Name: _____________________________________________ Group No: ____________

Yr & Sec: ________________________

BIOCHEMISTRY
ENZYMES AND FACTORS THAT AFFECT ENZYME ACTIVITY

THEORY
Metabolic processes provide energy for the maintenance of life-support systems of the organism.
They consist of degrading chemical compounds of relatively high potential energy. An example of such
degradation is the conversion of glucose to CO2 and H2 O. The energy evolved is collected, stored and
utilized by the cell for growth, reproduction, synthesis, repair of cellular materials and other functions which
keep life systems operational. These processes are made possible by the presence of catalytic substances
called enzymes.
As learned from basic Chemistry, catalysts alter the energy of activation, Ea, of a chemical reaction.
Increasing the Ea decreases the rate of a reaction (negative catalysis) and decreasing the Ea increases the
rate of reaction (positive catalysis).
Enzymes are complex organic substances produced by an organism to alter the rate of biochemical
reactions that take place in the cell. However, unlike inorganic catalysts, enzymes show a high degree of
specificity, with each enzyme catalyzing only one kind of reaction for only one kind of substrate. For
example, the hydrolysis of sucrose is catalyzed by invertase, which acts only on sucrose and no other
disaccharides. In contrast, HCI, in vitro, will hydrolyze a number of disaccharides and even polysaccharides
with facility.
At present, about 2,000 different enzymes have been isolated and characterized. In this experiment,
catalase, an enzyme derived from potatoes and which detoxifies peroxides produced in the body from the
metabolism of amino acids will be isolated and characterized.
All enzymes have a protein component and are affected by a number of physical and chemical
factors that denature proteins, e.g., extremes of pH and temperature, presence of metal and non-metal ions,
presence of alkaloidal reagents, etc. In addition, enzyme and substrate concentrations also affect enzyme
activity. In this experiment, the effect of temperature and pH on salivary amylase is determined.

OBJECTIVES
1. To isolate and determine the properties of catalase.
2. To determine the effect of sulfides on catalase activity.
3. To prove the presence of a protein factor in enzymes.
4. To determine the effect of temperature on enzyme activity.

MATERIALS
*1 pc. Small potato with peel 0.1 N NaCI
Filter paper 0.05 N HCI
6% H2 O2 0.05 N NaOH
*filter paper or cotton/gauze blender
Guaiac solution Funnel
Saliva *cheesecloth
Starch solution wooden splint
Acidified iodine solution (0.001 N)

PROCEDURES
I. A. Preparation of Catalase Enzyme
Grate a potato, including the peel, into a fine pulp. (A blender may be used for this.) Add
50 ml of water in a beaker and allow the mixture to stand for 10 minutes, swirling occasionally.
Strain through filter paper. (NOTE: If filtering through filter paper takes too long, use cotton or
gauze.) Use the filtrate for the following test:
Mix 5 ml of the filtrate and 5 ml of 6% H2 O2 in a test tube, immediately insert a glowing
wooden splint. What gas is indicated present?
Observation; The preparation of the catalase from potato is very effortful, however, it
does not show any results when we inserted at the glowing stick.

B. The Protein Nature of Enzymes

To 5 ml of the catalase extract, add 5 ml of 3M NaOH. Mix the two solutions by tapping
the tube against the palm of your hand. Add 5 drops of CuSO4 solution to the mixture. Observe
and record the color.
 Observation; There was a Violet Color in the solutions and is very visible in the saliva
solution. The Violet Color indicates that there is an apoenzyme in the saliva.

II. FACTORS THAT AFFECT ENZYME ACTIVITY

A. Preparation of enzyme. Extract 10-15 ml of saliva from a student donor and use it to
test for the effect of pH and temperature.
B. Effect of pH
Label 3 test tubes and mix the substances indicated below.
Tube Number 1% starch 0.1 M NaCI Acid/base/water saliva
solution added
1 10 ml 1 ml 1 ml 0.05 N HCI 2 ml
2 10 ml 1 ml 1 ml distilled 2 ml
water
3 10 ml 1 ml 1 ml 0.05 N 2 ml
NaOH

Mix well by shaking each test tube, and place in a water bath maintained at body
temperature (37°C). Recording the time at 3-minute intervals, test for the presence of starch
using 0.001 N iodine solutions. Record the time needed for the blue color of starch with
iodine to fail to appear. This means that starch has been completely hydrolyzed to glucose.
Time (minutes) Color of saliva extract with iodine
Test tube #1 Test tube #2 Test tube #3
3 Dark blue color Dark blue Dark blue
6 blue Dark blue Blue
9 blue Dark blue Blue
12 Light blue blued Light blue
15 Very light blue No blue Light blue
18 No blue No blue No blue

In what test tube is there evidence of unhydrolyzed starch after 18 minutes?

Observation; All the test tubes have complete hydrolysis, Test Tube 2 showed
hydrolysis after 12 minutes, Test Tube 1 and 3 after 15 minutes. Acidic or Basic pH
delays hydrolysis of starch. Salivary amylase is the enzyme.

C. Effect of Temperature
Label three test tubes , add 10 ml of 1% starch suspension and 1 ml 0.1 N NaCl solution.
Immerse tube 1 in a beaker of water at room temperature; tube 2 in a water bath at 37o C;
and tube 3 in a boiling water bath. Using a pipette, add exactly 2 ml of saliva into each
tube. Mix well and monitor in a manner similar to Part II-B. Note the length of time it
takes before the starch is completely hydrolyzed. Record the time needed before the blue
color with iodine no longer appears.

QUESTIONS:
1. What is the equation for the reaction catalyzed by catalase?
- 2 H2O2 catalase 2 H2O + O2

2. How does an enzyme alter the rate of chemical reaction?

- The positive catalyst lower energy of activation, so rate of reaction is faster which is the reverse
for negative catalyst.

3. What is the optimum pH and temperature for salivary amylase?

- The optimum pH for salivary analysis is 6.4 – 7.3 , optimum temperature is about 37 Celcius.

4. What effects do increase pH and temperature for salivary amylase?

a. Increased pH
- Ph higher than 7.3 denature apoenzyme component of enzyme, making it less efficient in
digesting carbohydrates.
 b. Increased temperature
- Higher temperature denatures protein component, making it less efficient in digesting
carbohydrates.

5. In summary, what factors affect enzyme activity?

- Factors affecting enzyme activity includes substrate concentration, enzyme concentration, pH,
and temperature.