I need methods to measure the amylase enzyme activity produced from Bacillus Subtilis
Hello Mustafa, several methods have been reported in literature. For instance, you could use the Bernfeld method which measures the rate at which maltose is released from starch. This is quantified by the ability of the maltose to reduce 3,5-dinitrosalicylic acid. One unit releases one micromole of β-maltose per min at 25°C and pH 4.8 under the specified conditions. You could also try the microplate-based starch–iodine assay protocol. Here, assay reactions are initiated by adding 40 ul of starch (Sigma S-2630) solution (2.0 g/L) and 40 ul of enzyme in 0.1 M phosphate buffer at pH 7.0 to microplate wells. To minimize evaporative loss during incubation, a plastic mat is used to cover the microplate in combination with using a temperature block equipped with a hot lid. After 30 min of incubation at 50 °C, where the assayed enzymes were most active, 20 ul of 1 M HCl was added to stop the enzymatic reaction, followed by the addition of 100 ul of iodine reagent (5 mM I2 and 5 mM KI). Following color development, 150 ul of the iodine-treated sample was transferred to a transparent Xat-bottomed 96-well microplate and the absorbance at 580 nm (A580) was measured using microplate reader (Bio-TEK Instruments, Winooski, VT, USA). Protocols will differ depending on whether the amylase is alpha, beta or glucoamylase. Please see the attached. Explore literature.
To measure your amylase activity, you will monitor the disappearance of amylase"s substrate. Starch reacts with iodine ( which is yellow) to form a blue compound ( Amax 620 nm ).The reaction is the basis of a colorimetric assay for amylase activity. The hydrolysis of starch can be measured through the use of of an enzyme test or assay. An enzyme assay will test for the simple presence of enzyme activity but can also be used to measure the reaction rate of an - catalyzed reaction. For more details consult https://msu.edu -ibs -EnzymeActivity04 ;;;https://www.sigmaaldrich.com -- biology
For measuring the amylase activity with DNS solution (Miller Method):
1. Prepare starch solution (10 mg/ml)
2. Prepare enzyme extract filtered with syringe filter
3. Prepare phosphate buffer solution (o.1(M), pH=7)
4. Add 1(ml) enzyme extract to test tube, then add 1(ml) of starch solution (it can be considered as a main sample)
5. Add 1(ml) enzyme extract to test tube, then add 1(ml) phosphate buffer (it can be considered as a blank or control sample)
6. Incubation of main and control sample at 37˚C for 30(min)
7. Add 2(ml) DNS solution to each test tube.
8. Transfer the tube to the water bath at boiling temperature for 15(min)
9. Keep the test tube at room temperature to cool the sample
10. Transfer the solution to the cuvette
11. Measure the absorption of the main sample at λ=540(nm)
It is necessary to mention that for calculating the enzyme activity the standard curve must be prepared by maltose solution.
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