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Enzyme Experiment

Abdelmoniem Abomosalam

The British University in Egypt

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Abstract

The purpose of this experiment is to investigate factors that affect enzyme-catalysed reactions.

Specifically, reaction conditions (Enzyme concentration, temperature, inhibitior ) are altered to

determine their effects on the rate of breakdown of starch into simple sugars by the enzyme

amylase. The addition of iodine solution was used as a measure of enzyme activity. Enzyme

activity was studied at temperatures 0 C, 37 C, 100 C. It showed that at 0 and 100, there was

almost no enzyme activity. Significant enzyme activity was seen at 37oC.Starch was tested using

0, 2,6,10 drops of amylase and it showed the effect of enzyme concentration was highest at 10

drops of amylase. The addition of sodium hydroxide (inhibitor) slowed down enzyme the

activity.
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Contents

Abstract ............................................................................................................................... 2

Introduction ......................................................................................................................... 5

Tertiary structure ............................................................................................................. 5

Enzymes as catalysts ....................................................................................................... 6

The effects of surrounding conditions on enzyme activity ............................................. 7

Enzyme Concentration ................................................................................................ 7

Substrate concentration ............................................................................................... 7

Competitive and non-competitive inhibitors .............................................................. 7

Temperature ................................................................................................................ 9

pH.............................................................................................................................. 10

Procedure. ..........................................................................................................................11

Part 1: Effect of Enzyme Concentration ........................................................................11

Part 2: Effect of Temperature ........................................................................................ 12

Part 3: Effect of inhibitors............................................................................................. 13

Results ............................................................................................................................... 14

Discussion ......................................................................................................................... 14

Conclusion ........................................................................................................................ 15

Bibliography ..................................................................................................................... 15
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List of Tables

Table 1: The effect of enzyme concentration on the rate of an enzyme-catalysed reaction ......... 14

Table 2:The effect of temperature on the rate of an enzyme-catalysed reaction .......................... 14

Table 3:The effect of inhibitor on the rate of an enzyme-catalysed reaction ................................ 14

List of figures

Figure 1 The effect of enzyme on activation energy....................................................................... 6

Figure 2: The effect of substrate concentration .............................................................................. 7

Figure 3: A competitive inhibitor competes with substrate molecules for the active site of the

enzyme ............................................................................................................................................ 8

Figure 4: Non-competitive inhibitors bind to the enzyme somewhere other than the active site.

This causes the active site to change shape .................................................................................... 9

Figure 5: The effect of temperature .............................................................................................. 10

Figure 6:Starch hydrolysis by amylase ..........................................................................................11

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Introduction

Tertiary structure

Enzymes are globular proteins. They have a precise shape- their tertiary structure. The

substance works on is called the substrate. There is part of the enzymes surface called the active

site into which the substrate fits. The active site and substrate are complementary- the substrate

fits into the active site, partly because it’s the right shape and partly because the chemical
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charges match. The specificity of an enzyme refers to its ability to catalyze just one reaction or

type of reaction. Only one substrate molecule will fit into the active site of the enzyme molecule.

Enzymes as catalysts

Enzymes are catalysts so they speed up chemical reactions without being altered themselves. For

any reaction to take place, the reactant molecules must collide and achieve the transition state,

one in which the existing bonds are strained. After this, the existing bonds will break and new

ones will form as new products are created.

Enzymes speed up reactions because they lower the activation energy needed to achieve the

transition state. This is achieved by enabling the reaction to take a slightly different pathway by

forming an enzyme-substrate complex. This complex alters the bonding in the substrate, enabling

the bonds to be broken more easily.

Figure 1 The effect of enzyme on activation energy

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The effects of surrounding conditions on enzyme activity

Enzyme Concentration

The higher the enzyme concentration, the greater the rate of reaction until the substrate

becomes the limiting factor

Substrate concentration

In a similar way to enzyme concentration, fig 2 shows that the more substrate there is, the

faster the enzymes will work until all the active sites are full all the time. After that no matter

what the substrate concentration, the reaction will not go any faster. All enzymes have a

maximum rate at which they can work.

Figure 2: The effect of substrate concentration

Competitive and non-competitive inhibitors

Inhibitors are substances that slow down or stop enzyme action:

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Competitive inhibitors are similar in shape to the substrate. They fit into the active site

but cannot be converted into the product, so they simply get in the way. The more competitive

inhibitors there are the less chance there is of successful collision between enzyme and substrate.

Non- competitive inhibitors bind to the enzyme away from the active site, but they alter

the shape of the enzyme so that the substrate cannot fit into the active site. Effectively, non-

competitive inhibitors switch off enzymes. If the inhibitor is removed the enzyme functions as

normal.

Figure 3: A competitive inhibitor competes with substrate molecules for the active site of the enzyme
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Figure 4: Non-competitive inhibitors bind to the enzyme somewhere other than the active site. This
causes the active site to change shape

The key difference between the two types of inhibitors is that the effects of competitive

inhibitors can be overcome by adding more substrate. It is simply a matter of increasing the

chances of a collision between enzyme and substrate compared with enzyme and inhibitor. With

non-competitive inhibitors, on the other hand, it does not matter how much substrate is added-

nothing can fit into the active site so there is no activity.

Temperature

Fig 5 shows the relationship between enzymes and temperature. As temperature

increases, the rate of reaction increases because all the molecules bound around faster and there

is more chance of a collision between the enzyme and the substrate. Therefore, more enzye-

substrate complexes form, resulting in a product being formed more quickly. However when the

temperature gets too high, the enzyme molecules vibrate more vigorously and the weaker bonds

such as hydrogen bonds break. The shape of the enzyme changes and the active site and substrate

are no longer complementary. The enzyme is denatured and this process is permanent.
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Figure 5: The effect of temperature

pH

Every enzyme has optimum pH. At optimum pH, the positive and negative charges on the

active site and substrate are complementary. Away from this pH, the H+ and OH- ions neutralize

the charges so that the enzyme and substrate are no longer complementary. In addition, extremes

of pH can denature enzymes (Boyle, 2015).

In this experiment the enzyme amalyse is studied. Amalye is responsible for the

hydrolysis of starch. In the presence of amylase, a sample of starch will be hydrolyzed to shorter

polysaccharides, dextrins, maltose, and glucose. The extent of the hydrolysis depends on how

long it is allowed to react – if the starch is hydrolyzed completely, the resulting product is

glucose.
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Figure 6:Starch hydrolysis by amylase

The presence or absence of starch in the solutions is tested using iodine (I2) . Iodine

forms a blue to black complex with starch, but does not react with glucose. If iodine is added to a

glucose solution, the only color seen is the red or yellow color of the iodine. Therefore, the faster

the blue color of starch is lost, the faster the enzyme amylase is working. If the amylase is

inactivated, it can no longer hydrolyze starch, so the blue color of the starch‐iodine complex will

persist.

Also, the presence of glucose in the samples is tested using Benedict’s reagent. When a

blue solution of Benedict’s reagent is added to a glucose solution, the color will change to green

(at low glucose concentrations) or reddish‐orange (at higher glucose concentrations). Starch will

not react with Benedict’s reagent, so the solution will remain blue.

Procedure.

Part 1: Effect of Enzyme Concentration

1. Label five test tubes 1 5. Place 4 mL of 1 % starch in each of the first four test tubes.

Place 4 mL of amylase solution in the fifth tube. Place all of the tubes in the 37°C water bath for
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5 minutes. Obtain 5 clean droppers and label them 1 5. (To avoid contamination of these

solutions, you will use a separate dropper for each mixture.)

2. Tube 1 will be your control and it will not contain any enzyme. Remove the tubes from

the water bath momentarily, and quickly add 3 drops of the warmed amylase solution to tube 2,

add 6 drops amylase to tube 3, and add 10 drops amylase to tube 4. Mix the tubes quickly by

shaking them gently, and immediately put the test tubes back in the 37°C water bath. Record the

time at which you added the enzyme to the test tube.

3. Transfer four drops of each reaction mixture (tubes 1‐4) using a separate clean dropper

for each to separate wells on a spot plate. (Keep the test tubes in the water bath.) Add one drop of

the iodine reagent to each. Record your observations. Use the visual color reference table to

assess the enzyme activity.

4. Repeat step 3 after the mixtures have had five minutes of reaction time.

5. Repeat step 3 after the mixtures have had ten minutes of reaction time.

6. In the test tubes where hydrolysis occurred, the presence of glucose can be confirmed

with Benedict’s reagent. Add 3 mL of Benedict’s reagent to each test tube and put them in the

boiling water bath for 3‐4 minutes. The appearance of a green to orange or red precipitate

indicates the presence of glucose. If the solution remains blue with no solid, then no glucose is

present and no hydrolysis of the starch has taken place.

Part 2: Effect of Temperature

1) Place 4 mL of 1 % starch solution in each of three clean test tubes. Place 4 mL of amylase

solution in 3 separate clean test tubes (so you will have a total of six test tubes: 3

containing starch and 3 containing enzyme).

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2) Place a starch tube and an amylase tube in the 37°C water bath. Place one tube of each in

an ice‐ water bath, and one of each tube in a boiling water bath. Keep the tubes in their

baths for 10 minutes to allow them to reach the temperature of their baths.

3) Read and record the temperature of the ice‐water bath. Pour the amylase solution into the

starch solution, mix, and put the tube back into the bath. Record the time.

4) Repeat step 3 for the 37°C bath.and for the boiling water bath.

5) For each mixture, after 10 minutes of reaction have elapsed, transfer 4 drops of the

mixture to a spot plate. Add 1 drop of iodine reagent to each sample. Record the color

and activity level of the enzyme in each case.

Part 3: Effect of inhibitors

1) Place 4 mL of amylase solution in each of three test tubes. To the first tube, add 10 drops

of 1 % NaCl solution. Add 10 drops of 95 % ethanol to the second test tube. Add 10

drops of either AgNO3 or Pb(NO3)2 solution to the third test tube.

2) In each of three separate clean test tubes, place 4 mL of 1 % starch.

3) Place all six test tubes in the 37°C water bath for 5 minutes. When the 5 minutes is up,

pour a tube of starch solution into each of the enzyme‐inhibitor mixtures. Mix each tube

and return the tubes to the water bath for 15 minutes. Meanwhile, rinse out three

droppers.

4) After 15 minutes, transfer 4 or 5 drops of each mixture (using a clean dropper each time)

to a spot plate. Add 1 drop of iodine reagent to each sample. Record your observations

and the enzyme activity level in each case.

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Results

Table 1: The effect of enzyme concentration on the rate of an enzyme-catalysed reaction

Tube # of drops of Color Amount of Enzyme activity

amylase starch remaining level
1 No amylase Dark blue All None (0)
2 2 blue low low (1)
3 6 Light brown Moderate Moderate (2)
4 10 gold None High(3)

Table 2:The effect of temperature on the rate of an enzyme-catalysed reaction

Tube Temperature Amount of Enzyme activity

(oC) starch remaining level
1 0 All None (0)
2 37 None High (3)
3 100 All None (0)

Table 3:The effect of inhibitor on the rate of an enzyme-catalysed reaction

Sample Iodine's reagent Amount of Enzyme activity

observation starch remaining level
Starch-amylase Light brown Some Moderate (2)
mixture with 10 colour
drops of NaOH

Discussion

The greater the concentration of enzyme, the greater the chance of a starch molecule colliding

with an active site of an amylase molecule. The more frequent these collisions, the faster the rate

of reaction of enzyme-substrate complexes and the faster the rate of reaction.

Increasing the temperature from 0 to 37 causes an increase in the rate of reaction. The optimum

temperature for this enzyme lies somewhere around 37. Increasing the temperature above 37

decreases the rate of reaction.

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Sodium hydroxide prevented the enzyme amylase activity in comparsion with its activity in the

absence of sodium hydroxide in the previous experiment

Conclusion

In this experiment, the effect of temperature, enzyme concentration, and inhibitor is

tested. The results showed that the enzyme activity was highest at temperature 37oC, absence of

inhibitor and high concentration of enzyme. Under these conditions, starch is hydrolyzed

successfully to its simplest sugar glucose. Starch concentrations were inferred qualitatively using

iodine solution.

Bibliography

Boyle, M. (2015). My Revision Notes: AQA A Level Biology. Hodder Education.

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