Hi,

Here I suggest a brief protocol:

1. You have to carry out the hydrolysis of the substrate (starch, I guess) by contacting a suitable dilution of the enzyme. Then, determine for the amount of product generated by DNS. Remember, you need the initial reaction. So, follow the kinetic during the first 20 min (approx.). Of course, you should do the experiment at a suitable condition pH, temperature, cofactor concentration (e.g, some divalent cation), etc.

2. To plot the product (glucose equivalent...I am supposing you use glucose for the calibration curve) versus time. Then, by regression determine the initial reaction rate and express it in uM/min (micromolar per minute). You should check that the enzyme dilution used allowing you a linear range during the assay ( Reaction rate is proportional to enzyme concentration...If you add the twice of enzyme, reaction rate increased 2-fold and so on)

3. Then multiply the reaction rate (uM/min) by the reaction volume of the assay (L, in liter). Thus, you will have IU (umol/min). Afterward, divide by the mass of protein contacted during the assay (protein concentration X volume of enzyme added). In this way, you will have IU/mg. Do not forget the dilution of the enzyme when calculating the enzyme concentration.

All the best.

Carlos

Hi,

Laneesha Moozhikkal

This link can be helpful for the calculation of alpha amylase activity.

http://www.microbiology.biology.upatras.gr/en/protocols/111-determination-of-a-amylase-activity.html