EXPERIMENT 2 ENZYME ASSAYS AND FACTORS AFFECTINGENZYME ACTIVITY

TABLE OF CONTENTS Bil. 1 2 3 4 5 6 7 Title Summary Pictorial Methodology Results Discussions Calculations Conclusions References Page 2 3 5 8 10 13 14

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EXPERIMENT 2 ENZYME 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 2 ENZYME 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 30C 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 2 ENZYME 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 2 ENZYME ASSAYS AND FACTORS AFFECTINGENZYME ACTIVITY

RESULT Table 1: Glucose Standard Curve Concentration of Glucose (mg/L) 100 200 500 1000 2000 Absorbance (nm) 0.328 0.339 0.327 0.350 0.317

Absorbance (nm) Vs Concentration of Glucose (mg/L)

0.36

0.35

Absorbance, nm

0.34

0.33 y = -6E-06x + 0.3367 R = 0.132

0.32

0.31

0.3 0 500 1000 1500 2000 2500

Concentration of Glucose (mg/L) Figure 1: Standard curve calibration

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EXPERIMENT 2 ENZYME ASSAYS AND FACTORS AFFECTINGENZYME ACTIVITY

Table 2: Effect of substrate on enzyme activity Concentration of Substrate (% w/v) 0.5 1.0 2.0 3.0 Absorbance (nm) 0.396 0.404 0.385 0.417

Absorbance, nm Vs Concentration of Substrate (%w/v)

0.42 0.415 0.41 Absorbance (nm) 0.405 0.4 0.395 0.39 0.385 0.38 0 0.5 1 1.5 2 2.5 3 3.5 Concentration of Substrate (% w/v) y = 0.0054x + 0.3918 R = 0.1941

Figure 2: Absorbance versus Substrate concentration

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EXPERIMENT 2 ENZYME ASSAYS AND FACTORS AFFECTINGENZYME ACTIVITY

Table 3: Effect of pH on enzyme activity pH Value 5 7 9 11 Absorbance (nm) 0.383 0.404 0.405 0.394

Absorbance (nm) Vs pH
0.41

0.405 y = 0.0017x + 0.3829 R = 0.1823

Absorbance I(nm)

0.4

0.395

0.39

0.385

0.38 4 5 6 7 8 pH Value 9 10 11 12

Figure 3: Absorbance versus pH

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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 2 ENZYME 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 2 ENZYME 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) V2 = 100ml 100mL-50ml =50ml For 0.5% (w/v) M1V1 = M2V2 (1.0g)(50ml) = (0.5g)(V2) V2 = 100ml 100mL-50ml =50ml
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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) 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) V2 = 1000ml 1000mL-100ml =900ml

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EXPERIMENT 2 ENZYME ASSAYS AND FACTORS AFFECTINGENZYME ACTIVITY

For 0.1% (w/v) M1V1 = M2V2 (2.0g)(100ml) = (0.1g)(V2) V2 = 2000ml 2000mL-100ml =1900ml

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EXPERIMENT 2 ENZYME 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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REFERENCES 1. 2. Dutta.R, Fundamental of Biochemical Engineering, Springer, New York, 2008, pg 31 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-affectingenzyme-activity

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