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Effect of Substrate Concentration on Enzyme Kinetics
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Materials Required:


1.   10ml of 0.5%, 1%, 2%, 3%, 4%, 5% Soluble Starch solution.
2.   10ml of ∞ Amylase solution
3.   10ml of  2N Sodium hydroxide (NaOH)
4.   15ml of DiNitro Salicylic acid (DNS)
5.   Distilled Water – 150ml


Reagent preparation:


1)    α – Amylase solution:

 

  • 0.5% starch solution- 0.5g starch soluble in 100ml deionised water.
  • 1% starch solution- 1g of starch soluble in 100ml deionised water
  • 2% starch solution- 2g of starch soluble in 100ml deionised water
  • 3% starch solution- 3g of starch soluble in 100ml deionised water
  • 4% starch solution- 4g of starch soluble in 100ml deionised water
  • 5% starch solution- 5g of starch soluble in 100ml deionised water

 

2)    2N NaOH Solution:

 

        8g NaOH in 100ml distilled water.

3)    DNS Solution:


            1g of DNS is dissolved in 50ml of distilled water.  30g of sodium potassium tartarate tetrahydrate is added in small lots. The solution turns milky yellow in colour. Then 20ml of 2N NaOH is added, which turns the solution to transparent orange yellow colour. The final volume is raised to 100 ml with the distilled water. This solution is stored in an amber coloured bottle.


Procedure

 

  1. Make different concentration of starch soluble (0.5%, 1%, 2%, 3%, 4% and 5%,).
  2. Take 12 clean and dry boiling tubes. Label tube as control “C” and Test “T” for each concentration.  Add 0.5ml (500µl) of starch solution to all the tubes.
  3. Preincubate the starch solutions of all concentrations and α  amylase solutions for 10 minutes at 37°C.
  4. Add 0.5ml (500µl) of α Amylase enzyme to the tubes labeled T of respective concentration.
  5. Incubate all the tubes for at 37°C for 10 minutes
  6. After incubation, Immediately add 2N NaOH to test tubes containing test solution and then to the test tubes containing control solution. Mix the solutions in each test tubes.
  7. Pipette out 0.5ml (500µl) of α Amylase to the test tube containing control solutions.  Mix the solutions well.
  8. Add 1 ml of DNS reagent to all tubes. Mix the solutions in the test tubes well.
  9. Keep in boiling water bath for 5 minutes at 100°C and cool it.
  10. Dilute all the tubes by adding 9.5 ml of distilled water.
  11. Mix the solutions in each test tubes by using vortex mixer.
  12. The absorbance of test solutions was read at 540nm against the Control.

 

 

 

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