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  Section: Biotechnology Methods » Enzymology
 
 
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Determination of the Effect of pH on the Activity of Human Salivary α-Amylase

 
     
 
Principle
a-amylase catalyses the hydrolysis of α-174 linkage of starch and produces reducing sugars. Maltose reduces 3.5 DNA into 3 amino –5 nitro salicylic acid, an orange-red complex. Read the OD at 540 nm.

Enzymes are active over a limited pH range only and a plot of activity against pH usually produces a bell-shaped curve. The pH value of maximum activity is known as optimum pH and is characteristic of the enzyme. The variation of activity with pH is due to the change in the state of ionization of the enzyme protein for other compounds of the reaction mixture.
 

Reagents

  1. DNS reagent
  2. 1% starch: 1 gm of starch dissolved in buffer of different pH
  3. Crude solution
  4. Buffer solution
    (i) Acetate buffer pH 4.0, 5.0, and 5.6
    (ii) Acetate acid: 11.5 mL/L (0.2 M)
         Sodium acetate: 16.4 gm/L (0.2 m)
         Phosphate buffer – pH, 6.0, 7.0, and 8.0
    (a) Na2 HPO4, 2H2O – 35.61 gm/L
    (b) Na2 HPO4, H2O – 27.67 gm/L


 
Procedure
Take 7 test tubes and add 0.5 mL of any pH buffer to the first tube and 0.5 mL of substrate of any pH, which serves as a blank. Then add 0.5 mL of enzyme to each tube, except for the blank. Add 0.5 mL of substrate of pH values of 4.0, 5.0, 5.6, 6.0, 7.0, and 8.0 to the respective test tube. Incubate all the test tubes for exactly 20 minutes at room temperature. Add 1 mL of DNS reagent to each test tube and keep all tubes in a boiling water bath for exactly 5 mL. Then add 10 mL of distilled H2O to each tube and read OD at 540 nm.

Plot the graph by taking different pH values along the x-axis and activity on y-axis.

Result: The optimum pH of human salivary α-amylase is ________ µM/ mL/min.
 
     
 
 
     




     
 
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