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beta-Amylase

(1,4-a-D-Glucan maltohydrolase; EC 3.2.1.2)

ß-Amylase is an exoenzyme that releases successive maltose units from the nonreducing end of a polysaccharide chain by hydrolysis of a-1,4-glucan linkages. The shortest normal saccharide attacked is maltotetraose (Myrback, K. and G. Neumuller, The Enzymes, Vol. I, Part 1, p 653, 1950). Since it is unable to bypass branch linkages in branched polysaccharides such as glycogen or amylopectin, their hydrolysis is incomplete and a macromolecular limit dextrin remains.

The enzyme is useful in structural studies of starch and glycogen and can remove contaminating &223;-glucosidase. It has a molecular weight of 206,000 and an optimum pH 4.0 to 5.0. The enzyme is sulfhydryl sensitive and inhibited by heavy metals ions, p-mercuribenzoate, iodoacetamide and urea. Ascorbate inhibition has been attributed to cupric ion reduction and subsequent formation of an inactive cuprous enzyme complex (Rowe, A.W. and C.E. Weill, Biochim. Biophys. Acta, 65, 245, 1962).





The amount of enzyme which liberates, from soluble starch, one micromole of maltose per minute at pH 4.8 at 37°C.


  1. 0.0016 M Sodium acetate, pH 4.8.
  2. 2 M Sodium hydroxide (NaOH).
  3. Dinitrosalicylic acid color reagent: Dissolve 1.0 g 3,5-dinitrosalicylic acid in 20 ml 2 M NaOH. Add slowly, 30.0 g sodium potassium tartrate tetrahydrate. Dilute to a final volume of 100 ml with distilled water.
  4. 1% Starch: Dissolve 1.0 g soluble starch in 100 ml 0.02 M sodium phosphate buffer, pH 6.9, containing 0.006 M NaCl. Bring to a gentle boil to dissolve. Cool and make volume to 100 ml with distilled water.
  5. Maltose stock solution: 5 mM/ml. Prepare by dissolving 180 mg maltose in 100 ml distilled water and incubate at 37°C for 10 minutes prior to assay.
  6. Enzyme (§-amylase) solution. Dilute to a concentration of 1-10 µg/ml. Prepare at least 3 different concentrations within this range. Must be prepared fresh immediately prior to assay.



  1. Set spectrophotometer to 540 nm and water bath to 37°C.
  2. Using the maltose stock solution prepare a maltose standard curve with dilutions ranging from 0.3 to 5 mM/ml. Use distilled water as blank.
  3. Pipette 0.5 ml of respective enzyme dilutions into a series of numbered test tubes. Include one blank with 0.5 ml distilled water.
  4. Incubate tubes at 37°C for 10 minutes to achieve temperature equilibration.
  5. At timed intervals, add 0.5 ml starch solution (at 37°C) and incubate exactly 5 minutes at 37°C.
  6. Exactly 5 minutes following the addition of starch, add 1 ml dinitrosalicylic acid reagent to each tube at timed intervals.
  7. Incubate all tubes in a boiling water bath for 5 minutes.
  8. Cool to room temperature and add 10 ml distilled water.
  9. Read absorbance at 540 nm after zeroing the spectrophotometer with the blank.
  10. Determine mM or maltose liberated from standard curve.



Activity (U/mg) = (micromoles maltose liberated)
(mg Enz. used in sample)(5 min.)
 

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