I have been trying to determine Km and Vmax of alpha amylase with the MM plot but I cannot reach the saturation point.
My substrate concentration (starch) goes from 0.1 to 3 mg/ml and in my last experiment I tried with U=0.1 for the amylase but my plot still behaves as a linear plot.
I dont know what am I doing wrong. Does any one can share a protocol where you managed to construct the MM plot for this enzyme, please?
It's possible that the starch Km for your particular amylase is very high. You may have to go to higher concentrations, if it is feasible, to get curvature in your plot. You could also try a different substrate, a different source of starch, a purer starch, or a different set of conditions (buffer, salt, pH, temperature).
Adam B Shapiro Thanks for your kind response. I have not considered that the Km could be very high, Im very new working with enzymes. I will try repeating the experiment as you suggest, increasing the substrate concentration and let you know if it worked.
Make sure the substrate you are using is of high purity. The same goes for the enzyme. Low-quality reagents produce unreliable results.
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