I have produced cellulase enzyme by solid state fermentation using rice straw as substrate and fungus Aspergillus flavus. I use CMCase assay method to determine enzyme activity. Now, how do i calculate enzyme activity in U/ml.
My assay method was 1% CMC as substrate in 0.05 M phosphate buffer. I used 1.8ml 1% CMC and 0.2ml Enzyme extract and incubated at 60°C for 30min. Then add 3ml of DNS and incubated at 100°C for 5min. and measured Optical density at 575nm. I made a standard curve. How do i calculate in U/ml from standard curve.
To begin, you need to decide upon a definition of the Unit of enzyme activity, and the exact conditions of the measurement. Try to choose a definition that is consistent with most of the relevant literature.
A typical definition is the amount of enzyme that produces 1 micromole of product per minute under the stated conditions.
Your standard curve is in mg/ml, so you would need to convert it to micromoles to use it to measure enzyme activity in those units. This conversion will require a volume measurement and the molecular weight of the measured substance. The relevant volume for the conversion is the volume of the enzyme reaction, not the volume of the final measurement.
The slope of the enzyme reaction progress curve during the initial rate phase is in absorbance units per unit time (minutes). Use the standard curve to convert the absorbance to micromoles, and you arrive at micromoles per minute.
Adam B Shapiro yes, i forget to mention, One unit of CMCase activity was expressed as 1 µmole of glucose liberated per ml enzyme per min.
So what is the formula to calculate this?
Divide the rate of product formation in absorbance units per minute by the slope of the standard curve in absorbance units per micromole to get micromoles per minute.
Notice that the units are micromoles, not micromolar. You could use micromolar units instead, then multiple by the reaction volume in liters.
I worked with AaCel9A endoglucanase from the cellulase family. Activity assay was determined by incubating the enzyme for 3 min with 1%(CMC) in phosphate buffer (0.05 M, pH 7.0) at 65 C using NS reagent as a color-developing agent. The reducing ends of sugars were measured spectrophotometrically at 540nm, with glucose as a standard. One unit (U) of activity was defined as the amount of enzyme that produces 1 μmol of glucose equivalent per minute under the above conditions.
for further info refer to this article;
A Comparative Study of Activity and Stability of the Free and the Immobilized Endoglucanase from Alicyclobacillus Acidocaldarius.
Article A Comparative Study of Activity and Stability of the Free an...
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