Enzymology

Experiment 4

Aim of the Experiment

Effect of pH on amylase activity.

Principle

• The effect of pH on α-amylase activity will be studied

• 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.

Procedure

Follow the procedure given in the table.

Calculation

Enzyme activity = OD (test) x concentration of St (µmoles) x dilution of enzyme

OD (st) x incubation time (3 min)

= µmoles maltose formed / min/ ml enzyme.