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Bromelain

(EC 3.4.22.4)

Stem and fruit bromelain catalyze the hydrolysis of protein substrates as well as synthetic substrates. The fruit Bromelain is an acidic protein. The enzyme is apparently homogenous as judged by rechromatography and electrophoresis on cellulose acetate. It catalyzes synthesis of acylamino acid anilides, is inhibited by mercurials and the activity is restored by cysteine. The stem enzyme is a basic protein with a molecular weight of approximately 33,000. It is more basic and 1.5 times larger in size when compared to papain. The stem Bromelain has one reactive sulfhydryl group per molecule as determined by titration with p-chloromercurbenzoate. This sulfhydryl group is essential for catalytic activity (T. Murachi and M. Yasui, Biochemistry, 4, 2275, 1965)





The amount of enzyme that when added to the the reaction mixture will yield a rate of 1 absorption unit per minute at 25°C.


  1. 15 mM p-nitrophenyl Na-benzyloxycarbonyl-L-lysinate (CLN), in acetonitrile containing 20% (v/v) water. The substrate is suspended in acetonitrile, the required amount of water is added and the volume is adjusted with acetonitrile.
  2. Buffer: 10 mM Sodium acetate-acetic acid containing 0.1 M KCl, 1 mM L-cysteine, pH 4.6. Prepare fresh.
  3. Bromelain (0.2-2.0 mg/ml) in buffer.



  1. Set water bath to 25°C and spectrophotometer to 25°C and 340 nm.
  2. Place 3 ml of buffer into quartz cuvette and place in spectrophotometer.
  3. Bromelain (2-100 µl) is added and thoroughly mixed with the flattened tip of a glass rod 2.5 mm in diameter.
  4. The solution is incubated for 1 minute to allow activation of the enzyme and then 50 µl of CLN solution is added with stirring.
  5. The increase in absorbance at 340nm is measured for a few minutes. A linear increase should occur at least up to 0.4 absorbance unit.
  6. The slow spontaneous hydrolysis of the substrate should also be determined in a blank reaction where 50 µl of buffer is added instead of enzyme solution.



Activity (U/mg) = (ΔE340nm/min)(Total Vol.)(Enz. Diln.)
(Enz. Vol.)(mg Enz./ml)
 

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