Title: Assay of Alpha Amylase in Terms of Reducing Sugar Produced and Calculation of Enzyme Unit
Aim: To determine the activity of alpha amylase by measuring the amount of reducing sugars produced and calculating the enzyme unit.
Introduction:
Alpha amylase is an enzyme that catalyzes the hydrolysis of starch into reducing sugars such as maltose and glucose. The activity of this enzyme can be quantified by measuring the amount of reducing sugars released using the dinitrosalicylic acid (DNS) assay. The enzyme unit is calculated based on the amount of enzyme required to release a specific amount of reducing sugar per minute under standard conditions.
Theory:
The enzymatic hydrolysis of starch by alpha amylase generates reducing sugars, which can be quantified using DNS reagent. Upon reaction, DNS undergoes a color change proportional to the concentration of reducing sugars. The absorbance is measured spectrophotometrically at 540 nm, and the enzyme activity is calculated in terms of units.
Principle:
The assay is based on the reduction of 3,5-dinitrosalicylic acid (DNS) by reducing sugars produced in the enzymatic reaction. The resulting product has an orange-red color, and its absorbance is measured at 540 nm. One unit of alpha amylase is defined as the amount of enzyme required to release 1 µmol of reducing sugar per minute under standard assay conditions.
Requirements:
Alpha amylase enzyme solution
Soluble starch (1% w/v)
Sodium phosphate buffer (pH 6.9, 50 mM)
DNS reagent
Sodium potassium tartrate
Test tubes
Water bath (boiling)
Spectrophotometer
Pipettes and micropipettes
Standard glucose or maltose solution for calibration
Procedure:
Take 1 mL of 1% starch solution in a test tube.
Add 1 mL of sodium phosphate buffer (pH 6.9, 50 mM).
Pre-incubate the mixture at 37°C for 5 minutes.
Add 1 mL of alpha amylase enzyme solution to the reaction mixture, and Incubate at 37°C for 10 minutes.
Add 2 mL of DNS reagent to each test tube.
Boil the tubes in a water bath for 5 minutes to develop the color.
Cool the tubes to room temperature under running tap water.
Dilute the samples if necessary.
Measure absorbance at 540 nm using a spectrophotometer.
Use a standard curve prepared with known concentrations of glucose or maltose to determine the concentration of reducing sugars.
Calculation of Enzyme Unit:
One unit of alpha amylase is defined as the amount of enzyme that releases 1 µmol of reducing sugar per minute under assay conditions.
Observation:
The intensity of the orange-red color increases with the amount of reducing sugars produced.
Higher enzyme concentrations result in greater absorbance values at 540 nm.
Result: The activity of alpha amylase was determined based on the amount of reducing sugars produced. The enzyme unit was calculated accordingly.
Conclusion: The assay successfully quantified the activity of alpha amylase by measuring the reducing sugars released during starch hydrolysis. The enzyme activity was calculated in terms of enzyme units, which is essential for enzyme kinetics and industrial applications.
