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

 
     
 

Principle
α-amylase catalyses the hydrolysis of a-1.4 linkage of starch and produces reducing sugars. The liberated reducing sugars add an orange-red color complex. Read the OD at 540 nm.

The effect of temperature on an enzyme-catalyzed reaction indicates the structural changes in the enzyme. Molecules must possess a certain energy of attraction before they can react and the enzyme functions as a catalyst lowering the energy of attraction. Energy of an enzyme-catalyzed reaction can be determined by measuring the minimum velocity at different temperature. The energy is more active at d/f temperature. Below this temperature, the enzyme activity decreases and above this temperature, the enzyme becomes denatured or loses the structure required for catalytic activity.

Reagents

  1. DNS reagent
  2. 1% starch
  3. Phosphate buffer
  4. Crude enzyme (1:20 dilute)
 

 
Procedure
Take 6 clean and dry test tubes. Pipette out 0.5 mL of enzyme to each, except for the first tube, which serves as a blank. Add 0.5 mL of substrate to each tube. Incubate each for 20 minutes at respective temperatures (13°C, 25°C, 36°C, 50°C, and 60°C). Add 1 mL of DNS reagent to each test tube and keep all test tubes in a boiling water bath for exactly 5 minutes. Add 10 mL of distilled water to each tube and read the OD at 540 nm.

Plot the graph by taking temperature on the x-axis and activity on the y-axis.

Result:
The optimum temperature of the human salivary a-amylases is 36°C.
 
     
 
 
     




     
 
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