Date :

EFFECT OF DIFFERENT pH ON ACTIVITY OF β-AMYLASE ENZYME EXTRACTED FROM

SWEET POTATO

Aim: To study the effect of different pH on the activity of β-amylase enzyme extracted from sweet potato.

Requirements: Sweet potato, funnel, conical flask, cotton, filter paper, plate, distilled water, mortar and
pestle, DNSA reagent, boiling water bath, 0.2% NaCl, different pH of acetate buffer (0.2M) – pH 4.2, 4.6,
4.8, 5.0, 5.2, 5.4, 16 test tubes, 210ml pipettes.

Principle: Activity of β-amylase is analysed by the maltose that is produced by its activity on starch.
Maltose being a reducing sugar, reduces 3,5-dinitrosalicylic acid to 3-amino-5-nitro salicylic acid which
is orange brown in colour. The colour intensity is read at 520 nm.
Each enzyme has it's optimum pH at which it shows maximum activity, above or below which the activity
declines. Very high or low pH affects the ionic properties of amino acids at the active site of the enzyme,
thus affecting its activity. Optimum pH is decided by the type of amino acids that make up the active site
of the enzyme.

Procedure: Prepare the following set of tubes as shown in the chart.

Calculations: Henderson Hasselbalch equation is used to prepare buffers

 pH Volume of CH3COOH (ml) Volume of CH3COONa (ml) Total volume

4.2 0.784 0.216 1.000

4.4 0.696 0.304 1.000

4.6 0.591 0.409 1.000

4.8 0.477 0.523 1.000

5.0 0.365 0.635 1.000

5.2 0.266 0.734 1.000

Observation Table :

pH Acetate 2% 0.2% Instructions Enzyme Instructions Corrected

buffer Starch NaCl Extract Abs (520
(ml) Solution Solution (ml) nm)
(ml) (ml)

Blank 1.0 1.0 1.0 Keep all test 1.0 Keep all tubes at
(D/W) tubes at 37°C (D/W) 37°C for 30
for 5 minutes. minutes. Then
4.2 1.0 1.0 1.0 1.0 take 1 ml of
reaction mixture
into
4.4 1.0 1.0 1.0 1.0 corresponding
tubes having 1ml
DNSA reagent.
4.6 1.0 1.0 1.0 1.0 Keep in a
boiling water
bath for 5
minutes, colour
4.8 1.0 1.0 1.0 1.0
change occurs.
Cool the test
tubes and add
5.0 1.0 1.0 1.0 1.0 8ml d/w. Take
absorbance at
520 nm.
5.2 1.0 1.0 1.0 1.0

5.4 1.0 1.0 1.0 1.0

Graph: Plot a graph of Absorbance at 520 nm on Y-axis v/s pH on X-axis

Result: The enzyme β-amylase shows maximum activity at pH .

Thus pH is the optimum pH for β-amylase enzyme activity.

Viva questions :-

Q - What are the examples of enzymes with optimum pH?

Ans -
Enzyme Optimum pH

Lipase (pancreas) 8.0

Lipase (stomach) 4.0-5.0

Lipase (castor oil) 4.7

Pepsin 1.5-1.6

Trypsin 7.8-8.7

Urease 7.0

Invertase 4.5

Maltase 6.1-6.8

Amylase 6.7-7.0

Amylase (malt) 4.6-5.2

Catalase 7.0

Q- Explain the nature of the graph of effect of pH on enzymes?

Ans-The most favorable pH value - the point where the enzyme is most active - is known as the optimum
pH. Extremely high or low pH values generally result in complete loss of activity for most enzymes.

The inflection points on each side of the curve represent the pKa of the critical R-groups in the
enzyme.The inflection points on each side of the curve represent the pKa of the critical R-groups in the
enzyme. http://plantphys.info/plant_physiology/enzymekinetics.shtml

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