Lab # 5

Date: March 23, 2023

Topic: Denature of enzyme

Aim: To investigate the effect of varying conditions of temperature and substrate concentration

on the activity of amylase.

Material:

1. Starch solution 11. Hotplate/stirrer

2. Amylase 12. Stop Watch

3. Iodine in KI 13. Labels

4. Test Tubes 14. Rack

5. White tiles

6. Microplate

7. Pipettes

8. Beakers

9. Measuring cylinders

10. Thermometer

Method:

Effect of substrate concentration:

1. A clean petri-dish was used to create a 4x4 grid of 12/KI drops.

 2. 4 test tubes were then labeled, the next step was the addition of 1 ml of 3% amylase in

each test tube. Thereafter, 5 ml of 1% amylose, 5 ml of 2% amylose, 5 ml of 4% amylose

and 5 ml of 8% amylose were added in each test tube respectively one at a time, while the

timer was started. Afterwards, one drop from each test tube was removed and placed on a

petri dish while also one drop of the 12/KI solution was added on the petri dish with the

drops from the test tubes. The color change was observed on the petri dish.

*The pipet was rinsed with distilled water and shook after each use

3. Then for every 30 second intervals it was repeated and the color change was observed

and recorded in a data table. This was done until there was no longer a bluish purple color

change. The test tubes were gently shook occasionally during this process for faster

results.

4. The time taken for each test tube to lose all its starch (no longer have a bluish purple)

was recorded.

Effect of temperature:

1. A clean petri-dish was used to create a 4x4 grid of 12/KI drops.

2. 4 test tubes were then labeled, thereafter 1ml of 1% amylase was added in each test tube.

Then 5ml of 1% amylose was added to each test tube(it was mixed well) while each test
 tube was placed on a tube in ice, a tube in 37 degree water bath, a tube in 80 degree water

, and a tube at room temperature respectively. The time was started and recorded.

3. After 30 seconds, one drop from each test tube was removed and added on a petri dish

then one drop of 12/KI solution was added on each petri dish thereafter.

4. Then for every 30 second intervals it was repeated and the color change was observed

and recorded in a data table. This was done until there was no longer a bluish purple color

change, while gently shaking the test tube.

5. The time taken for each test tube to lose all the starch was recorded.

Results:

TABLE SHOWING RESULT OF AMYLASE ACTIVITY IN DIFFERENT

CONCENTRATIONS OF AMYLOSE
Time (s) Percentage of amylose %

0.5 1 2 4

30 No color change Color change Color change Color change

60 No color change Color change Color change Color change

90 No color change Color change Color change Color change

120 No color change No color change No color change Color change

150 No color change No color change No color change Color change

180 No color change No color change No color change Color change

210 No color change No color change No color change No color change

240 No color change No color change No color change No color change

270 No color change No color change No color change No color change

300 No color change No color change No color change No color change

360 No color change No color change No color change No color change

TABLE SHOWING RESULTS OF AMYLASE ACTIVITY ON AMYLOSE IN

DIFFERENT TEMPERATURES
Time Temperature C𝇈

Ice bath Room 43 90

(10) temperature
(27)

30 No color change No color change No color change No color change

60 No color change No color change No color change No color change

90 No color change No color change No color change No color change

120 No color change No color change No color change No color change

150 No color change Color change No color change No color change

210 No color change Color change No color change No color change

240 No color change Color change No color change No color change

270 No color change Color change No color change No color change

300 No color change Color change Color change No color change

330 No color change Color change Color change No color change

GRAPH SHOWING THE TIME AND CONCENTRATION RELATIONSHIP DURING
ENZYME ACTIVITY

GRAPH SHOWING HOW TEMPERATURE AFFECT DURATION OF THE ENZYME

ACTIVITY
Discussion:

Enzymes are defined as substances that act as a catalyst in living organisms, regulating the rate at

which chemical reactions proceed without itself being altered in the process. In this experiment

the enzyme “Amylase” was utilized to catalyze amylose. The enzyme amylase belongs to the

group glycosylases, which are starch degrading and the role of amylase is the hydrolysis of

internal α-1-4 glycosidic bonds in polysaccharides to form simpler sugar constitutes and limit

dextrins. The sequence of enzyme catalysis starts with the attraction of substrates to the active

site of an enzyme(enzyme-substrate complex) to accelerate the reaction to form products and

then as a result allow the products to dissociate. The iodine’s purpose is to determine the ability

of enzyme activity and whether the substrate continues to be broken down. Since this lab is

based on different concentrations and temperature this will reflect the ability of enzymes to

catalyze substrates and whether the enzymes become denatured, the iodine indicates the

enzyme’s remaining abilities under different conditions.

Furthermore, as the amylose concentration increased the rate of enzyme activity also increased

since the enzyme became saturated with substrate this resulted in an increased reaction level.

However, too much concentration would also slow down the enzyme activity because of its

excess substrates. Based on the results of the experiment it was evident that as the concentration

level increased so did the rate of reaction therefore the concentration with the maximum activity

was at 1% and 2% amylose. The more accurate result would have been the 0.5% since it was the

lowest concentration therefore it would have been dissociated first resulting in a more minor rate

of reaction, this is a result of human error. The concentration with minimum enzyme activity was

at 4% since it had more substrate to take care of as well as it had the most rate of reaction during

the experiment. The other part of the experiment had to do with different temperatures and their
effects on enzyme activity. As it relates to the relationship with temperature and enzyme, the rate

of enzyme-catalyzed reaction will increase as the temperature increases. As such higher

temperature resulted in more enzyme activity however, if the temperature continued to increase

the enzyme’s active site would shrink, resulting in drastic decrease in activity, this may be what

was observed in the experiment for the 90oC condition. An even higher temperature would result

in the enzyme becoming denatured thus being no longer able to function. From the graph it was

apparent that 27oC condition had the highest enzyme activity while the 90oC had the lowest

enzyme activity, nonetheless, the lowest temperature did not result in any color change during

the experiment this may be due to the fact that enzymes tend to function more slowly in colder

environments or in the ice bath from the experiment.

Source of error:

A source of error to this lab would be if the pipette was not properly washed after each use and

this could result in the contamination of the other results.

Another source of error could be the inaccuracy of the amount of iodine placed in each grid at

every 30 second intervals.

Limitation:

A limitation to the two experiments would be the sensitivity of iodine to light which could have

affected the iodine's ability to detect enzyme activity and, therefore, inaccurate results .

Conclusion: In conclusion it was determined that 1% and 2% concentration as well as 27°C

conditions of solutions had the most enzyme activity whereas the 4% concentration and 90°C

conditions had the least enzyme activity.

 References:

Encyclopædia Britannica, inc. (2023, March 27). Enzyme. Encyclopædia Britannica.

Retrieved April 2, 2023, from https://www.britannica.com/science/enzyme

Amylose. Amylose - an overview | ScienceDirect Topics. (n.d.). Retrieved April 2, 2023,

from
https://www.sciencedirect.com/topics/materials-science/amylose#:~:text=Amylose%20is%
20an%20unbranched%20linear,enzymes%20(Basit%2C%202000).

Libretexts. (2021, January 15). 19.5: Effect of concentration on enzyme activity. Chemistry
LibreTexts. Retrieved April 2, 2023, from
https://chem.libretexts.org/Courses/Saint_Francis_University/Chem_114%3A_Human_Ch
emistry_II_(Muino)/19%3A_Enzymes_and_Vitamins/19.05%3A_Effect_of_Concentration
_on_Enzyme_Activity#:~:text=Initially%2C%20an%20increase%20in%20substrate,the%2
0concentration%20of%20an%20enzyme.

BBC. (n.d.). Effect of temperature, substrate concentration and ph on reaction rate -

enzymes - edexcel - GCSE biology (single science) revision - edexcel - BBC bitesize. BBC
News. Retrieved April 2, 2023, from
https://www.bbc.co.uk/bitesize/guides/z88hcj6/revision/2#:~:text=As%20the%20temperatu
re%20increases%20so,enzyme's%20active%20site%20changes%20shape.

Khan Academy. (n.d.). Enzymes review (article). Khan Academy. Retrieved April 2, 2023,
from
https://www.khanacademy.org/science/ap-biology/cellular-energetics/environmental-impac
ts-on-enzyme-function/a/hs-enzymes-review#:~:text=Factors%20affecting%20enzyme%20
activity&text=Temperature%3A%20Raising%20temperature%20generally%20speeds,has
%20an%20optimum%20pH%20range.
Enzyme Activity (Observation, Recording & Reporting)
Procedure & Report Format (5 marks)
- Procedure: past tense/passive voice/3rd person [1]
- Procedure: clear, logical sequence of steps [1]
- Correct spelling, grammar and punctuation [1]
- Acceptable scientific reporting [2]
Observations & Results (7 marks)
- Accurately tabulate all relevant data [2]
- Plot data on line graph for simple and clear interpretation [3]
- Valid titles (below object, printed, all caps, underlined) [1]
- Correct units shown [1]

Enzyme Activity (Analysis & Interpretation)

Discuss substrate concentration and temperature on enzyme activity (5 marks)

- Briefly discuss the type of enzyme being used to catalyze amylose [2]
- Give a brief description of enzyme action [1]
- Describe the significance of I2/KI solution in the experiment [2]
Analysis of results (5 marks)
- Discuss how concentration generally affects enzyme activity [1]
- Based on results from lab discuss which concentration demonstrated maximum activity [1]
- Based on results from lab discuss which concentration demonstrated minimum activity [1]
- Based on results from lab discuss temperature range that showed maximum activity [1]
- Based on results from lab discuss temperature range that showed minimum activity [1]
Sources of error/Limitations/Assumptions (2 marks)
- any two [2]
- less than two