Group 2

Chiong, John Ezra M. Gomez, Abraham III A.

Cometa, Rem Ashley G. Frando, Sandra Rose Y.

Delmendo, Aldrin M. Kahulugan, Paul Jarvin J.

Fernandez, Kriezelle Adona Joyce D.

Experiment 6

I. A. Enzymes and Factors that Affect Enzyme Activity

Mix 5mL of the filtrate and 5 mL of 6% H2O2 in a test. Immediately insert a

glowing wooden splint. What gas is indicated present?

Observation: The splinter did not burn but remained glowed due to the presence of
O2

B. The Protein Nature of Enzyme

To the 5ml of the catalase extract, add 5mL 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 colour.

Observation: colour change to violet

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 if pH and temperature.

B. Effect of pH
Label 3 test tubes and mix the substances indicated below.

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 0.001N iodine solution. Record the time needed for the
blue colour of starch with iodine to fail to appear. This means that starch has
been completely hydrolyzed to glucose.
 In what test tube is there evidence of unhydrolyzed starch after 18 minutes?
Observation:

What is the optimum pH of salivary amylase? pH 6.8

Conclusion:
After mixing the contents each test tube is placed in water bath maintained at
37oC.The time when the blue colour of starch with iodine fails to appear indicate
when starch has been completely hydrolysed to glucose.

C. Effect of Temperature
Label 3 test tubes, add 10mL of 1% starch suspension and 1mL of 0.1N NaCl
solution. Immerse Tube #1 in a beaker of water at room temperature, Tube #2 in
a water bath at 37˚C, and Tube #3 in a boiling water bath. Using a pipette, add
exactly 2mL of saliva into each test 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 colour with iodine no longer
appears.

QUESTIONS:

1. Write the equation for the reaction catalyzed by catalase.

Catalase + H2O2 Catalase + 2 H2O + O2

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

Enzymes will make chemical reactions go faster. Enzymes are catalysts which will lower
the activation energy of a chemical reaction.
Activation energy is the energy needed to get a reaction going. By lowering the amount
of energy needed to start a reaction, the reaction can go more quickly.

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

 The optimum temperature of salivary amylase ranges from 32°C to 37°C. This applies to
the human body since salivary amylase is suitable to function within these temperatures.
The optimum pH for the enzymatic activity of salivary amylase ranges from 6 to 7. Above
and below this range, the reaction rate reduces as enzymes get denaturated. The
enzyme salivary amylase is most active at pH 6.8.

4. What effects do a.) increased pH and b.) increased temperature have on the activity of
salivary amylase?
a.) Increased pH:
The optimum pH for the enzymatic activity of salivary amylase ranges from 6 to 7.
The enzyme salivary amylase is most active at pH 6.8. Our stomach has high level of
acidity which causes the salivary amylase to denature and change its shape. So the
salivary amylase does not function once it enters the stomach.
b.) Increased temperature:
Increasing temperature by more than a couple degrees (say 5 or so) would decrease
salivary amylase’s ability to function properly (decrease its enzymatic “activity”)
because the protein structure it is made of would start to breakdown (due to the
increase in thermal energy). Because the shape of an enzyme is critical to its
function (form fits function), the breakdown of the protein would lose the shape it
needs to work.
5. In summary, what factors affect enzyme activity?
 Temperature
o Higher temperatures mean molecules are moving faster and colliding more
frequently. As the temperature of an enzymatically catalyzed reaction increases,
so does the rate (velocity) of the reaction.
o However, when a temperature increases beyond a certain point, the increased
energy begins to cause disruptions in the tertiary structure of an enzyme;
denaturation occurs.
o Change in tertiary structure at the active site impedes catalytic action, and the
enzyme activity quickly decreases as the temperature past this point.
o Optimum temperature in humans is 37 degrees Celsius. Denaturation begins at
temperature of 40 degrees Celsius.
 pH
o Small changes in pH (less than one
unit) can result in enzyme
denaturation and subsequent loss of
catalytic activity.
o Each enzyme has a characteristic
optimum pH, which usually falls
within the physiological pH range of
7.0-7.5. This is the pH at which
enzymes exhibit maximum activity.
o A variation from normal pH can also
affect substrates, causing either protonation or deprotonation of groups on the
 substrate. The interaction between the altered substrate and the enzyme active
site may be less efficient than normal or even impossible.

 Substrate concentration
o As substrate concentration increases, the
point is eventually reached where enzyme
capabilities are used to their maximum
extent. The rate remains constant from this
point on.

 Enzyme concentration
o If the amount of substrate present is kept
constant and the enzyme concentration is
increased, the reaction rate increases
because more substrate molecules can be
accommodated in a given amount of time.
o The greater the enzyme concentration, the
greater the reaction rate.