I have immobilized an enzyme on the immobilization resin Dowex. How can I determine the number of enzyme units present in the powder form. Is it possible that enzyme assay is performed with powder and enzyme units are reported in units/g instead of units/ml. For example, to perform laccase enzyme assay, 10μL enzyme sample is required and results are reported in units/ml. What if I take 10mg of sample and report the results in units/g by performing the same procedure. If it is possible, then kindly provide the reference document or any other way to perform enzyme assay on powdered immobilized enzyme.
I don't think you can because you don't know how much protein you have used. protein (enzyme) concentration is important for following calculation such as activity and etc, why not to resuspend it in a suitable buffer then measure protein concentration and assay it?
Hope this helps
April 1, 2021
Dear Usama,
Knowing the amount of protein immobilized on the Dowex is not the most important parameter as some of the enzyme protein may have been inactivated during the immobilization process. So you need to measure the activity, not the amount of bound protein.
One approach would be to determine the dry wt of the Dowex/enzyme complex, e.g., mg/mL Dowex/enzyme suspension. For the enzyme assay, add an accurately-measured volume of suspension to your buffer, followed by the substrate. It is important to keep the resin/enzyme in suspension, so incubate with stirring (very small stir bar) at a suitable temperature. At periodic intervals (e.g., 0.5- or 1 min), withdraw measured volumes of the liquid over time and measure the amount of product formed/min. This can be used to calculate the # enzyme units/mL (or per dry wt) of the Dowex/enzyme complex.
I hope this information helps you.
Bill Colonna Ames, Iowa, USA [email protected]
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