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Sunday, November 22, 2009

How to find out optimum PH of enzyme activity

How to determine optimum pH of Amylase

Principle: - Optimum pH of an enzyme is the PH at which the reaction velocity is maximum. Each enzyme has a characteristic PH at which it reacts at maxi, velocity. The optimum PH is determined by carrying out an enzyme reaction at various PH values.
In the case of amylase the activity is determined in terms of reducing sugar produced at the end of the reaction. These sugars reduce 3, 5 dinitrosalicylic acid to 3- amino – 5 – nitro salicylic acid and the co lour developed is measured calorimetrically at 540 nm. Reducing sugar +3, 5 PNS alkali (NaOH) 3 – amino – 5 – nitro salicylic acid + oxidized sugar
Requirements: - Prepare series of test containing 1ml starch solution [substrate]. 1ml of buffer of different PH. 1ml of 0.5% Nail and 1ml of enzyme extract. Prepare blank in the same manner omitting enzyme extract. Incubation all the tubes at 370 C for 30 mins. After incubation add 1ml of DNS reagent and 1ml of NaOH to all tubes Add 1ml of enzyme extract to the blank, Heat all the tubes in a boiling water bath for 10mins. And then allow to cool. Dilute all the solution to 10 times with distilled water, Mix the contents thoroughly and read the extinction in each tube calorimetrically at 540 nm. Plot graph of Extinction against PH and determine optimum PH.

Preparation of reagent:- Amylase extract: Suspend 5gms of ground sweet potato in 20ml of chilled distilled water keep the suspension in w/d for 30mins. Centrifuge and use.

3, 5 dinitrosalicylic acid: - Dissolve 1gm of 3, 5 dinitro salicylic acid and 30 gms of Na-K- temperature in distilled water. Add to it 20ml of 2N NaOH and make the volume to 100ml.
 


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