Micro porous starch was prepared from potato starch by alpha amylase catalysis combined with ultrasonic treatment. Twenty milliliters of starch slurries (40, 60, and 80% w/w) were treated with 0.05% v/v alpha amylase (enzyme activity >100 000 U/g), or a combination of 0.05% v/v alpha amylase with ultrasound at 100 W (20% power), 200 W (40% power), and 300 W (60% power) for 10, 30, and 50 min concurrently or sequentially.
You can use soluble starch as a substrate and DNS as a stop reagent. There is a famous Bernfeld method for amylase activity. It is important that after you add stop solution (DNS), you boil your samples for 5 minutes at 100 degrees C. After that cool your samples in cold water and dilute them with distilled water. The absorbance is measured at 540 nm. Also every sample should have its own blank that is prepared in the same way as essay solution but you add DNS to the enzyme first and then you add starch solution. Blank is also boiled. I am sending you Bernfeld method and links to the two papers to see buffer used and procedures.
http://www.doiserbia.nb.rs/img/doi/0352-5139/2011/0352-51391100098B.pdf
http://www.doiserbia.nb.rs/img/doi/0352-5139/2014/0352-51391300155G.pdf
Glucose releasement by any commercial method. First a calibration curve with diiferent amiunts of glucose and then you can carry out your reaction and measure the glucose releasement by action of alfa amilase, then enter the values in the curve and calculate activiy of the enzyme....
You can use soluble starch as a substrate and DNS as a stop reagent. There is a famous Bernfeld method for amylase activity. It is important that after you add stop solution (DNS), you boil your samples for 5 minutes at 100 degrees C. After that cool your samples in cold water and dilute them with distilled water. The absorbance is measured at 540 nm. Also every sample should have its own blank that is prepared in the same way as essay solution but you add DNS to the enzyme first and then you add starch solution. Blank is also boiled. I am sending you Bernfeld method and links to the two papers to see buffer used and procedures.
http://www.doiserbia.nb.rs/img/doi/0352-5139/2011/0352-51391100098B.pdf
http://www.doiserbia.nb.rs/img/doi/0352-5139/2014/0352-51391300155G.pdf
Hi Hady,
There are sensitive kits by Megazyme that you can use for the measurement of alpha-amylase activity that involve a colourimetric oligasaccharide substrate. These substrates are specific for the assay of alpha-amylase even in crude enzymatic mixtures in the presence of beta-amylase or other exo-acting enzymes.
Alpha-amylase hydrolyses the colourimetric substrate and liberates a p-nitrophenolate group that can be detect using a spectrophotometer at 400 nm.
Below are the links to the 2 kits, you can use either of them:
Ceralpha kit: https://secure.megazyme.com/Alpha-Amylase-Assay-Kit
Amylase SD kit: https://secure.megazyme.com/Alpha-amylase-SD-Assay-Kit
Please find attached two scientific publications that used these methods for the measurement of alpha-amylase.
https://secure.megazyme.com/Alpha-amylase-SD-Assay-Kit
Luis Enrique Trujillo Toledo and Nikola Gligorijević thanks for sharing these ideas.
Hady Bodie Bah Claudio Cornaggia Nikola Gligorijević Can you help me in standard curve plotting? I mean for standard curve I will take maltose instead of test sample. After that should I follow the same procedure as it is adding both enzyme and starch solution and DNS reagent ?
Dear Monalisa,
You do not need to do this. You can just make maltose solutions in different concentrations in the same final volumes as you would have in your test samples prior to adding DNS reagent. Then after you add DNS, everything else should be the same. The point of adding everything in each test sample during the amylase activity assay is to subtract everything that may contribute to absorbance at 540 nm besides DNS. As for your standard curve, you only have maltose diluted in sample buffer so blank for this is only buffer and that is it. I hope my answer is clear to you. Best of luck!
Nikola Gligorijević You mean I should add DNS solutions to different concentration of maltose and will take reading?
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