INVESTIGATION ON ENZYME ACTIVITY AND KINETICS

UNIVERSITI TEKNOLOGI MARA FAKULTI KEJURUTERAAN KIMIA BIOPROCESS ENGINEERING LABORATORY (CBE 661) NAME AND MATRIC NO

: MUHAMMAD ARSHAD BIN ABDUL RASHID (2014683386)

GROUP

: 5

EXPERIMENT

: LAB 5 (INVESTIGATION ON ENZYME ACTIVITY AND KINETICS)

DATE PERFORMED

: 29 SEPTEMBER 2015

SEMESTER

: 5

PROGRAMME CODE

: EH242 5E

SUBMIT TO

: PN SUHAILA BT MOHD SAUID

No. 1. 2. 3. 4. 5. 6 7. 8. 9. 10. 11. 12. 13.

Title Abstract/Summary Introduction Aims Theory Apparatus Methodology/Procedure Results Calculations Discussion Conclusion Recommendations Reference Appendices TOTAL

Allocated Marks 5 5 5 5 5 10 10 10 20 10 5 5 5 100

Marks

Remarks: Checked by:

Rechecked by:

…………………………….........

……………………………..

Date:

Date: 1

Table of Contents

NO.

TITLE

PAGES

1

Abstract

3

2

Introduction

4

3

Objectives

5

4

Theory

5

5

Apparatus

9

6

Methodology/Procedure

9

7

Results

11

8

Calculations

18

9

Discussion

20

10

Conclusion

22

11

Recommendations

23

12

Reference

23

13

Appendices

24

2

Abstract Enzymes are protein catalysts that speed up chemical reaction in living organisms. This investigation tested the effects of temperature and pH has on enzyme activity. The 2% starch solution was treated with different temperature (30 ͦ C, 40 ͦ C, 50 ͦ C and 60 ͦ C), pH (5,6, 7, 8 and 9) and the substrate concentration (0.5%, 1.5%, 2.0%, 2.5% and 3.0%). Data was collected by determine the enzyme activity and the absorbance value at λ=540 nm. The objective for this experiment is determination of the effects of temperature on the enzymatic activity and changes in enzyme concentration of an enzyme-catalyzed reaction. It is also describe the relationship between substrate concentration and the maximum velocity of an enzyme. After conducting the experiments we can see that different temperature, pH and substrate concentration give different enzyme activity and the absorbance value. From the pH experiment we can see the optimum pH for amylase is at pH 6, the optimum temperature we fail to determine due to some error while conducting the experiment while the higher substrate concentration the lower the enzyme activity. This experiment has shown that enzymes must have certain environmental conditions present in order for them to function properly. With this knowledge, one can successfully perform experiments using enzymes in the future by making sure that the environmental conditions present are optimum for the enzyme that is being used.

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Introduction

Cells function largely because of the action of enzymes. Life is a dynamic process that involves constant changes in chemical composition. These changes are regulated by catalytic reactions, which are regulated by enzymes. At one time, the cell was actually conceived of as a sac of enzymes. It was believed that if we knew all of the reactions and their rates of action, we could define the cell, and indeed, life itself. Few biologists continue to think of this as a simple task, but we know that life as we know it could not exist without the function of enzymes. Ideally, we would examine enzymes within an intact cell, but this is difficult to control. Consequently, enzymes are studied in vitro after extraction from cells. Enzymes are protein molecule that acts as biological catalysts. Without changing of the overall process, they increase the rate of reactions. Enzymes are long chains of amino acids bound together by peptide bonds. Besides that, they are seen in all living cells and controlling the metabolic processes in which they converted nutrients into energy and new cells. Other than that, enzymes also help in the breakdown of food materials into its simplest form. The reactants of enzyme catalyzed reactions are termed substrates and each enzyme is quite specific in character, acting on a particular substrates to produce a particular products. The central approach for studying the mechanism of an enzyme-catalyzed reaction is to determine the rate of the reaction and its changes in response with the changes in parameters such as substrate concentration, enzyme concentration, pH, temperature and known as enzyme kinetics. The substrate concentration, is one of the important parameter that affecting the rate of a reaction that catalyzed by an enzyme. However, studying the effects of substrate concentration is elaborated by the fact that during the course of an in vitro reaction, substrate changes due to the conversion of substrate to product. In this experiment we can see how substrate concentration, pH and temperature effect the enzyme activity. Amylase is a type of enzyme. Amylase has an active site organized in subsites, each of which accommodates a glucose residue (Talamond, Noirot & de Kochko, 2005). It breaks down starch to glucose, giving food that sweet taste. An example of amylase in the natural world is in bananas. When they are green, the amylase has yet to break down the starch, but by the time they’ve turned brown, the reaction has been completed. This is why brown bananas taste sweeter than their green counterpart.

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Objectives 

Determination of the effects of temperature on the enzymatic activity and changes in enzyme concentration of an enzyme-catalysed reaction.



Describe the relationship between substrate concentration and the maximum velocity of an enzyme.



Estimation of Michaelis-Menten parameters, effect of pH and temperature on enzyme activity and kinetics of inhibition.

Theories Enzymes are protein molecules that act as biological catalysts by increasing the rate of reactions without changing the overall process. They are long chain amino acids bound together by peptide bonds. Enzymes are seen in all living cells and controlling the metabolic processes in which they converted nutrients into energy and new cells. Enzymes also help in the breakdown of food materials into its simplest form. The reactants of enzyme catalyzed reactions are termed as substrates. Each enzyme is quite specific in character, acting on a particular substrates to produce a particular products. The central approach for studying the mechanism of an enzymecatalyzed reaction is to determine the rate of the reaction and its changes in response with the changes in parameters such as substrate concentration, enzyme concentration, pH, temperature etc .This is known as enzyme kinetics.

One of the important parameters affecting the rate of a reaction catalyzed by an enzyme is the substrate concentration. During enzyme substrate reaction, the initial velocity V0 gradually increases with increasing concentration of the substrate. Finally a point is reached, beyond which the increase in V0 will not depend on the substrate concentration. When we plot a graph with substrate concentration on the X axis and corresponding velocity on Y axis. It can be observed from the graph that as the concentration of the substrate increases, there is a corresponding increase in the V0. However beyond a particular substrate concentration, the velocity remains constant without any further increase. This maximum velocity of an enzyme catalyzed reaction under substrate saturation is called the Vmax , Maximum velocity.

5

Figure 4.1: Graph of initial velocity against substrate concentration (Nelson, D.L et. al, n.d) Michaelis – Menten Equation

Leonor Michaelis and Maud Menten postulated that the enzyme first combines reversibly with its substrate to form an enzyme-substrate complex in a relatively fast reversible step:

Eqn.1 In the next step, this ES complex is breaks down in to the free enzyme and the reaction product, P:

Eqn.2 Since the second step is the rate limiting step, the rate of overall reaction must be proportional to the concentration of the ES that reacts in the second step. The relationship between substrate concentration, substrate and Initial velocity of enzyme, V0 has the same general shape for most enzymes (it approaches a rectangular hyperbola). This can be expressed algebraically by the Michaelis-Menten equation. Based on their basic hypothesis that the rate limiting step in enzymatic reactions is the breakdown of the ES complex to free enzyme and product, Michaelis and Menten derived an equation which is;

6

Eqn.3

The necessary terms in this reaction are S, V0, Vmax, and Km (Michaelis constant). All these terms can be measured experimentally. Lineweaver – Burke Plot

In 1934, Lineweaver and Burke made a simple mathematical alteration in the process by plotting a double inverse of substrate concentration and reaction rate.

Eqn.4 For enzymes obeying the Michaelis-Menten relationship, the “double reciprocal” of the V0 versus S from the first graph, yields a straight line. The slope of this straight line is KM /Vmax, which has an intercept of 1/Vmax on the 1/V0 axis, and an intercept of -1/KM on the 1/[S] axis. The doublereciprocal presentation, also called a Lineweaver-Burk plot. The main advantage of LineweaverBurk plot is to determine the Vmax more accurately, which can only be approximated from a simple graph of V0 versus S.

Figure 4.2: Lineweaver-Burk plot. (Nelson, D.L et. al, n.d) 7

The enzyme α Amylase can catalyze the hydrolysis of internal α -1,4-glycosidic bond present in starch with the production of reducing sugars. In the study of substrate concentration on enzyme kinetics, the enzyme is kept constant where as the concentration of Starch is taken in increasing order. As the substrate concentration increases, the amount of products produced in every successive tube also increases. This was explained by Michealis and others that an enzyme catalyzed reaction at varying substrate concentrations is diphasic i.e. at low substrate concentration the active sites on molecules (enzyme) are not occupied by substrate and the enzyme rate varies with substrate molecules concentration (phase1). As the number of substrate molecules increases, the enzyme attains the saturation level, since there is no more reaction sites remaining for binding. So the enzyme can work with full capacity and its reaction rate is independent of substrate concentration. (Phase II). This Enzyme – substrate reaction can be determined by measuring the increase in reducing sugars using the 3, 5 Dinitro salycilic acid reagent. In an alkaline condition, the pale yellow colored the 3, 5- dinitro salicylic acid undergo reduction to yield orange colored 3- amino -5-nitrosalicylic acid. The absorbance of resultant solutions is read at 540nm. The intensity of color depends on the concentration of reducing sugars produced.

α Amylase Starch

Maltose + glucose

Figure 4.3: The enzyme-substrate reaction example. (vlab.amrita.edu, 2011)

8

Apparatus 1. Alpha Amylase enzyme 2. Starch 3. pH buffer solution (pH 4-9) 4. DNSA Reagent 5. Beaker 6. Measuring cylinder 7. Cuvette 8. Falcon tube rack 9. Falcon tube 10. Micropipet and tips 11. Label sticker 12. Schott bottle 13. Vortex mixer 14. Water bath 15. Spectrophotometer 16. Hotplate

Procedure

i. Preparation of 2% Starch Solution

a) 4 g of soluble starch is mixed in approximately 50 ml of cold water. b) While stirring, the slurry is added to approximate 100 ml of gently boiling water in a large beaker. c) Then the final volume of 200ml is topped up and mix well. ii. Effect of pH on the activity and stability of amylase enzyme. a) Five test tubes is labelled with pH 5, 6, 7, 8 and 9. In each tube, 1 mL of 2% starch solution is placed and 1 mL of the appropriate buffer is added (at corresponding pH) to each tube. b) Five additional clean test tubes is added and 2 mL of amylase solution was put in each tube.

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c) All 10 tubes is placed in the 37°C water bath for about 5 minutes to allow the temperature to equilibrate. d) The content of each amylase test tube is poured into each starch test tube and mixed on vortex mixer. e) The tubes is returned to the 37°C water bath. f) The hydrolysis reaction is proceed for exactly 10 minutes. g) The amylase activity is determined using the method given in Appendix 1. h) Graph of pH vs. enzyme activity is plotted.

iii. Effect of temperature on the activity and stability of amylase enzyme. a) One test tube is labelled with 30ºC. In the tube, 1 mL of 2% starch solution and 1 mL of pH=7 buffer is placed to the tubes. b) Additional clean test tub is added and 2 mL of amylase solution is put in the tube. c) Both tubes is placed in the 30°C water bath for about 5 minutes to allow the temperature to equilibrate. d) The contents of the amylase is poured test tube into starch test tube and mix them on vortex mixer. e) Return the tubes to the 30°C water bath. f) Let the hydrolysis reaction proceeded for exactly 10 minutes. g) The amylase activity is determined using the method given in Appendix 1. h) Step a-g is repeated at 4 different temperatures ranging from 30-70 ºC. i) Graph of temperature vs. amylase activity is plotted.

iv. Effect of substrate concentration on the activity of amylase enzyme. a) Starch solutions of varying concentration (0.5, 1.5, 2.0, 2.5, and 3.0% w/v) is prepared as the substrate. b) Each tube is labelled with starch concentration and place 1ml of each starch solution into the test tubes. c) 1 mL of pH=7 buffer is added to the tubes. d) Five additional clean test tubes is added and put 2 mL of amylase solution in each tube. e) All tubes is placed in the 37°C water bath for about 5 minutes to allow the temperature to equilibrate.

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f) The content of each amylase is poured test tube into starch test tube and mix them on vortex mixer. g) Return the tubes to the 37°C water bath. h) Let the hydrolysis reaction proceed for exactly 10 minutes. i) The amylase activity is determined using the method given in Appendix 1. j) Graph of starch concentration against amylase activity is plotted. Appendix 1 (Demonstration of Enzyme Activity) a. After 10 minutes (the time of hydrolysis reaction), the reaction is stopped by adding 4 ml of DNS reagent. b. Then it is boiled for 10 minutes and then left to cool to room temperature. c. The absorbance of the samples is measured at λ=540 nm. d. The absorbance value is compared with glucose standard curve prepared to obtain the glucose concentration. e. The enzyme activity is calculated. Note: Enzyme activity is the amount of glucose formed in reaction mixture per unit time.

Appendix 2 (Glucose Standard Curve Preparation) a. The standard solutions of glucose is prepared at five different concentrations ranging from 01000mg/L by serial dilution. b. 1 ml of each glucose solution is added in test tubes. c. 1 ml of DNS reagent is added in each tube and mix for few seconds on vortex mixer. d. The test tubes is placed in water bath (T=100°C) for 10 min and then left to cool at room Results The obtained results that being recorded from the experiment of investigation on enzyme activity and kinetics. Glucose Standard Curve Table 7.1: The values of absorbance optical density (OD) at five different concentration of glucose

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Glucose

Absorbance Optical

No.

Concentration (g/L)

Density (OD) (nm)

1

200

0.378

2

400

0.668

3

600

0.801

4

800

1.224

5

1000

2.052

The data on the table 7.1 above is used to plot the standard curve of absorbance optical density (OD) (nm) against glucose concentration (g/L). These obtained results were recorded based on the observation through the spectrophotometer which already set up at 540 nm.

Absorbance Optical Density (OD)

A graph of Absorbance Optical Density (OD) against Concentration 2.5 2

y = 0.0018x R² = 0.89

1.5

1 0.5 0 200

400

600

800

1000

Concentration (g/L)

Figure 7.1: The standard curve of absorbance optical density (OD) (nm) against glucose concentration (g/L) The effect of pH to enzyme activity Table 7.2: The values of absorbance optical density (OD) at five different pH Values.

No. 1.

pH Value

Absorbance Optical Density (OD) (nm)

5

2.680

12

2.

6

5.170

3.

7

2.625

4.

8

2.546

5.

9

2.350

The table 7.2 above showed the reading of absorbance optical density (OD) (nm) which affected by five different pH Value.

Absorbance Optical Density (OD) (nm)

A graph of absorbance Optical Density (OD) (nm) against pH Value 6 5 4 3 2 1 0 5

6

7

8

9

pH Value

Figure 7.2: The effect of absorbance Optical Density (OD) (nm) reading at different pH Values The plotted graph as shown in figure 7.2 showed the five different pH Values which affect the absorbance Optical Density (OD) (nm) reading. The absorbance Optical Density (OD) (nm) reading was rapidly increasing from the pH 5 until pH 6 which known as the optimum pH for the amylase enzyme and the absorbance Optical Density (OD) values is 5.170 nm. Table 7.3: The values of enzyme activity at different pH pH

Absorbance

Glucose

Glucose

Enzyme

value

reading (nm)

concentration,

released (mol)

activity, V

X (g/mL) 5

2.680

0.0015

(mol/min) 8.26×10-6

8.26×10-7

13

6

5.170

0.0029

1.59×10-5

1.59×10-6

7

2.625

0.0015

8.09×10-6

8.09×10-7

8

2.546

0.0014

7.80×10-6

7.80×10-7

9

2.350

0.0013

7.26×10-6

7.26×10-7

The table 7.3 above showed the calculated values that obtained according to the effect of five different pH values against the reading of the absorbance optical density (OD) (nm).

A Graph of enzyme activity against pH 0.0000018 0.0000016 0.0000014 0.0000012

Enzyme 0.000001 activity, V 0.0000008 (mol/min)

0.0000006 0.0000004 0.0000002 0 0

2

4

6

8

10

pH

Figure 7.3: The effect of enzyme activity against pH values Based on the observation, the graph that plotted in the figure 7.3 showed the higher values of enzyme activity was occurred at pH 6 or optimum pH. The enzyme is more reproducible during the optimum pH.

The effect of temperature on the activity and stability of amylase enzyme Table 7.4: The values of absorbance optical density (OD) at four different temperatures (ͦ C).

No.

Temperature (ͦ C)

Absorbance Optical Density (OD) (nm)

14

1.

30

2.630

2.

40

5.350

3.

50

7.290

4.

60

8.070

The table 7.4 above showed the effect of four different temperatures on the reading of the absorbance optical density (OD). The temperatures that required are in the range between 30 ͦ C to 70 ͦ C only.

Absorbance Optical Density (OD) (nm)

A graph of absorbance Optical Density (OD) (nm) against Temperature (ͦ C) 9 8 7 6 5 4 3 2 1 0 30

40

50

60

Temperature (ͦ C) Figure 7.4: The reading of absorbance Optical Density (OD) (nm) against temperature (ͦ C) The plotted graph in figure 7.4 showed the reading of absorbance Optical Density (OD) (nm) kept increasing continuously against four different temperature which not exceed 70 ͦ C. The values of absorbance Optical Density (OD) is more higher if the surrounding temperature not almost at boiling temperature. Table 7.5: The values of enzyme activity at four different temperatures Temperature

Absorbance

Glucose

Glucose

Enzyme

(˚C)

reading (nm)

concentration,

released

activity, V

X (g/mL)

(mol)

(mol/min)

15

30

2.63

0.0015

8.33×10-6

8.33×10-7

40

5.32

0.0030

1.67×10-5

1.67×10-6

50

7.29

0.0041

2.28×10-5

2.28×10-6

60

8.07

0.0045

2.50×10-5

2.50×10-6

The table 7.5 above showed the effect of four different temperatures on the values of enzyme activity for amylase enzyme.

A Graph of Enzyme activity, V (mol/min)against Temperature (˚C ) 0.000003 0.0000025 0.000002

Enzyme activity, V 0.0000015 (mol/min) 0.000001 0.0000005 0 0

10

20

30

40

50

60

70

Temperature (˚C)

Figure 7.5: The values of enzyme activity at four different temperatures The graph that plotted as shown in the figure 7.5 showed the values of enzyme activity that affected by the four different temperatures at range between 30 ͦ C to 70 ͦ C only. The values of enzyme activity kept increasing as the enzyme is more reproducible under the optimum temperature that not exceeds 70 ͦ C. The higher temperature at which the enzyme is operating at is well above 100oC, and then thermal deactivation can occur which this condition also known as denaturation.

The effect of substrate concentration on the activity of amylase enzyme Table 7.6: The values of enzyme activity at different substrate concentration 16

Substrate

Absorbanc

Glucose

Glucose

Enzyme

1/V

1/S

concentratio

e reading

concentration

released

activity, V

n (%)

(nm)

, X (g/mL)

(mol)

(mol/min)

0.5

4.06

0.0023

12.27x10-6

12.27x10-7

8.15×105

2.00

1.5

3.39

0.0019

10.55x10-6

10.55x10-7

9.48×105

0.67

2.0

3.26

0.0018

9.991x10-6

9.991x10-7

1.00×105

0.50

2.5

2.66

0.0015

8.326x10-6

8.326x10-7

1.20×105

0.40

3.0

2.18

0.0012

6.661x10-6

6.661x10-7

1.50×105

0.33

The table 7.6 above showed the effect of five different substrate concentrations on the values of enzyme activity for the amylase enzyme.

Enzyme activity, V (mol/min)

Graph of enzyme activity against substrate concentration 14 12 10 8 6

4 2 0 0

0.5

1

1.5

2

2.5

3

3.5

Substrate concentration (%)

Figure 7.6: The values of enzyme activity against substrate concentration Based on the observation, the graph that plotted in figure 7.6 showed the effects of substrate concentration against the values of enzyme activity. The values kept decreasing since the amount of substrate concentration kept increasing as well. This condition showed that the enzyme activity a bit slow or being inhibited by the increasing of substrate concentration.

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A Graph of 1/V against 1/S 12 10

y = 3.8889x + 1.2327 R² = 0.4161

8

1/V 6 4 2 0 0.33

0.83

1.33

1.83

2.33

1/S

Figure 7.7: The values of 1/V that affected by the 1/S Overall the graph that plotted in figure 7.7 showed the 1/S can affect the values of 1/V. Based on the observation, the 1/V values were keeping decreased as the 1/S values were keeping longer. The equation that being presented in this graph is 𝑦 = 3.8889𝑥 + 1.2327 and 𝑅 2 = 0.4161.

Calculation 1. Determination of glucose concentration, X (g/mL) From the standard curve of glucose that had been plotted as shown in figure 7.1, the linear equation of the curve is presented as: 𝑌 = 0.0018𝑋 Where; X = protein concentration and Y = absorbance reading. Therefore, to calculate protein concentration, 𝑋=

𝑌 0.0018

𝑋=

2.660 0.0018

𝑋=

1477.78 1𝐿 1 × 10−3 × × 𝐿 1000𝑚𝑙 𝑚𝑖𝑙𝑙𝑖

𝑋 = 0.0015 g/mL

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2. Determination of glucose released (mol) MW of glucose = 180.1559 g/mol; Volume of enzyme (amylase) = 1 mL 𝑔 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 ( ) 𝑚𝐿 Moles of glucose released (mol) = × 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑒𝑛𝑧𝑦𝑚𝑒 (𝑚𝐿) 𝑀𝑊 𝑜𝑓 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 𝑔 0.0015 𝑚𝐿 Moles of glucose released (mol) = 𝑔 × 1 𝑚𝐿 180.1559 𝑚𝑜𝑙 Moles of glucose released (mol) = 8.326 × 10−6 mol 3. Determination of enzyme activity, V (mol/min) Duration of hydrolysis reaction: 10 minutes 𝐸𝑛𝑧𝑦𝑚𝑒 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (𝑚𝑜𝑙/𝑚𝑖𝑛) =

𝐸𝑛𝑧𝑦𝑚𝑒 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (𝑚𝑜𝑙/𝑚𝑖𝑛) =

𝑚𝑜𝑙 𝑜𝑓 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 𝑟𝑒𝑙𝑒𝑎𝑠𝑒𝑑 𝐻𝑦𝑑𝑟𝑜𝑙𝑦𝑠𝑖𝑠 𝑟𝑒𝑎𝑐𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒

8.326 × 10−6 𝑚𝑜𝑙 10 𝑚𝑖𝑛

𝐸𝑛𝑧𝑦𝑚𝑒 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (𝑚𝑜𝑙/𝑚𝑖𝑛) = 8.326 × 10−7 mol/min 4. Equation for Michaelis-Menten: 𝑉=

𝑉𝑚𝑎𝑥 [𝑆] 𝐾𝑚 + 𝑆

Double reciprocal; 1 𝐾𝑚 1 1 = + 𝑉 𝑉𝑚𝑎𝑥 𝑆 𝑉𝑚𝑎𝑥 𝑦 − 𝑎𝑥𝑖𝑠 =

1 𝑉

𝑥 − 𝑎𝑥𝑖𝑠 =

1 𝑆

𝐼𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡 =

𝑆𝑙𝑜𝑝𝑒 =

1 𝑉𝑚𝑎𝑥

𝐾𝑚 𝑉𝑚𝑎𝑥 19

From graph, the linear equation obtained is: 𝑌 = 3.8889𝑥 + 1.2327 Finding value of Vmax, maximum enzyme activity: 1 𝑉𝑚𝑎𝑥

= 1.2327

𝑉𝑚𝑎𝑥 = 0.8112 mol/min Finding value of Km, Michaelis constant 𝐾𝑚 = 3.8889 𝑉𝑚𝑎𝑥 𝐾𝑚 = (3.8889) × (0.8112) = 3.1547

Discussion As the concentration of substrate increases, the rate of reaction also increases until the point saturation occurs. It means as you increase the concentration, rate keeps increasing and then one point comes when the maximum rate is achieved and there is no free enzyme to bind with substrate and all the active sites of enzyme are bound to the substrate. So after that point, increasing the concentration won’t have any effect. The maximum for each enzyme is usually given by Km value (michealis menten graph or the other one called Lineweaver burke plot). The Km value is the rate constant or it can be explained as how much substrate concentration is required by an enzyme to reach to the half of maximum rate or velocity of enzyme. Each enzyme has different Km values. Wherever the Vmax occurs and it intersects the curve drawn for substrate concentration and velocity (or rate of reaction), that point is the saturation point or maximum substrate concentration to have maximum rate of the reaction.

From the graph, we can see the results follow what is stated in theory. As the concentration increase, the enzyme activity decrease. This is because when there is too much of substrate, the enzyme don’t have enough space to growth. So, to get optimum production of product, we need to provide balance amount of substrate and enzyme

The enzyme reaction have effect with change of temperature. Based on the plotted graph, as the temperature increase, the absorbance Optical Density (OD) also increase. Enzyme usually 20

have its limit on temperature. When the temperature is very high, it will be denatured thus the production of product decrease. From this experiment we can see that amylase enzyme still can grow up to 60oC but the reaction of enzyme becomes slower. If this experiment is proceed with higher temperature maybe the enzyme activity will decrease because starting from 50oC the enzyme activity not have much increase. This is because the enzyme start to denatured.

pH can give several effect on structure and activity of an enzyme. For example, pH can have an effect of the state of ionization of acidic or basic amino acids. Acidic amino acids have carboxyl functional groups in their side chains. Basic amino acids have amine functional groups in their side chains. If the state of ionization of amino acids in a protein is altered then the ionic bonds that help to determine the 3-D shape of the protein can be altered. This can lead to altered protein recognition or an enzyme might become inactive. Changes in pH may not only affect the shape of an enzyme but it may also change the shape or charge properties of the substrate so that either the substrate cannot bind to the active site or it cannot undergo catalysis. The most favorable pH value - the point where the enzyme is most active - is known as the optimum pH.

Figure 9.1: Effect of pH on reaction rate (Anonymous, n.d) In this experiment, the pH is increases from pH of 5 up to pH 9. But, starting from pH 6, the enzyme activity decreases from 1.59×10-6 for pH 6 to 8.09×10-7 for pH 7. After that, the enzyme activity decrease until pH 9 with value of 7.26×10-7. Based on this result of experiment, the optimum pH that is with highest velocity is sample pH 6 with velocity 0.2488. The lowest velocity is pH 1.

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There is fluctuation of value of absorbance that give the fluctuation of value of velocity might because of the sample is contaminated. The other factor might be the time for taking reading of absorbance for every pH sample is not fixed. Besides that, while taking reading of absorbance, the water vapor from condensation of the sample is still there, so, it might affect the absorbance reading.

Conclusion The data shown on the graph from the experiment shows that catalase functions of pH, substrate concentration and temperature give different effect on enzyme activity. Table 7.3 shows that from pH 5 to pH 6, the enzyme activity increases and at pH 7 the enzyme activity start to decrease. The enzyme activity keep on decreasing when the pH is 8 and 9. When the enzyme activity increase the absorbance reading also increase. By looking at figure 7.2 and figure 7.3, one can see that as the pH of the solution rose to a pH of 6, catalase became more efficient and was able to better carry out its function. These results help support the idea that as a solution becomes more acidic than the optimum pH of an enzyme, the enzymes present in the solution will denature, and in turn will not be able to function properly. This will result in lower reaction rates, which is shown in figure 7.2 and 7.3. At very high and very low temperatures we expected the absorbance or enzyme activity be low. The highest absorbance should have appeared at room temperature, because most human enzyme activity occurs at room temperature but in this case using enzyme amylase, we still cannot find the optimum temperature as we can see in figure 7.4 and figure 7.5 the graph did not shows any decreasing in enzyme activity. For me I think maybe there is an error during conducting experiment or maybe the amylase have high optimum temperature. For the substrate concentration, we can see in figure 7.6 that as the percent of substrate concentration increase, the enzyme activity increase.The information gathered throughout this experiment is very useful for the future. This experiment has shown that enzymes must have certain environmental conditions present in order for them to function properly. With this knowledge, one can successfully perform experiments using enzymes in the future by making sure that the environmental conditions present are optimum for the enzyme that is being used. A limitation of the procedure was that we were unable to test for the presence of catalase in the extract before beginning the experiment. If we were able to test for the presence of catalase in the extract, we could have ensured that the decomposition of hydrogen peroxide resulted from enzyme catalysis and not from the natural spontaneous decomposition of the chemical. Instead, 22

we were forced to assume that catalase was present in the extract, an assumption that may, or may not have, been correct.

Recommendations

 This experiment must be carried out under the laminar flow hood for sterility to prevent any contamination from the surrounding directly attached to the culture.  Properly, wash hand with plenty of water and appropriate soap after handling the culture which can expose to the health.  Worn the appropriate gloves that provided and disinfect the work area with Ethanol (70% ethanol for swabbing for sterility) before handling the culture.  Avoid the parallax error to be occurring during the measuring volume or amount of reagent and solution by using provided apparatus. This can affect the concentration of solution which indirectly interrupts the absorbance optical density (OD) values.  The amylase is easier to expose against the surrounding contaminants especially through the air.  Always ensure the cuvette must be wiped cleanly to prevent any scratch that would affect the spectrophotometer reading on absorbance optical density (OD).  Dispose of all contaminated materials after taking the reading of absorbance optical density (OD) by using the spectrophotometer in appropriate containers. Reference Anonymous, (n.d). Effect of pH on enzyme. Retrieved on October 15, 2015 from http://academic.brooklyn.cuny.edu/biology/bio4fv/page/ph_and_.htm

David L. Nelson, Michael M. Cox , Lehninger principles of biochemistry, 4th edition.

vlab.amrita.edu,. (2011). Effect of Substrate Concentration on Enzyme Kinetics. Retrieved 15 October 2015, from vlab.amrita.edu/?sub=3&brch=64&sim=1090&cnt=1 Talamond, Pascale, Michel Noirot, and Alexandre De Kochko. “The Mechanism of Action of αamylase

from

Lactobacillus

Fermentum

on

Maltooligosaccharides.”Journal

of

Chromatography B (2005): 42-47. Science Direct. Web.

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Appendix

Figure 12.1: Absorbance Optical Density (OD) of Glucose

Figure 12.2: The effect of substrate concentration

Figure 12.3: The effect of pH values

Figure 12.4: The effect of temperature

Figure 12.5: Spectrophotometer

Figure 12.6: Test tube 24

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