I am measuring glucoamylase activity in commercial amylase (Spirizyme, Novozymes) based on the starch-iodine method, according to the method described by Oliveira et al. (2019): Article Flexible and expeditious assay for quantitative monitoring o...
In more detail:
Activity (U/ml) = (Mcontrol - Massay )/ (time * Volume) * Dilution factor
where hydrolysis time was 10 min (T=45oC) and volume of enzyme (diluted in buffer of pH 4.9) was 0,5ml.
The calibration curve received (mgstarch vs absorbance at 580nm): ABS= 0,2772 * M(mg)
I am presenting some represetative results as follows:
ABS ABS Dilution M(mg) M(mg) Activity
control assay factor control assay U/ml
0,486 0,155 5000 1,75 0,544 1208
0,486 0,191 7000 1,75 0,689 1489
0,486 0,223 10000 1,75 0,804 1897
0,486 0,324 20000 1,75 1,17 2337
I notice a stepwise increase in activity with an increased dilution factor.
How is this explained? How can I approximate an accurate value for the activity?
I do not know the buffer you used, but sodium acetate is recommended at a pH of 4.3. What standards and the dilutions and diluents. Please see the attached document for details.
Be careful it is not related to the method.(If it is just the last result the highest the better)
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