Title: Assay of Alpha-Amylase Enzyme Based on Substrate Consumption
Aim: To determine the activity of the alpha-amylase enzyme by measuring the consumption of the substrate (starch) over time.
Introduction:
Alpha-amylase is an enzyme that catalyzes the hydrolysis of starch into maltose and dextrins. Its activity can be assessed by measuring the amount of substrate consumed during the reaction. This method provides insight into the enzyme kinetics and efficiency, which are crucial in various industries, including food, pharmaceuticals, and biotechnology.
Theory:
The enzyme alpha-amylase acts on starch, breaking it down into smaller sugar molecules. The rate of enzyme activity depends on the availability of the substrate and follows Michaelis-Menten kinetics. By measuring the decrease in starch concentration over time, the enzyme activity can be quantified. The reaction is monitored using iodine, which forms a blue-black complex with starch. As the starch is hydrolyzed, the intensity of the blue-black color diminishes, indicating substrate consumption.
Principle:
Alpha-amylase hydrolyzes starch into maltose and dextrins:
Starch + H₂O → Maltose + Dextrins
The consumption of starch is measured using the iodine-starch complex. A decrease in blue-black coloration indicates the extent of substrate hydrolysis. The reaction progress can be quantified using a spectrophotometer at 540 nm.
Requirements:
Alpha-amylase enzyme solution
Starch solution (1% w/v)
Iodine solution (I₂-KI)
Buffer solution (pH 6.5)
Test tubes
Pipettes and micropipettes
Water bath (37°C)
Spectrophotometer
Stopwatch
Procedure:
Prepare a 1% starch solution in buffer and heat to dissolve completely.
Label test tubes for different time intervals (0 min, 5 min, 10 min, 15 min, 20 min).
Add 2 mL of starch solution to each test tube.
Pre-warm the tubes in a 37°C water bath for 5 minutes.
Add 0.5 mL of alpha-amylase solution to each tube and mix well.
Start the reaction by adding the enzyme and begin timing.
At each time interval, stop the reaction by adding 1 mL of iodine solution to the respective test tube.
Note the color change at each time interval.
Measure absorbance at 540 nm using a spectrophotometer.
Result:
A gradual decrease in blue-black color intensity over time indicates starch consumption.
Absorbance values at 540 nm can be plotted against time to study the rate of starch hydrolysis.
Conclusion:
The consumption of starch over time indicates the progressive activity of alpha-amylase. The rate of starch hydrolysis provides insights into enzyme kinetics and efficiency, which are essential for industrial applications and enzyme-based processes.