Both alpha and beta amylases use starch as substrate. It is well known that end product of alpha amylase is glucose, while that of beta amylase is maltose. Both glucose and maltose are reducing sugars and DNSA reagent can be used to quantify both these reducing sugars (rather all reducing sugars). I am looking for a simple spectrophotometric protocol that can help me in determining contribution made by each of these amylases in the breakdown of starch in plants during various developmental stages.
I think you can do so by estimating beta amylase activity in the presence of alpha amylase, by adopting selective inhibition of alpha amylase by using phytic acid as described by M N Gupta in his article published in J. Nanosci. Nanotechnol. 13, 5028–5033 in 2013 and subsequently without inhibition and deduce their individual contribution.
you can estimate the final product separately. After incubation, you can use GOD-POD METHOD specifically for glucose and Somogyi METHOD for reducing sugars. Then you can subtract glucose value of reducing sugar value and shall have an approximate lactose value
Dear Dr. Yadav and Dr. Rosa,
Thank you.
We have problem with salts of phytic acid (namely calcium salt). We shall see if phytic acid is available with any of our suppliers. It might work as it can chellate calcium ions (which are important for alpha amylase activity).
Can you please elaborate on GOD-POD method? Can we use simply glucose oxidase? But the products would be gluconic acid and hydrogen peroxide. Woud gluconic acid react with DNSA or Somogyi reacgent?
The questioner seems to have some problem with terminology. The usual understanding is that amylases, both alpha and beta, produce maltose and maltotriose as the main sugar products. Alpha amylase can act at any location along a suitable polygycan chain but beta amylase is used to describe the enzyme which acts stepwise from the non-reducing end of the chain. Glucose is produced from amyloglucosidases not amylases
Dear Dr. Butterworth,
Firstly, thanks a lot for correcting me.
Yes, it is true that alpha-amylase produce maltose and maltotriose, but it is well known in literature that alpha-amylase also produces dextrins as well as glucose (besides maltose and maltotriose). You have correctly pointed that alpha amylase unlike beta amylase can act at any location (i.e. alpha-Amylase catalyzes the hydrolysis of internal alpha-1,4-glucan links in polysaccharides containing 3 or more alpha-1,4-linked D-glucose units). It is sure that beta amylase can not produce glucose (it largely produces maltose and little amount of maltotriose). But, dextrins, maltose, maltotriose as well as can react with DNSA or Somogyl reagents (as they have free reducing groups).
You can as well quantify alpha and beta amylase using assay kits with substrates of high specificity. A very good example is the betamyl 3 assay kit for beta amylase and alpha amylase reagent method using ceralpha method. These are produced by megazyme international.
You can determine alpha and beta-amylase by using a mixture of 3 ml soluble starch (2% v/v) and 3 ml extract was incubated for 60 min at
30C. After incubation, an equal volume of alkaline color reagent was added to 1-ml incubation mixture, mixed and heated for five min in a boiling water bath. The absorbance at 546 nm was measured against a
blank (1 ml H2O plus 1 ml alkaline reagent). The standard curve was obtained by using different concentrations of maltose in the range of 0-1.5 umol/ l. The alkaline color reagent was prepared by dissolving
1 g of 3,5-dinitrosalycylic acid in a mixture of 40 ml 1 N NaOH solution and 30 ml H2O. Solid potassium sodium tartrate was added and dissolved. The mixture was brought to a final volume of 100 ml . Beta-amylase was determined as described above but soluble starch was replaced by amylopectin (Steup 1988).
Go to the Megazyme website. You will see a very specific method for alpha-amylase (Ceralpha Method) and a highly selective method for beta-amylase (Betamyl-3). DNSA is a useless method for measurement of any polysaccharide endo-hydrolase, especially for alpha-amylase. Under the conditions of the DMSA method, starch is hydrolysed so the measured values are many fold overestimated and the assay blank is through the roof. DNSA method is even useless for assays employing beta-linked polysaccharides such as for endo-cellulase and endo-xylanaes. In a recent paper (A Comparison of Polysaccharide Substrates and Reducing Sugar Methods for the Measurement of endo-1,4-b-Xylanase; Barry V. McCleary and Paraic McGeough. Applied Biochem and Biotechnology, we showed just how inaccurate this method is for measuring endo-xylanase.
@Stjepan Krešimir Kračun
This is a very elaborate response. A lot believe the action of alpha amylase on starch produces glucose in addition to the alpha dextrins and short chain oligosaccharides, which is why glucose is mostly monitored using DNSA or GOD/GPx method. This is a chemistry that needs to be carefully studied again. Although, glucoamylase (amyloglucosidase) produces glucose, it is known to act majorly on the alpha-1,6- bonds whereas, alpha amylase exerts its action on the alpha-1,4- bonds which is known to be their distinguishing factor.
Maybe a careful consideration of their mechanisms of action, specificity of their bond cleavage and products formed could further help in the naming and classification and perhaps update on the CAZy structure.
In the mean time, to distinguish between the action of Alpha and Beta amylases, I concur to the specific methods of assaying as suggested by @Frank Abimbola ogundolie and @Barry V McCleary.
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