I have given the protocol for the cyclodextrin glygosyl
transferase assay:
One ml of appropriately diluted enzyme sample was incubated
at 60 °C for 15 min with 5 ml of 1% (w/v) gelatinized
soluble starch in 50 mM, 7.0-pH Tris–HCl buffer. Reaction
was terminated by boiling the reaction mixture for 3 min
and reaction volume was made to 10 ml with distilled water.
Two ml of above reaction mixture was withdrawn and mixed
with 3 ml of Tris–HCl buffer, 5 ml of 125 mM Na2CO3, and
0.5 ml of phenolphthalein (25 mg phenolphthalein/100 ml
absolute alcohol). Absorbance was measured at 550 nm. The
percent decrease of sample was calculated with respect to
control containing 5 ml of buffer, 5 ml of sodium carbonate
and 0.5 ml of phenolphthalein.
where Acontrol = absorbance of control and Atest =
absorbance of sample.
The amount of β-cyclodextrin (β-CD) produced was estimated
from the standard graph of 0–500 μg/ml β-CD concentration
against % decrease in absorbance. One unit of CGTase was
defined as the amount of enzyme required to produce 1 μm of
β-CD/min.
Please can you suggest me the formula for the defination
given in the last line
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Answer / Kumkum Panday
The formula given in the last line defines one unit of CGTase (Cyclodextrin Glycosyl Transferase) as follows:
1. Calculate the percent decrease in absorbance for both the sample and control using the following equation: Percent_Decrease = ((Acontrol - Atest) / Acontrol) * 100%
2. Plot a standard graph of % Decrease against β-CD concentration (0-500 μg/ml).
3. Determine the slope (S) of the line that best fits the graph. The slope represents the change in percent decrease for a given change in β-CD concentration.
4. One unit of CGTase is defined as the amount of enzyme required to produce 1 μm (millimole) of β-CD per minute.
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