I am about to calculate alpha-amylase activity for some fungal samples. I followed the protocol of Sigma-Aldrich (https://www.sigmaaldrich.com/DE/en/technical-documents/protocol/protein-biology/enzyme-activity-assays/enzymatic-assay-of-a-amylase) and incubated my samples in 0.2 % starch solution for 3 minutes.
According to the protocol, the equation for the U/ml value is:
U/ml = (mg of maltose released * dilution factor) / (ml of enzyme applied)
I think I also have to consider the incubation time of 3 minutes, and divide my result from the equation by 3?
Thanks in advance for help.
This is a case where a non-standard unit definition is being used. According to the Sigma document the unit is:
Unit Definition: One unit will liberate 1.0 mg of maltose from starch in 3 minutes at pH 6.9 at 20 °C.
The 3 minutes is already accounted for by the definition, so you don't have to divide by it.
The unit definition for the Amylase is : One unit will liberate 1.0mg of Maltose from starch in 3 minutes, at pH 6.9 @ 20C. So you do not have to compensate for the 3 minute incubation time in the calculation.
I agree, acording to the sigma company defintion for amylase activity, the time (3 minutes) was involved in the calculation of activity, therefore you dont need to divide the result on 3
It would be better to express the enzymatic activity in the SI unit katal (mol maltose released per s) or a suitable fraction thereof (nkat or fkat). A non-standard definition of a non-standard unit: shudder.
Yes, at least an International Unit should be used.
Amylases hidrolyze glycosidic bonds. When a general substrate is used, as starch or glycogen, the hydrolysis will be done for ALL enzymes capable to hydrolyze alpha 1-4 and alpha 1-6 bonds between glucoses. Then every broken bond will produce a new reducing sugar. So, if you measured reducing sugars, to calculate the amylase activity, you will be able to know how many glycosidic bonds were hydrolyzed, by time unit (min). 1 unit of amylase is the enzyme required to release 1 micromol of reducing sugar (from starch or glycogen substrate) by minute.
A maltose standard curve will be necesary to get the molar extinction coefficient, to convert absorbance (ie. 550 nm by DNS method) to equivalent moles of maltose, as reducing sugar.
Hector
Adam B Shapiro Hi, I have a question about the way to prepare control samples. In my experiments, I put enzymes into starch solution after incubating the solution at the same conditions as samples, and then centrifuge to remove starch before adding DNS. The results showed that the difference between control and samples was not high, so I always have low amylase activity even when the color of samples is strong. Do you think that there is any problem in my protocol? Thank you!
Why do you put enzyme into the control samples?
What may be happening is that when you add the enzyme to the pretreated starch control sample, it immediately produces a lot of glucose, similar to the amount in the other samples.
To measure the activity of amylase in your samples, you need a negative control that does not have any amylase activity so that you can measure the background absorbance. The easiest way to prepare this control is simply to leave out the enzyme.
If the reason that you put the enzyme into the control samples is because you are concerned that the enzyme itself (aside from its amylase activity) may contribute to the background, you can measure this by preparing a control with enzyme but no starch.
Adam B Shapiro Thank you so much for your advice! I'll try to test the control samples by just adding amylase and buffer.
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