Does anybody has the idea why the alpha-amylase (from porcine pancreas - A3176 SIGMA) could not be dissolved in distilled water or in-vitro fluids without causing floating flakes or lint?
The method is in ICUMSA (International Commission of Uniform Methods of Sugar Analysis). Not much point in reinventing the wheel!
11/15/16
Dear Marcin,
I've not worked w/ porcine a-amylase recently; however, according to information on Sigma's website, it is soluble in water, e.g., ~1 mG/mL. It also can be dissolved in 25 mM Tris-HCl @ pH 7.5 w/ 0.1 M KCl, or in 1 mM PO4 buffer, pH 7.3 w/ 30 mM CaCl2. Sometimes the presence of salts will aid the solubilization process. There is more information about the enzyme at www.sigmaaldrich.com.
The enzyme preparation is not pure amylase, but amylase + some impurities, such as lactose (90% by weight, according to Sigma) and probably other proteins. It may be that the amylase is actually dissolving, but the impurities are not, so that you see floating flakes. One way to find out is to centrifuge the preparation to remove the insolubles, then check for soluble protein at 280 nm on a spectrophotometer. Alternatively, add some of the solution to starch and allow it to incubate for a time. If amylase is present, you can see this by checking for reducing sugars with DNSA, or for the disappearance of starch using I2/KI (iodine) reagent.
I hope this information helps you.
Bill Colonna, Center for Crops Utilization Research, Iowa State University, Ames, IA, USA [email protected].
You will have to remember, that many enzymes have an activity if 1000s or 100000s of U/mg so they are regularly blended with an inert excipient like lactose (in your case). In addition, they may come with a coenzyme like ATP.
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