BASICS OF ENZYMOLOGY

Experiment 3

Principle

· Enzymes are affected by changes in pH. The optimum pH value is defined as the pH at which the enzyme rate of reaction (enzyme activity) reach the maximum activity (V max)

· Deviation in pH from the optimum cause decrease in enzyme catalytic activity

· Extremely high or low pH values generally result in complete loss of activity for most enzymes.

· The enzyme stability is depending on the optimum pH. Each enzyme has a region of optimum pH for stability.

· The optimum pH can be determined by incubating the enzyme in different incubation media containing different pH buffer range from 1.5 –10. The enzyme activity will be calculated at each pH at which the enzyme will be incubated. Plot a curve of enzyme rate of reaction (enzyme activity) against the different pH at which the enzyme catalytic reactions are incubated.

· From the curve, the optimum pH which give the maximum activity of the enzyme will be determined.

Materials required

• 0.1 M Sodium phosphate buffer (Different pH)

• 1% Starch

• 2 N Sodium hydroxide

• Sodium potassium tartrate tetrahydrate

• Dinitrosalicylic acid color reagent

• Standard Maltose Stock Solution

• Amylase enzyme

Procedure

Calculation

Enzyme Unit = µmoles maltose formed / min/ 0.5 ml enzyme (x 2)

= µmoles maltose formed / min/ ml enzyme used

Conclusion

From the results observed ,change in the pH cause effect on function of amylase enzyme, ultimately it reflects in the results of amylase enzyme activity.

Reference Material

Recipe to make phosphate buffer of different pH:www.aatbio.com/resources/buffer-preparations-and-recipes/phosphate-buffer-ph-5-8-to-7-4

Questions

1. What is the meaning of pH?

2. What is the composition of 0.1 M phosphate buffer?

3. What are the other parameters, that affect amylase enzyme activity?