.1-0.12 FIP units/ml. Before use the solution is equilibrated to 35 °C.
  6. Enzyme Reference Solution: Dissolve a suitable amount of bromelain FIP standard in enzyme-activation solution to obtain a solution with an activity of apparently 0.1-0.12 FIP units/ml. Before use the solution is equilibrated to 35 °C.

    Bromelain FIP Reference Standard is issued by: Centre for Standards FIP, Harelbekestraat 72, B-9000 Ghent, Belgium.

  7. Protein precipitation reagent: 0.11 M TCA, 0.364 M sodium acetate, 0.688 M acetic acid. Dissolve 9.0 g TCA (trichloroacetic acid) p.a., 24.8 g sodium acetate x 3H2O and 19.5 ml acetic acid in distilled water to a final volume of 500 ml.
  8. Filter paper control: Determine the suitability of the filter paper by filtering through the paper 5 ml of protein precipitation reagent. Measured at 275 nm against an unfiltered aliquot of the reagent the absorbance of the filtered reagent should not exceed 0.04.

Procedures

Transfer into test tubes Assay Blank
Substrate solution (C)
equilibrate to 35 °C in a water bath
2.5 ml     2.5 ml
Enzyme solution (E & F) pre-equilibrated to 35 °C
mix, incubate for exactly 10 min (stop watch)
2.5 ml  ----
Protein precipitation reagent (B) 5.0 ml  5.0 ml
Enzyme solution (E & F) 
mix, incubate for 30 min at 35 °C,
filter through filter paper;
measure absorbance of filtrate
against blank (1 cm cuvette, 275 nm)
---- 2.5 ml

 

Calculation

The specific activity of the enzyme is calculated as:
m´ x E x A
m x E´

m  = mass in milligrams of the substance to be examined,
m´= mass in milligrams of Bromelain FIP standard,
E = absorbance of the filtrate of the substance to be examined,
E´= absorbance of the filtrate of the Bromelain FIP standard
A = activity of Bromelain FIP standard in FIP units/mg

Availability

Standard qualities
Bromelain 5.0 FIP units/mg ≈ 2500 GDU/g 1560 CDU/mg
Bromelain 4.0 FIP units/mg ≈ 2000 GDU/g ≈ 1250 CDU/mg
Bromelain 3.2 FIP units/mg ≈ 1600 GDU/g ≈ 1000 CDU/mg
Bromelain 2.4 FIP units/mg ≈ 1200 GDU/g ≈   750 CDU/mg

Customized qualities are available upon request.

References

  1. International Union of Biochemistry. Nomenclature Committee: Enzyme Nomenclature. Academic Press, Inc., London 1984.
  2. Godfrey, T., Reichelt, J.: Industrial Enzymology. Macmillan Publishers Ltd., Surrey (U.K.), 1983.
  3. Barman, T.E.: Enzyme Handbook, Vol. II. Springer, New York-Heidelberg-Berlin-Tokyo, 1985.
  4. Lauwers, A.; Scharpé, S.: Pharmaceutical Enzymes, drugs and pharmaceutical sciences., Volume 84, Marcel Dekker, Inc., New York-Basel-Hong Kong, 1997.
  5. Stellmach, B.: Bestimmungsmethoden Enzyme für Pharmazie, Lebensmittel-chemie, Technik, Biochemie, Biologie, Medizin. Steinkopff-Verlag, Darmstadt, 1988.

 

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