Exp 3 Biochemistry Enzyme activity

EXPERIMENT 3: INVESTIGATION ON ENZYME ACTIVITY AND THE FACTORS THAT AFFECTS IT

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1.0 Introduction Enzymes are globular proteins which act as biological catalysts. This means that they speed up the rate of reaction by lowering the activation energy, which is the energy required to break bonds. Enzymes are a complex tertiary and sometimes quaternary shape and catalyse reactions by forming a complex (known as the enzyme substrate complex) at a specific region of the enzyme called the active site. Enzyme + substrate

enzyme substrate complex

product

Enzymes are specific; any individual enzyme can usually only catalyze one particular reaction. The rate of enzyme activity is influenced by time, enzyme concentration, temperature, pH, and the presence of inhibitors.The first factors that affect the rate of enzyme activity are temperature. As the temperature rises, reacting molecules have more and more kinetic energy. This increases the chances of a successful collision and so the rate increases. There is a certain temperature at which an enzyme's catalytic activity is at its greatest (see graph). Above this temperature the enzyme structure begins to break down (denature) since at higher temperatures intra- and intermolecular bonds are broken as the enzyme molecules gain even more kinetic energy.

Figure 1: The effect of temperature on enzyme activity.

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The next factors are pH. Each enzyme works within quite a small pH range. There is a pH at which its activity is greatest (optimum pH). This is because changes in pH can make and break intra- and intermolecular bonds, changing the shape of the enzyme and, therefore, its effectiveness.

Figure 2: The effect of pH on enzyme activity. Other factors are inhibitors. Some substances reduce or even stop the catalytic activity of enzymes in biochemical reactions. They block or distort the active site. These chemicals are called inhibitors, because they inhibit reaction. Inhibitors that occupy the active site and prevent a

substrate

molecule

from

binding

to

the

enzyme

are

said

to

be active

site-

directed (or competitive, as they 'compete' with the substrate for the active site). Inhibitors that attach to other parts of the enzyme molecule, perhaps distorting its shape, are said to be nonactive site-directed (or non-competitive). Next one is concentrations. Changing the enzyme and substrate concentrations affect the rate of reaction of an enzyme-catalysed reaction. Increasing substrate concentration will increases the rate of reaction. This is because more substrate molecules will be colliding with enzyme molecules, so more product will be formed. However, after a certain concentration, any increase will have no effect on the rate of reaction, since substrate concentration will no longer be the limiting factor.The enzymes will effectively become saturated, and will be working at their maximum possible rate. While, by increasing the enzyme concentration, it will increase the rate of reaction which as more enzymes will be colliding with substrate molecules.

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EXPERIMENT 3: INVESTIGATION ON ENZYME ACTIVITY AND THE FACTORS THAT AFFECTS IT

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However, this too will only have an effect up to a certain concentration, where the enzyme concentration is no longer the limiting factor.

Figure 3 & 4: The effect of concentration on enzyme activity. In regards to substrate concentration, enzyme kinetics follow the Michaelis-Menton Model. The model takes the form of an equation describing the rate of enzymatic reactions, by relating reaction rate

to

, the concentration of a substrate S. Its formula is given by :

.

Here,

represents the maximum rate achieved by the system, at maximum (saturating)

substrate concentrations. The Michaelis constant reaction rate is half of

is the substrate concentration at which the

.

Figure 5: Example of Michaelis-Menton graph.

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1.1 Objective 1) To determine the effects of temperature on the enzymatic activity and changes in enzyme concentration of an enzyme-catalysed reaction. 2) To describe the relationship between substrate concentration and the maximum velocity of an enzyme. 3) To estimate the Michaelis-Menten parameters, effect of pH and temperature on enzyme activity and kinetics of inhibition.

2.0 Material 1. 1% starch solution 2. Amylase solution 3. 0.2 M phosphate buffer 4. DNS reagent 5. Glucose standard 6. 0.1 M Lead (II) nitrate Pb(NO3)2 7. Distilled water

2.1 Procedure 2.1.1

Determination of time and enzyme concentration

Enzyme was prepared into 0.2 ml and mixed with 0.3 ml of 0.2 M phosphate buffer at pH 7.0 followed by 0.5 ml of 1 % (w/v) of starch solution.

The similar sample was prepared as step (a) according to the table 1 for determination of optimum time for amylase activity.

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Allowed the mixture for duration according in Table 1 at 30◦C using an incubator with shaking at 150 rpm.

After 30 min, glucose concentration was determined by using DNS method.

The optical density was determined at 540 nm.

The standard curve was prepared for glucose by using the concentration of pure glucose in the range of 0-1000 mg/L.

1U (unit) of amylase activity is defined as the amount of enzyme required to release 1µg of reducing sugar per min under the conditions stated.

Test tube no

Amylase solution (ml)

Phosphate buffer (0.2 M), ml

1 % starch solution, ml

Duration (min)

1

0.2

0.3

0.5

30

2

0.2

0.3

0.5

60

3

0.2

0.3

0.5

90

4

0.2

0.3

0.5

120

5

0.2

0.3

0.5

150

Table 1: Determination of time and enzyme concentration

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2.1.2

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Effect of temperature on the amylase activity

The test tube was prepared according to the Table 2 and duplicate for each test tube. Incubate the sample from 30 to 60 minutes.

Allow the mixture for 30 minutes at temperature based on Table 3. After 30 minutes, the glucose concentration was determined by using DNS method at 540 nm.

Then, repeat the same analysis for second sample which is at 60 minutes.

Test tube no

Temperature OC

1

Amylase solution 1-5% (ml) 0.2

Phosphate buffer (0.2 M), ml 0.3

1 % starch solution, ml 0.5

2

0.2

0.3

0.5

30

3

0.2

0.3

0.5

40

4

0.2

0.3

0.5

50

5

0.2

0.3

0.5

100

Ice water Bath

Table 2: Determination effect of temperature on the amylase activity

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2.1.3

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Effect of pH on the amylase activity

The optimum pH for the enzyme was determined by incubating the reaction mixtures prepared using the buffer solution of pH from 5 to pH 9. The buffer systems (0.2 M) used in the experiment is stated in Table 3.

Make a duplicate for each of the test tube. Incubate the sample from 30 to 60 minutes. Shake at 150 rpm.

mined After 30 minutes, glucose concentration was determined by using DNS method.

The absorbance obtained for each test tube was recorded and repeat incubation for 60 min.

Test tube no

Temperature OC

Amylase solution 1-5% (ml)

Phosphate buffer (0.2 M), ml

1 % starch solution, ml

1

0.2

2

0.5

Ice water Bath

2

0.2

4

0.5

30

3

0.2

7

0.5

40

4

0.2

9

0.5

50

5

0.2

12

0.5

100

Table 3: Determination effect of pH on the amylase activity

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2.1.4

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Effect of substrate concentration on the activity of amylase enzyme

The starch solution was prepared at concentration 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5 and 3.0 & (w/v) as the substrate. The substrate was placed into 0.5ml of various concentrations into the test tubes.

The reaction mixture was incubated for 30 min at 30◦C with shaking at 150 rpm.

The amylase activities were determine by using DNS method, and then plot the graph of substrate concentration against amylase activity.

2.1.5

Effect of inhibitor

2 ml of amylase solution, 2 ml of 1 % starch and 10 drops of 0.1 M Pb(NO3)2 was added into a dry test tube

Mix and incubate in water bath for 30 min. After 30 min, determine glucose concentration using DNS method at 540 nm. Record the absorbance obtains.

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EXPERIMENT 3: INVESTIGATION ON ENZYME ACTIVITY AND THE FACTORS THAT AFFECTS IT

3.0 Result 3.1 Concentration of Glucose for Standard Glucose Table 1: The absorbance and concentration of glucose for standard glucose Concentration of glucose (mg/L) 0.000 200.0 400.0 600.0 800.0 1000

Absorbance 0.132 0.145 0.194 0.208 0.270 0.373

Figure 1 : Absorbance against the concentration of glucose for standard glucose 0.4

Graph 1: Absorbance Vs Concentration of Glucose Standard

0.35

y = 0.0002x + 0.1065 R² = 0.905

Absorbance

0.3 0.25 0.2 0.15 0.1 0.05 0 0

200

400

600

800

1000

1200

Concentration of glucose standard

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3.2 Determination of Time and Enzyme Concentration Time (minutes)

Absorbance

Enzyme activities

30

0.620

0.0856

60

0.646

0.0450

90

0.701

0.0330

120

0.691

0.0244

Table 2 : The absorbance and enzyme activities at different duration

Figure 2: The enzyme activities against time 0.09

Graph 2: Enzyme Activities Vs Time

0.08

Enzyme Activities

0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 0

20

40

60

80

100

120

140

Time

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EXPERIMENT 3: INVESTIGATION ON ENZYME ACTIVITY AND THE FACTORS THAT AFFECTS IT

3.3 Effect of Temperature on the Amylase Activity

Table 3: The absorbance and enzyme activities for 30 minutes at different temperature Temperature

Absorbance

Enzyme activities

Ice water bath

0.836

0.1216

30

0.833

0.1211

40

0.816

0.1183

50

0.840

0.1223

100

0.797

0.1151

Figure 3: Enzymes activities against temperature for 30 minutes 0.123

Graph 3: Enzymes Activities Vs Temp 30 min

0.122

Enzyme Activities

0.121 0.12 0.119 0.118 0.117 0.116 0.115 0.114 0

20

40

60

80

100

120

Temperature

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EXPERIMENT 3: INVESTIGATION ON ENZYME ACTIVITY AND THE FACTORS THAT AFFECTS IT

3.4 Effect of Temperature on the Amylase Activity

Table 4: The absorbance and enzyme activities for 60 minutes at different temperature Temperature

Absorbance

Enzyme Activities

Ice water bath

0.754

0.0540

30

0.687

0.0484

40

0.687

0.0484

50

0.708

0.0501

100

0.734

0.0523

Figure 4: Enzymes activities against temperature for 60 minutes

0.055

Graph 4: Enzyme Activities Vs Temp 60 min

0.054

Enyme Activities

0.053 0.052 0.051 0.05 0.049 0.048 0.047 0

20

40

60

80

100

120

Temperature

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EXPERIMENT 3: INVESTIGATION ON ENZYME ACTIVITY AND THE FACTORS THAT AFFECTS IT

3.5 Effect of pH on the Amylase Activity

Table 5: The absorbance and enzyme activities for 30 minutes at difference pH pH

Absorbance

Enzyme Activities

0

0.301

0.0324

6

0.191

0.0141

7

0.158

0.0086

8

0.158

0.0086

9

0.182

0.0126

Figure 5: The enzyme activities against pH for 30 minutes 0.035

Graph 5: Enzyme Activities Vs pH for 30 min

0.03

Enzyme Activiyies

0.025 0.02 0.015 0.01 0.005 0 0

1

2

3

4

5

6

7

8

9

10

pH

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EXPERIMENT 3: INVESTIGATION ON ENZYME ACTIVITY AND THE FACTORS THAT AFFECTS IT

3.6 Effect of pH on the Amylase Activity

Table 6: The absorbance and enzyme activities for 60 minutes at difference pH pH

Absorbance

Enzyme Activities

0

0.396

0.0241

6

0.308

0.0168

7

0.233

0.0105

8

0.251

0.0120

9

0.197

0.0075

Figure 6: The enzyme activities against pH for 60 minutes

0.03

Graph 6: Enzyme Activities Vs pH for 60 min

Enzyme Activities

0.025

0.02

0.015

0.01

0.005

0 0

1

2

3

4

5

6

7

8

9

10

pH

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3.7 Effect of Substrate Concentration on the Activity of Amylase Enzyme

Table 7: The absorbance and concentration of glucose at difference concentration of substrate Concentration of substrate

Absorbance

Enzyme Activities

0.25

0.214

0.0179

0.5

0.326

0.0366

0.75

0.219

0.0188

1.0

0.262

0.0259

1.5

0.238

0.0219

(%)

Figure 7: The enzyme activities against concentration of substrate

0.04

Graph 7: Enzyme Activities Vs Concentration of Substrate

0.035

Enzyme Activities

0.03 0.025 0.02 0.015 0.01 0.005 0 0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Concentration of substrate (%)

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3.8 Effect of Inhibitor

Table 8: The absorbance and enzyme activities at 30 minutes Test tube

Absorbance

Enzyme Activities

1

0.824

0.1196

2

0.836

0.1216

3

0.833

0.1211

4

0.816

0.1183

5

0.840

0.1223

Figure 8: The enzyme activities against inhibitor Graph 8: Enzymes Activities Vs Inhibitor 0.1225 0.122

Enzyme Activities

0.1215 0.121 0.1205 0.12 0.1195 0.119 0.1185 0.118 0.81

0.815

0.82

0.825

0.83

0.835

0.84

0.845

Inhibitor

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4.0 Discussion Enzymes are proteins that act as catalysts for biological reactions. Enzymes, like all catalysts, speed up reactions without being used up themselves. This occurs by lowering the activation energy of a reaction. All biochemical reactions are catalyzed by enzymes. Most enzymes have optimum activity at a neutral pH and at body temperature. Enzymes are also very specific, it only act on one substrate or one class of related substrate molecules. Typically, only one kind of substrate will fit into the active site. This experiment was conducted to investigate the factors that may affect enzyme activity. Enzyme activity means the ability of an enzyme that catalyzes a specific chemical reaction under certain conditions. Temperature, pH, concentration of substrate and inhibitor were tested. There are several factors that can affect this enzyme activity. The glucose standard graph was plotted as shown in figure 1. Based on figure 1, the linear equation will get y=0.0002x + 0.1065. Theoretically according effect of concentration, enzyme activities calculated with concentration of glucose and time. During catalysis, the first step is the substrate (S) binding to the enzyme (E), giving an enzyme-substrate complex (ES). This is an equilibrium reaction, and will be favored by a high concentration of enzyme and/or substrate. After the substrate is bound, the reaction takes place, and then the product is released. Based on figure 2 shows that the enzymatic activities are decrease with the duration of sample incubated with shaking at 150 rpm is an increase. The longer an enzyme is incubated with its substrate, the greater the amount of product that will be formed.

Enzyme catalyst reactions are reversible. Initially, there is little or no product present, and therefore the reaction proceeds only in the forward direction. However, as the reaction continues, so there is a significant accumulation of product, and there is a significant rate of back reaction. As a result, the rate of formation of product slows down as the incubation proceeds, and if the incubation time is too long, then the measured activity of the enzyme is falsely low. This is why at 90 minutes to 120 minutes; the product formed was getting low. FOOD BIOCHEMISTRY

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Other factor that may affect the enzymatic activity is temperature. As the temperature rises, reacting molecules have more kinetic energy. This increases the chances of a successful collision and so the rate increases. Theoretical, all reactions are faster at a higher temperature. However, enzyme-catalyzed reactions become slower or stop if the temperature becomes too high, because enzymes become denatured at high temperatures. Therefore, enzymes have an optimum temperature that corresponds to maximum activity. (At higher or lower temperatures, the activity of the enzyme is lower.) The optimum temperature is usually around body temperature (37°C).

As shown in Figure 3, the enzyme activities decreasing with high temperature, and then abruptly declines with further increase of temperature. The result a show was not followed the theory. This might be because of some errors. For example, the sample prepared was not been pipette properly because the pump was not functioning. Therefore the amount that supposed to be ready was incorrect. Most animal enzymes rapidly become denatured at temperatures above 40°C. This is why the graph was slightly decreased at temperature 40⁰C. This experiment was repeated again for 60 minutes. The data obtains can be shown in figure 3. The enzyme activities product for 30 minutes was much higher than at 60 minutes. Supposedly at 60 minutes, the enzyme activities produced was more because the longer the time for incubation, the greater the amount of product formed. Improper preparing the sample could be contributed to this undesirable result. Changes in pH also may affect the enzymatic activity. Since enzymes are proteins, they are very sensitive to changes in pH. Each enzyme has its own optimum range for pH where it will be most active. Amylase is an enzyme that catalyses the hydrolysis of starch into sugars. It is present in the saliva of humans and some other mammals, where it begins the chemical process of digestion. In this case, amylase optimum pH is at 6. Referring to figure 6, it was show not bell-shaped. The result a show was not followed the theory.

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The amount of enzyme activities produced was higher at pH 0 where amylase most active. As the pH increase, the enzyme activities produced was decreased. Compared to figure 5, which the incubation duration was 30 minutes, the optimum pH was at pH 0. Besides that, the enzyme activities produced at pH 7.0 was the lowest. The enzymes activities produced for 30 minutes of incubation process was more than for 60 minutes incubation. This result was not followed the theory since the longer the time for incubation, the greater the amount of product formed. This condition happened again because improper in preparing the sample to be investigated. Figure 7 shows the enzyme activities against the concentration of substrate. According to Michaelis-Menten kinetics, enzyme-substrate reactions are actually comprised of two elementary reactions. The first is the when the substrate forms a complex with the enzyme and then in the second, the complex decomposes to product and enzyme. As the substrate concentration is increased, each enzyme is able to locate and react with more substrate molecules and the observed enzyme activity increases. When this maximum velocity had been reached, all the available enzyme has been converted to ES, the enzyme substrate complex. This point is designated as Vmax. However the data obtained was fluctuated. This result is away from the theory. Therefore the Vmax cannot be identified. 0.1 M Pb(NO3)2 is an inhibitor. The function of inhibitor is to reduce the rate of enzyme activity, usually by binding with the enzyme and interfering with the formation of the enzymesubstrate complex. There are three common types of enzyme inhibition; competitive, noncompetitive and substrate inhibition. Competitive inhibition occurs when the substrate and a substance resembling the substrate are both added to the enzyme. Non-competitive inhibitors are considered to be substances which when added to the enzyme alter the enzyme in a way that it cannot accept the substrate. In this experiment 0.1 M Pb(NO3)2 caused the reaction rate to be decreased. The figure 8 shows that as the incubation time increased the enzyme activities produced will be increased too.

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5.0 Conclusion Enzyme activity means the ability of an enzyme that catalyses a specific chemical reaction under certain conditions. There are several factors that can affect enzyme activity, from the experiment that was conducted temperature, pH, concentration of substrate and inhibitors were tested to determine the factors affecting enzyme activities. Enzyme-catalyzed reactions become slower or stop if the temperature becomes too high, because enzymes are denatured at high temperatures. As for the pH, the result shows the longer the time for incubation, the greater the amount of product formed, but this was against the theory due to error during conducting the experiment. According to the Michaelis-Menten kinetics, the Vmax cannot be identified because the data obtained was fluctuated. By using an inhibitor the incubation time increased the enzyme activities produced will be increased too. The precaution is to avoid from making parallax error for most of the measured content for an accurate final results.

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6.0 References 1) Raymond Chang, Chemistry 10th Ed, McGraw Hill 2) Nelson, J., Chemistry: The Central Science, 3rd Ed, Prentice Hall ( 1985). 3) Eed, John (2013) "Factors Affecting Enzyme Activity,"ESSAI: Vol. 10, Article 19.Available at:http://dc.cod.edu/essai/vol10/iss1/19. 4) Reece, Jane B., Lisa A. Urry, Michael L. Cain, Steven A. Wasserman, Peter V. Minorsky, and Robert B. Jackson. Campbell Biology. 9th ed. Boston: Benjamin Cummings/Pearson Education, 2011. 5) Petersen, Chris E., and Barbara J. Anderson. Investigation in the Biology 1151 Laboratory. Champaign, IL: Stipes L.L.C., 2005.

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