Generally enzyme unit is defined as, "amount of enzyme required to release μM of product per min or require to catalyze uM of substrate per min." But I am using 50 ul of purified enzyme (0.01 μM concentration) with 50 ul of substrate (total reaction mixture is 100 ul now) followed by incubation for 5 min and estimation of reducing sugars by DNS method. Now I am able to get mg of sugar released per min from standard curve but I am confused about how to define the unit in this case as I am using fixed/ known concentration of enzyme.
Dear Umma Hamd.
you must always convert to U/ ml, using equation of DNS method.
Thank Hind Khudhair. I know when ever we are working with batch fermentation than it is easy to calculate enzyme unit in U/ml/min. But as I have mentioned earlier if I am using known concentration (0.01 uM) and if i calculate per ml it means i should multiply with 20 to get for 1 ml (50*20= 1000 ul). But what about the enzyme conc? should i define it in unit definition or not? I am confused about it.
Hi Umma.
the Final volume including DNS, so how much did you add of DNS?, and by using protein concentration , you can calculate specific activity of your enzyme.
The specific activity of an enzyme is another common unit. This is the activity of an enzyme per milligram of total protein (expressed in μmol min−1mg−1). Specific activity gives a measurement of the activity of the enzyme.It is the amount of product formed by an enzyme in a given amount of time under given conditions per milligram of total protons.
Hi,
The enzyme activity (U) in this case can be defined as μmol of released sugar (probably glucose) per unit time (min). Then, to calculate the enzyme concentration, the enzyme activity (U) is divided by working volume (mL). The enzyme concentration cannot be defined as mg/mL. In order to measure the enzyme purity, it must be measured based on specific activity (U/mg-protein) or also with consideration of volume (U/ml.mg-protein)
you have mentioned that you use the purified enzyme for your calibration curve, if the purity of the enzyme is close to 95-100% then you can recalculate your sample enzyme activity per mg of the enzyme in the sample.
Dear All
I need to use 90 KLU/L enzyme to hydrolyze the oil. May I know how can I calculate it as a gram? because my Enzyme is in powder and I need to weigh the required amount to prepare enzyme solution.
Regards
Reza
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