I do not see how there can be a way without separating the two enzymes. That should be doable by IEF and MW in media with different pH. I think these two enzymes have different pH optimiums for their media.

I think it is not possible to evaluate kinetic parameters without knowing the amount (and ratio) of both enzymes in the medium and without inhibiting one of them separately. You should know their kinetics first in order to predict (sometimes) the consequences of mixture kinetics, but not the other way around.

Use the respective properties of the two enzymes to selectively inhibit one while the other is active. this paper may be useful; Inhibition of Glucoamylases from a Rhizopus Sp. and Aspergillus saitoi by Aminoalcohol Derivatives

J Biochem (1985) 98(2): 341-347.

how about using inhibitors for one process specifically and carrying the other

but the problem with this, what are you accessing? is it the end product? or the product of the enzyme intermediates only. It depends on your question and composition too.

i think try stop flow technique you might be able to trace it even with out any inhibitors real time if you were able to separate the enzymes by size on HPLC column

You should try with starches crosslinked to various degree as substrates or with specific inhibitors to differentiate among glucoamylase and alpha-amylase

I would compare the kinetcs of isolated individual enzymes alpha- and gluco- amylases. Then I would prepare diferent mixtures containig diferent proportions of alpha- and gluco- amylases and observe the kinetics of oligosaccharides and glucose release. Finally I would trry sspecific inhibitors

The problem is that you modify the substrate for alpha-amylase by the action of glucoamylase and vice versa. Consequently, the action of one enzyme in its isolated form on starch is not the same as in combination with the other enzyme. So much for inhibition of the individual enzymes. Moreover, what "kinetic parameters" do you expect to obtain? It is no Michaelis-Menten kinetics with a high MW substrate (and the number of substrate molecules initially increases by the hydrolysis). Thus, you cannot obtain kcat or KM. If you can distinguish between the products by alpha-amylase and glucoamylase, you might extract units per mL of your mixture (as you obviously don't have protein/enzyme concentration numbers). Very crude...

You might try using some different substrates, for example a small oligosaccharide to measure the glucoamylase activity and a polysaccharide to measure alpha-amylase activity. Megazyme offers a variety of substrates to measure carbohydrase activity. Their website is: www.megazyme.com. Do you know if your alpha is a G4 or G6 type amylase, i.e. cleaves 4 or 6 glucose units? This might guide you in choosing the appropriate substrates. Another idea to consider is the use of pH. Do the two enzymes have the same optimum pH? If not, can you assay each at their optimum pH while slowing down the activity of the other. These suggestions may not give you totally accurate kinetic parameters, but it should help you differentiate the kinetics of the two enzymes.

Hi, you cold see the paper entitled

A solid state fermentation based bacterial α-amylase and fungal glucoamylase system and its suitability for the hydrolysis of wheat starch

Sanjeev K Soni, ,

Arshdeep Kaur,

Jugal Kishore Gupta

they made a nice work on both enzymes activities at different temperature and in presence or absence of 10mM CaCl as stabilizer

In my opinon you can try to measure the enzymatic activity on two different pH, i.e. 3 and 8 because the optimum pH reported for the two enzyms are very different. Anyhow some more information shold be noted, i.e. from where the enzymes came, what is the substrate concentration you use. Also if in the market is available a selective substrate for the glucoamilase you can try a titration, that is always usefull. Moreover alfa-amylase is Ca++ dependent, so what happen adding EDTA in the substrate solution?