Practical 4.3
Investigation of
the Effect of Temperature
on Enzyme Activity (Book 1A P. 11-12)
Objective: To investigate the effect
of temperature on enzyme activity
Biological
principle and experiment design: (14 marks)
Enzyme activity is affected by temperature. When
temperature increases, the kinetic (1) energy of the enzyme molecules and
the substrate molecules increases, so that the chance of formation of
Enzyme-substrate complex (1) increases. At
high
temperature, on the other hand, the
vibrations (1) of the amino acid molecules within enzyme proteins
become more
vigorous,
leading to the breakdown of the weak bonds maintaining the 3D conformation (1)
of the active site (1) of enzyme. The enzyme is said to be denatured (1).
To investigate the effect of temperature on the
catalytic activity of enzyme, amylase, which catalyzes the hydrolysis of starch,
can be used:
amylase
starch (1) maltose (1)
water (1)
The amylase activity can be deduced by measuring
how quickly starch (the substrate) disappears (1).
The presence of starch can be shown by iodine (1)
solution. When all starch molecules are
completely broken down, the reaction mixture no
longer shows blue-black
(1).
The independent variable in this experiment is the reaction
temperature (1).
The dependent variable is the activity of amylase (1), which can be
measured as the reciprocal of the reaction time (the time of disappearance
of starch), i.e. 1 / reaction time.
Results: (7
marks)
|
Samples
|
Temp (OC)
|
Time of disappearance of blue-black
colour (t) (min)
|
Reaction rate (1/t) (min-1)
|
|
A + 1
|
4
|
?
|
?
|
|
B + 2
|
2X
|
?
|
?
|
|
C + 3
|
40
|
?
|
?
|
|
D + 4
|
60
|
?
|
?
|
|
E + 5
|
80
|
?
|
?
|
Time (1); Correct calculation of reaction time (1)
Title (1), Choice
and labels of axis (1), units (1), Correct
plot – Low
temperature to optimum temperature (1) + Temperature for denaturation (1)
Result
Interpretation: (7 marks)
1
Describe and explain for the result of the above graph.
At low temperature, the reaction rate is
very low (1). This is because the enzyme is inactive at low temperature (1).
The reaction rate increases with
increasing temperature (1), as the kinetic energy of the enzyme and substrate
molecules increase with the increasing temperature (1), which increases the
chance of the formation of the enzyme-substrate complex (1).
The reaction rate is highest at X oC,
the optimum temperature for the enzyme (1). When temperature is higher than
optimum temperature, enzymes denature. The loss of the 3D-conformation causes
the reaction rate decrease and become zero (1).
Discussion:
(9 marks)
2 Explain
why it is necessary to place the test tubes into the water bath for 10 minutes
before mixing the solutions. (1)
To
ensure that the amylase and the starch solution have reached the pre-set
temperature before mixing together / the reaction (1).
3 Explain
why we have to use a clean dropper to transfer the mixtures
at each 2-minute interval.
(1)
To prevent the remains in the dropper from
interfering with the experimental results (1).
4 Predict and explain the effects of the following treatments
on the experimental results.
(a) Heating
the mixture of starch and amylase from 0oC to 37oC. (3)
Starch
will be broken down (1), leading to the disappearance of blue-black colour (1).
This is because amylase becomes more active
as the temperature increases from 0oC to 37oC
(b) Cooling
the mixture of starch and amylase from 100oC to 37oC. (3)
The
blue-black colour will not disappear (1) as starch will not be broken down (1).
Amylase has been denatured at 100oC. It’s
activity cannot be reversed even the temperature drops to 37oC (1).
5 Besides measuring the time of disappearance of
starch molecules, what other methods can be used to measure the activity of
amylase? (1)
To measure
the rate of appearance of maltose molecules (1)
Conclusion: (3 marks)
Amylase
is inactive at low temperature (1) and its activity increases with increasing
temperature (1). It loses its activity when the temperature is too high (1).
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