2010 SPM Selangor Trial Biology Paper 3
An experiment was carried out to study the effect of enzyme concentration on the activity of amylase on starch. 3 test tubes P, Q and R were filled with 2 ml of 0.5 %, 1.0 % and 1.5 % of amylase respectively.

2 ml of 1% starch suspension was added into each test tube and the contents were stirred.

Every 2 minutes a drop of the mixture from each test tube was mixed with a drop of iodine solution on a white tile.

All test tubes are immersed in a water bath at temperature of 37^oC during the experiment as shown in Diagram 1.

Table 1 shows the results of the experiment.

a) Record the time taken for the starch to be hydrolysed completely in the boxes provided in Table 1.

(b)(i) State two different observations made from Table 1.

Observation 1:In test tube P/0.5% amylase enzyme, the time taken for the starch to be hydrolysed completely is 18 minutes.

Observation 2: In test tube R/1.5% amylase enzyme, the time taken for the starch to be hydrolysed completely is 10 minutes.

(ii) State the inferences from the observation in 1(b)(i).

Inference from observation 1:In test tube P, the concentration of enzyme is low, so rate of enzyme reaction is slow

Inference from observation 2. In test tube R, the concentration of enzyme is high, so rate of enzyme reaction is fast

(c) Complete Table 2 based on this experiment.

Variables	Method to handle the variables
Manipulated variable: Enzyme concentration	Use different concentration of enzyme, that is 0.5%, 1.0% and 1.5% enzyme amylase solutions.
Responding variable: The time taken for the starch to hydrolyse completely	Measure and record the time taken for the starch to hydrolyse using a stopwatch.
Controlled variable: Volume of enzyme// concentration// volume of starch // temperature of water bath	Fix the volume of enzyme amylase at 2 ml // Fix the concentration of starch suspension at 1% // Fix the temperature of the water bath at 37oC. (Write one only and must correspond)

(d) State the hypothesis for this experiment.

As the concentration of enzyme amylase increases, the time taken for the starch to hydrolyse completely decreases / the rate of enzyme amylase activity increases.

(e)(i) Construct a table and record all the data collected in the experiment.

Enzyme concentration (%)	The time taken for starch to hydrolyse completely (minute)	Rate of enzyme amylase activity (minute-1)
0.5	18	0.06
1.0	12	0.08
1.5	10	0.10

f)Plot a graph of rate of enzyme amylase activity against enzyme concentration on graph paper.

g) Based on the graph, state the relationship between enzyme concentration and the rate of enzyme amylase activity. Explain your answer.

The higher the concentration of enzyme solution, the higher the rate of reaction, cause by more enzyme molecules in the test tube. More chances of collision occur between enzyme and starch / enzyme substrate- complexes are more formed.

h) Based on the experiment, define operationally what enzyme is.

Enzyme /amylase hydrolyses starch in 37^o C into a product that make the iodine solution remains unchanged / yellow, and the rate of reaction is affected by the enzyme concentration.

i) The experiment is repeated with test tube R immersed in water bath of 65^o C. Predict the time taken for starch to hydrolyse completely in R. Explain your answer.

30-40 minutes / longer than 30 minutes / longer than the time taken when kept at 37^oC. This is because in high temperature, enzymes are denatured / the active sites for the reaction are destroyed.

Answer to Bioscore pg 78-79/ Hands on pg 42

2. Plan an experiment to study the effect of enzyme concentration on the activity of an enzyme.

Objective	To study the effects of enzyme concentration on the activity of salivary amylase.
Problem Statement	What are the effects of enzyme concentration on the activity of salivary amylase?
Hypothesis	The higher the enzyme concentration, the higher the rate of reaction until it reaches a maximum rate.
Variables	Manipulated: Enzyme concentration Responding: Time taken for the complete hydrolysis of starch//Rate of reaction //Activity of enzyme Fixed : Substrate concentration// temperature// pH
Materials & Apparatus	Materials :1% starch suspension, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.2% amylase, iodine solution and distilled water. Apparatus : 5 ml syringe, 1 ml syringe, test tubes, glass rod, stopwatch, white tile with grooves, measuring cylinders and dropper.
Technique	Measure and record the time taken for the complete hydrolysis of starch using a stopwatch. // Calculate the rate of reaction using the formula : (1/time taken for the complete hydrolysis of starch).
Procedure	1. 6 test tubes were labelled as A, B, C, D, E and F and put in a water bath at 37oC. A : 1.0 ml 0.2% amylase B : 1.0 ml 0.4% amylase C : 1.0 ml 0.6% amylase D : 1.0 ml 0.8% amylase E : 1.0 ml 1.0% amylase F : 1.0 ml 1.2% amylase 2. Each test tube was filled with different volumes of amylase as given above. 3. A few drops of iodine were dropped separately on the grooves of a white tile. 4. 4 ml of 1% starch suspension was poured into test tube A. 5. The stopwatch was started. A small amount of mixture was added to the drop of iodine on the white tile immediately. 6. The colour change in iodine was observed. 7. The iodine test was carried out at an interval of 30 seconds until there was no more change in iodine colour (colour remained yellow). 8. The time taken when the colour of iodine remained yellow was recorded. 9. Steps 4-8 were repeated for test tubes B, C, D, E and F. 10. All observations were recorded. 11. A graph of rate of reaction (1/time) against enzyme concentration was plotted.
Results	Concentration of amylase (%) Time taken for the complete hydrolysis of starch (minutes) Rate of reaction (1/time) (min-1) 0.2 0.4 0.6 0.8 1.0 1.2
Conclusion	Hypothesis is accepted. The higher the enzyme concentration, the higher the rate of reaction until it reaches a maximum rate.

Procedure for page 34 (Hands-on)

Procedure:
1. 6 test tubes were labelled as A, B, C, D, E and F and put in a water bath at 37oC.
2. Each test tube was filled with different volumes of saliva and distilled water as given.
3. A few drops of iodine were dropped separately on the grooves of a white tile.
4. 4 ml of starch solution was poured into test tube A.
5. The stopwatch was started. A small amount of mixture was added to the drop of iodine on the white tile immediately.
6. The colour change in iodine was observed.
7. The iodine test was carried out at an interval of 30 seconds until there was no more change in iodine colour (colour remained yellow).
8. The time taken when the colour of iodine remained yellow was recorded.
9. Steps 4-8 were repeated for test tubes B, C, D, E and F.
10. All observations were recorded.
11. A graph of rate of reaction (1/time) against enzyme concentration was plotted.

Answer to Written Practical Question (Hands on Pg 40)

1a)

Set	Time taken (min)
A	20
B	10
C	3
D	6
E	30

b)(i) Observation 1-The time taken for complete hydrolysis of starch in set C/at 37oC is 3 minutes.
Observation 2-The time taken for complete hydrolysis of starch in set E/at 50oC is 30 minutes.
(ii) Inference 1 -The time taken for complete hydrolysis of starch in set C is fastest because 37oC is the optimum temperature for enzyme activity.
Inference 1 - The time taken for complete hydrolysis of starch in set E is the slowest because at too high temperature, that is 50oC, enzyme are denatured.

c)

Variable	Method to handle the variable
Manipulated variable Temperature	5 sets of apparatus using water baths of different temperatures, 15oC, 25oC, 37oC, 45oC and 50oC are set up.
Responding variable Time taken taken for complete hydrolysis of starch	Measure and record the taken for complete hydrolysis of starch using a stopwatch
Fixed variable pH// enzyme concentration// substrate concentration	Fix the pH to be the neutral throughout the experiment Fix 1ml of saliva throughout the experiment Fix 5 ml of starch throughout the experiment

d) The higher the temperature, the higher the rate of enzyme reaction until an optimum temperature is reached.
e)

Temperature (oC)	Time taken for hydrolysis of starch (minutes)	Rate of reaction (min -1)
15	20	0.05
25	10	0.10
37	3	0.33
45	6	0.17
50	30	0.03

f) An increase in temperature will also increase the rate of reaction until the optimum temperature of 37oC is reached. The rate of reaction decreases after 37oC.
g)An enzyme/amylase hydrolyses starch into a product that make the iodine solution remains unchanged/yellow, and the rate of reaction is affected by temperature.
h)The rate of reaction will be zero because all enzymes are denatured at high temperature of 70oC.

ANSWERS TO PG 38-40

Section A
1. a) Saliva amylase breaks down starch into maltose.
b)(ii) The rate of reactions in sets P and Q are slow because enzymes are less active at low temperatures. The rate of reaction in set R is the highest because it is in its optimum temperature of enzme activity. Starch is hydrolysed very quickly. There is no breakdown of starch in set S as enzymes are denatured after they have been boiled.
c) The reaction in set R will stop as salivary amylase is not active in acidic medium.
d) Increase the temperature to 40oC.
e)(i) Wash the clothes in water of temperatures between 37oC and 40oC.
(ii) A temperature range of between 37oC and 40oC is an optimum temperature for enzymes to break down stains.

2. a) To study the effect of pH on enzyme activity.
b) A: Clear solution
B: Cloudy suspension
C: Cloudy suspension
D: Clear solution
c) In test tube A, enzyme pepsin breaks down albumen because pepsin works effectively in an acidic medium. Neutral medium is not suitable for the action of pepsin as shown in test tube B. There is no breakdown of proteins in test tube C as there are no enzymes present.
d) It is because a temperature of 37oC is an optimum temperature for enzyme activity.
e) There is no albumen/substrate, so no reaction takes place.

Section B
3a)

Characteristic	Explanation
Enzymes are biological catalysts and enzyme reactions are reversible	.Enzymes speeds up the rate of metabolism in the body. · Metabolic reactions are reversible · Enzymes remain unchanged at the end of reaction
Enzyme action is highly specific	· Acts on specific substrate only · Has an active site which is complimentary to certain substrate molecule only · Acts on ‘lock and key hypothesis’
Enzymes are sensitive to pH	· Normally function best at neutral eg amylase · Some enzymes work best in acidic condition. For example, pepsin · Some enzymes work best in alkaline condition. For example, trypsin.
Enzymes are sensitive to temperature	· Work best at temperatures between 35oC and 40oC. · Enzymes are inactive at low temperatures. · They are denatured at high temperatures.

b)

Acidity or alkalinity of solutions affect rate of reaction.

Each enzyme has an optimum pH so as to function at its maximum rate.

For example, pepsin at pH 3, salivary amylase at pH 7, trypsin at pH 8.5

Change in pH alters the charges on the active sites of an enzyme.

Alterations in the ionic charges also changes configuration of substrate.

Excess hydrogen ions attach to active sites of enzymes when pH is low.

Substrate is not able to bind to form enzyme-substrate complex.

Free hydroxyl ions attach to active sites when pH is high.

Extreme changes in pH alter the structures of enzymes and substrates.

4a) An enzyme has a specific three-dimension shape. As it is a protein molecule, the polypeptide chain fold to form an active site. Active site of enzyme complements the structure of the substrate it acts on. Substrate molecule fits into active site of enzyme like a key fits into a lock. Substrate is represented by the key while the lock represents the enzyme. When substrate fits into active site of enzyme, an enzyme-substrate complex is formed. Hydrogen and ionic bonds hold the enzyme-substrate complex. Reaction takes place to convert the substrate into products. The products have different shapes. The product then leaves the active sites.

b)Food processing industry
In meat industry, protease is used to tenderise meat.
In fish industry, protease is used to remove the skin of fish.
In dairy industry, lipase is used for ripening of cheese, rennin coagulates milk in the making of cheese, lactase is used to hydrolyse lactose.
In baking industry, amylase is used to breakdown starch in the making of bread, glucose oxidase is used for the stability of bread dough.
In brewing industry,zymase is used to hydrolyse sugar into ethanol.
In seaweed products, cellulase is used to hydrolyse cellulose to extract agar-agar from seaweeds.

Medical field
Trypsin is used to remove blood clots and clean wounds.