I need to know if I can estimate the amylase enzymatic kinetics. I have data of absorbance and concentration and I have a standard curve of glucose (DNS) and starch (iodine solution).
DNS and iodine (Fuwa?) methods present you with two different interpretation: based on DNS you can calculate the rate of reducing sugar produced whilst iodine is for the amount of starch degraded. Both essentially can be use to measure amylase kinetics. It depends on which amylase you are working on: alpha-amylase or glucoamylase. Glucoamylase is exo-enzyme thus it produces glucose. Alpha-amylase is endo-enzyme and its product of reaction is a mixture of maltose, dextrin, and few glucose. For glucoamylase definitely you can use DNS data. For alpha, iodine method is the actual fair assessment but we do can use DNS method, in my case, I used maltose as the standard.
The kinetic parameters are usually estimated from the Lineweaver–Burk plot (1/v vs 1/s) by varying the substrate concentration at the temperature of the maximum activity. Km and Vmax were determined from the intercepts at x and y axes, respectively.
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