EXPERIMENT 2ENZYME ASSAYS AND FACTORS AFFECTINGENZYME ACTIVITY
TABLE OF CONTENTS
Bil. Title Page
1 Summary 2
2 Pictorial Methodology 3
3 Results 5
4 Discussions 8
5 Calculations 10
6 Conclusions 13
7 References 14
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EXPERIMENT 2ENZYME ASSAYS AND FACTORS AFFECTINGENZYME ACTIVITY
SUMMARY
For this second experiment entitled Enzyme Assays and Factors Affecting Enzyme
Activity, there are two main objectives which are to comprehend physical characteristic and
properties of enzymes and to study the factors affecting their activity. From the results of the
experiment, Graph 1 shows the glucose standard calibration curve did not follow the theory
because graph pattern obtained not linear line due to errors occurred in this experiment.
Theoretically, when the concentration of glucose increases the enzyme activity also increases.
While Graph 2 shows the effect of substrate concentration on enzyme activity, shows that as
the substrate concentration (starch solution) increase with the concentration 0.5, 1.0, 2.0 and
3.0 % (w/v), the concentration of enzyme activity is increase and nearly produce a linear line
neglecting the small increase the graph still follow the theory but due to at point substrate 1.0,
the rate decrease abnormally causing the defect in the graph. Then, Graph 3 shows the effect
of pH on the enzyme activity shows that, the optimum pH is at pH 9. The result of pH 9
shows that the enzyme activity reaction is actively produced at this pH which is still within
the range of the theory stated. As for the conclusions, there are few factors that affect the
enzyme activity which are the temperature, pH and the substrate concentration. As the
concentration of substrate increases, the enzyme activity also increases. However, after
period of time the reaction is decreases due to the saturation of enzyme. . In this experiment,
there are few errors might be occurred which might affect the result of experiment as some of
the result did not follow the theory. This may be due parallax error.
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EXPERIMENT 2ENZYME ASSAYS AND FACTORS AFFECTINGENZYME ACTIVITY
PICTORIAL METHODOLOGY
B. Effect of Substrate Concentration on Enzyme Activity
a) Prepare a starch solution of concentration 0.5, 1.0, 1.5, 2.0 and 3.0% (w/v) as the substrate.
Place 0.5 mL of the substrate of various concentrations into the test tubes.
b) Add 0.2 mL enzyme preparation and 0.3 mL, 0.2 M phosphate buffer at pH 7.0
c) Incubate the reaction mixture for 30 min at 30ºC with shaking of 150 rpm.
d) Determine the amylase activity using the method given in Appendix 1.
e) Plot the graph of product formed against substrate concentration.
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EXPERIMENT 2ENZYME ASSAYS AND FACTORS AFFECTINGENZYME ACTIVITY
C. Effect of pH on Enzyme Activity
a) Determine the amylase activity using method shown in Appendix 1.
b) Determine the optimum pH for the enzyme by incubating the reaction mixture at various
pH: 5.0, 7.0, 8.0, 9.0, 11.0.
c) Determine the amylase activity at all pH and plot a graph of product formed against pH.
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EXPERIMENT 2ENZYME ASSAYS AND FACTORS AFFECTINGENZYME ACTIVITY
RESULT
Table 1: Glucose Standard Curve
Concentration of Glucose (mg/L) Absorbance (nm)
100 0.328
200 0.339
500 0.327
1000 0.350
2000 0.317
0 500 1000 1500 2000 25000.3
0.31
0.32
0.33
0.34
0.35
0.36
f(x) = − 5.91210613598684E-06 x + 0.336693200663351R² = 0.131976570912927
Absorbance (nm) Vs Concentration of Glucose (mg/L)
Concentration of Glucose (mg/L)
Ab
sorb
ance
, nm
Figure 1: Standard curve calibration
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EXPERIMENT 2ENZYME ASSAYS AND FACTORS AFFECTINGENZYME ACTIVITY
Table 2: Effect of substrate on enzyme activity
Concentration of Substrate (% w/v) Absorbance (nm)
0.5 0.396
1.0 0.404
2.0 0.385
3.0 0.417
0 0.5 1 1.5 2 2.5 3 3.50.36
0.37
0.38
0.39
0.4
0.41
0.42
f(x) = 0.00535593220338961 x + 0.391796610169492R² = 0.194091121132014
Absorbance, nm Vs Concentration of Substrate (%w/v)
Concentration of Substrate (% w/v)
Abs
orba
nce
(nm
)
Figure 2: Absorbance versus Substrate concentration
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EXPERIMENT 2ENZYME ASSAYS AND FACTORS AFFECTINGENZYME ACTIVITY
Table 3: Effect of pH on enzyme activity
pH Value Absorbance (nm)
5 0.383
7 0.404
9 0.405
11 0.394
4 5 6 7 8 9 10 11 120.37
0.375
0.38
0.385
0.39
0.395
0.4
0.405
0.41
f(x) = 0.0017 x + 0.382900000000001R² = 0.182334384858065
Absorbance (nm) Vs pH
pH Value
Abs
orba
nce
I(nm
)
Figure 3: Absorbance versus pH
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EXPERIMENT 2ENZYME ASSAYS AND FACTORS AFFECTINGENZYME ACTIVITY
DISCUSSION
Based on Graph 1, the glucose standard calibration curve did not follow the theory
because graph pattern obtained not linear line due to errors occurred in this experiment.
Theoretically, when the concentration of glucose increases the enzyme activity also increases.
This calibration curve is used as a reference to the substrate and the pH effects towards
enzyme activity due to finding its concentration. Meaning that, when an amylase enzyme
reacts with substrate starch solution, it will produce glucose. Due to this matter, the glucose
standard calibration curve has been used for determining the concentration of glucose after
the reaction. The substrate that been used in this experiment is the starch solution, while the
enzyme that been used is the amylase enzyme.
Basically, a very small amount of enzyme can consume a large amount of substrate.
The rate of reaction depends directly on the amount of enzyme present at a specific time at
unlimited substrate concentration. If two folds increase the amount of enzyme the reaction
rate is doubled. By increasing the enzyme molecules an increase is the number of active sites
takes place. At higher concentration of the enzyme the inhibitors will fall short. More active
sites will convert the substrate molecules into products, in the given period of time. After a
certain limiting concentration, the rate of reaction will no longer depend upon this increase.
At low concentration of substrate the reaction rate is directly proportional to the substrate
available. If the enzyme concentration is kept constant and the amount of substrate is
increased, a point is reached when a further increase in the substrate does not increase the rate
of the reaction any more. This is because at high substrate level all the active sites of the
enzyme are saturated with substrate. Based on Graph 2, the effect of substrate concentration
on enzyme activity, shows that as the substrate concentration (starch solution) increase with
the concentration 0.5,1.0,2.0 and 3.0 % (w/v), the concentration of enzyme activity is
increase and nearly produce a linear line neglecting the small increase the graph still follow
the theory but due to at point substrate 1.0, the rate decrease abnormally causing the defect in
the graph. Hence, it is proved that, when the concentrations of substrate increase, the enzyme
activity also will increase due to the reaction activity.
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EXPERIMENT 2ENZYME ASSAYS AND FACTORS AFFECTINGENZYME ACTIVITY
Other factor that affecting the enzyme activity is the changes of pH. Varies pH
concentration has been used in this experiment in order to observe the effect of the enzyme
activity. In theory, the enzymes are very sensitive to pH and it also has a specific range of
activity. Enzymes are active only within a limited range of pH. But the limits may be wide,
pH 5 to 10. Within the range there will be an optimum at which the maximum activity is
attained. This pH can stop the enzyme activity by denaturating (altering) the three
dimensional shape of the enzyme by breaking weak bonds such as ionic and hydrogen. The
high and low of the pH value will extremely result in complete loss of activity for most
enzymes. Meaning that, pH is the factors of stability of enzymes. Basically, the optimum pH
of amylase enzyme is between 6.7 -7.0 (pancreas) and 4.6-5.2 (malt). By referring to Graph
3, the effect of pH on the enzyme activity shows that, the optimum pH is at pH 9. The result
of pH 9 shows that the enzyme activity reaction is actively produced at this pH which is still
within the range of the theory stated.
Based on the result obtained, it shows that some of the result did not follow the
theory. It is because there are several errors occurred in this experiment. Some of the
apparatus are contaminated because they were not been cleaned properly so the possibilities
of other chemicals on the apparatus used is higher which will affect the expected result of the
experiment. Besides, while measuring the amount or quantity of the chemical, student was
not in the right position in order to ensure the chemical that need to be used is enough. This is
called parallax error. Lastly, the surrounding temperature might affect the concentration of
the chemical. So we have to make sure the experiment running under appropriate condition.
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EXPERIMENT 2ENZYME ASSAYS AND FACTORS AFFECTINGENZYME ACTIVITY
CALCULATION
Calculation for Starch (Substrate) solution preparation
(Each of the substrate solution needs to be in 50ml)
For 3.0% (w/v): 3g of starch was dilute in 50ml deionized water
For 2.0% (w/v)
M1V1 = M2V2
(3.0g)(50ml) = (2.0g)(V2)
V2 = 75ml
75mL-50ml =25ml
(The amount of water that needs to be dispersed in water to obtain
50ml for each substrate solution with different concentration.)
For 1.0% (w/v)
M1V1 = M2V2
(2.0g)(50ml) = (1.0g)(V2)
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EXPERIMENT 2ENZYME ASSAYS AND FACTORS AFFECTINGENZYME ACTIVITY
V2 = 100ml
100mL-50ml =50ml
For 0.5% (w/v)
M1V1 = M2V2
(1.0g)(50ml) = (0.5g)(V2)
V2 = 100ml
100mL-50ml =50ml
Calculation for glucose solution preparation
(Each of the substrate solution needs to be in 100ml)
For 2.0% (w/v): 2g of glucose was dilute in 1L deionized water
For 1.0% (w/v)
M1V1 = M2V2
(2.0g)(100ml) = (1.0g)(V2)
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EXPERIMENT 2ENZYME ASSAYS AND FACTORS AFFECTINGENZYME ACTIVITY
V2 = 200ml
200mL-100ml =100ml (the amount of water that needs to be dispersed
in water to obtain 50ml for each substrate solution with different
concentration)
For 0.5% (w/v)
M1V1 = M2V2
(2.0g)(100ml) = (0.5g)(V2)
V2 = 400ml
400mL-100ml =300ml
For 0.2% (w/v)
M1V1 = M2V2
(2.0g)(100ml) = (0.2g)(V2)
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EXPERIMENT 2ENZYME ASSAYS AND FACTORS AFFECTINGENZYME ACTIVITY
V2 = 1000ml
1000mL-100ml =900ml
For 0.1% (w/v)
M1V1 = M2V2
(2.0g)(100ml) = (0.1g)(V2)
V2 = 2000ml
2000mL-100ml =1900ml
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EXPERIMENT 2ENZYME ASSAYS AND FACTORS AFFECTINGENZYME ACTIVITY
CONCLUSION
The objective of this experiment is to study the factors that affect the enzyme activity.
There are few factors that affect the enzyme activity which are the temperature, pH and the
substrate concentration. Based on the result obtained, as the concentration of substrate
increases, the enzyme activity also increases. However, after period of time the reaction is
decreases due to the saturation of enzyme. Based on the result, the optimum pH for amylase
to react is at pH 9. The pH playing a vital role for the stability of enzyme. Different enzyme
have different rate of reaction at different pH. In this experiment, there are few errors might
be occurred which might affect the result of experiment as some of the result did not follow
the theory. This may be due parallax error.
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EXPERIMENT 2ENZYME ASSAYS AND FACTORS AFFECTINGENZYME ACTIVITY
REFERENCES
1. Dutta.R, Fundamental of Biochemical Engineering, Springer, New York, 2008, pg 31
2. Factors Affecting Enzyme Activity, 2012, Encyclopaedia Britannican , (Online).
(Accesses on 17th September 2012). Available from World Wide Web:
http://www.britannica.com/EBchecked/topic/189245/enzyme/2123/Factors-affecting-
enzyme-activity
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