Principle: – B-Amylase catalyses the hydrolysis of B-1, 4 glycosides links in starch with the Production of reducing sugar. The amylase activity is determined in terms of amt. of reducing sugars produced at the end of the reaction. These sugars reduce 3, 5 dinictrosalieylic acid (DNS) to 3-amino – 5- nitro salicylic acid and the colour developed is measured calorimetrically at 540 nm.
The effect of temperature is studied by carrying out enzyme reaction of various temp. Each enzyme has a characteristic temperature at which it reacts with maximum velocity. Optimum temp. of an enzyme is the temp. at which reaction velocity is maximum.
Requirement: – 5 % starch soln, 0.5% Nacl, DNS reagent. Acetate buffer (PH 4.8), Amylase extract.
Procedure:- Prepare a series of test tubes as follows.
Incubate tubes at different temperature as mentioned for 30 mins. After incubation add 1ml of 3, 5 dinitrosaliaylic acid soln and 1ml of NaOH (bench reagent). Add 2ml of distilled water to each test tube; Heat all the tubes in a boiling water bath for 10 mins and then allow to cool. Dilute solution 10 times and determine at 540 nm. Plot graph of temp against extinction and determine optimum temperature.
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