I want to study the kinetics and thermodynamics properties of cloned, purified alpha amylase. It is active at 90°C. Can anyone suggest a good experimental guide line to set up the experiment to determine the Vmax, Km, Kcat etc. I have few questions.
1. Substrate is starch and I cannot determine its molecular weight. Is it ok to use it in percentage instead of uM?
2. What concentration of starch should I use for determination of V max and K cat?
Whatever I have read, most of the papers use percent starch as the substrate instead of uM.. for details of the assay. u can see this paper..
http://www.pakbs.org/pjbot/PDFs/42(5)/PJB42(5)3507.pdf
We have been performing enzyme kinetic and binding studies for many years, focusing on the action of alpha-amylase on native and hyrothermally treated starches. I recommed the attached papers.
Yes, you can only use percentage concentration, since it is very difficult to determine reliably the molecular wt of starch. Running experiments at 90 degrees C is not easy - this will (as I am sure you know) change the structure and properties of the starch, in that it will be gelatinised.
Article An investigation of the action of porcine pancreatic ??-amyl...
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