Experiment 2 Enzyme Assays and Factors Affectingenzyme Activity

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

Page 1


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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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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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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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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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

Page 6


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

Page 7


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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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.

Page 9


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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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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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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V2 = 1000ml

1000mL-100ml =900ml

For 0.1% (w/v)

M1V1 = M2V2

(2.0g)(100ml) = (0.1g)(V2)

V2 = 2000ml

2000mL-100ml =1900ml

Page 13


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.

Page 14


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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http://www.britannica.com/EBchecked/topic/189245/enzyme/2123/Factors-affecting-enzyme-activity

http://www.britannica.com/EBchecked/topic/189245/enzyme/2123/Factors-affecting-enzyme-activity


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Experiment 2 Enzyme Assays and Factors Affectingenzyme Activity