VARIATION OF ENZYMATIC ACTIVITY WITH

TEMPERATURE

INSTRUCTED BY: H.G.H.U RATHNASIRI

GROUP MEMBERS NAME: AHAMED M.N.I.

ARACHCHI K.O.K. :160037B INDEX NO: 180018L

AHAMED M.N.I. :180018L COURSE: BSc. ENGINEERING

AMBAWALAGE P.M. :180032A GROUP: G-1

CHATHURANGA R.M.N.A. :180099H DATE OF PER: 03/11/2022

DAYANATH W.M.S :180110L DATE OF SUB: 17/11/2022

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1. OBJECTIVES

• To identify the pattern of variation in enzymatic activity of amylase with

Temperature
• To determine the optimum Temperature for the amylase enzymatic activity

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2. INTRODUCTION
Enzymes are protein molecules that act as catalysts for biological reactions and are widely
used in various industries such as cheese, breweries, bread, fruit juices, and leather amongst
much more. Enzymes lower the activation energy by positioning molecules in the correct
orientation to enable the reaction.
The general theory is that enzymes contain a specifically shaped region called the “active
site” where the substrate binds and undergoes the reaction. Once the reaction is complete,
the product is detached from the active site. Many models such as the lock-and-key model
and induced fit model are prevalent to explain the mechanism of enzymes [1].
The enzymatic activity largely depends on the temperature and pH of the environment.
Temperature can affect the reaction rate in two ways; a rise in temperature corresponds to
a rise in the rate constant whilst high temperatures can cause enzyme denaturing [2]. As
such, there exists an optimum temperature where the enzymatic activity is maximum. This
experiment aims at analyzing the variation of enzymatic activity with temperature and
determining the optimum temperature for amylase.

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3. THEORY
Amylase catalyzes the decomposition of starch into maltose. Thus, a fixed concentration of
starch and amylase is allowed to occur under a series of temperature values for a fixed time
interval. The reaction is then halted by adding Iodine-containing acid. Iodine reacts with any
remnant starch to form a blue complex.
Assuming a proportional relationship between starch concentration and absorbance, the
remnant starch and consequently the reaction rate can be determined using absorbance
measurements.
4. CHEMICALS AND APPARATUS USED
4.1 CHEMICALS
1. 0.1 M HCl Solution
2. Iodine Solution: 5g/liter Iodine and 50g/liter KI
3. Amylase Solution: 13000 IU Amylase/lit
4. Starch Solution: 20g/liter Starch
4.2 APPARATUS
1. Test Tubes
2. 250 ml Volumetric Flask
3. Pipettes (25,10,5,1.0 ml)
4. Measuring Cylinder
5. Test Tubes
6. Spectrophotometer
7. Thermometer
8. Electronic Balance

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5. PROCEDURE
i. A Stopping Solution was prepared by measuring 200.0 ml of 0.1 N HCl and 20.0 ml of
Iodine solution using a measuring cylinder and mixed well. This solution was used to
stop the activity of Amylase in the samples.

ii. 10.0 ml of the stopping solution was added into each of 9 clean and dried test tubes
using a pipette. They were labeled them 1 to 9.

iii. 5 ml of Distilled Water and 1.0 ml of Starch Solution was added into a clean, dry test
tube. This was placed in a temperature-controlled water-bath at 80oC.

iv. After a temperature equalization time of 1 minute, 0.1 ml of Amylase Solution was
added to the test tube and a stopwatch was started whilst the mixture was being
stirred.

v. After 5 minutes, 0.5 ml of the sample was removed from the test tube and added to
Test Tube No. 1.

vi. The above steps were repeated for the remaining temperature values.

vii. The color intensity of the solutions was measured in test tubes No. 1 to 9 using a
Spectrophotometer.

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6
6. CALCULATIONS

Average Absorbance vs Temperature

0.25

0.2

Absorbance (AU) 0.15

0.1

0.05 20, 0.02

0
15 25 35 45 55 65 75 85
Temperature (oC)

Figure 1 - Average Absorbance vs Temperature

7. RESULTS
The minimum absorbance can be observed at an approximate Temperature of 20oC. This implies that
the remnant starch concentration is least at a temperature of 20oC or equivalently, the amylase
activity is highest at a temperature of 20oC

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8. DISCUSSION
8.1 Optimum Amylase Activity in Literature and Comparison of Experimental Value
Generally, Alpha Amylase exhibits optimum behavior at temperatures between 30-60oC as
indicated in Table 1. However, this largely depends on the source and purity of the amylase
sample. Similarly, Beta Amylase is said to exhibit optimum behavior at a temperature of 55oC
[4, p.326]. This too largely depends on the source and purity of the enzyme used.
Table 1 - Optimum Temperature for various Alpha Amylases

Source Optimum pH Optimum Temp. (oC)

Human Ptyalin [3,p.82] 5.6 - 5.9 37
Bovine Ptyalin [3,p.82] 6.5 - 6.6 45
Horse Ptyalin [3, p.82[ 6.2 50
Pancreatin [3,p.82] 7 - 7.2 30 - 50
Krill Amylase [3, p.83] 6.4 35
Tuber Amylase [3, p.87] 5.8 - 6.4 71.5
Almond Amylase [3, p.87] 5.5 52.5
Bacillus Amylase [3, p.88] 6 55 - 60

Now the observed value of 20oC in this experiment seems to not tally with the literature
values. This could be due to a variety of reasons such as incorrect placement of the sample
in the spectrophotometer, human error, and inadvertent mixing of the samples. However,
it is also apparent from Figure 1, that a local minimum does indeed occur between 35oC to
45oC. This is in close correspondence to literature and thus, is a positive outcome.
8.2 Enzymes used in the Food and Biochemical Industry
Enzymes have diverse applications in the food industry. For instance, mainly three enzymes
are used in the bakery industry; Amylase, Protease and Lipase. Amylase is responsible for
expanding the bread volume and enhancing the color of the product. Protease is responsible
for improving the mechanical properties for easier processing whilst Lipase helps improve
the product shelf life [5].
Meanwhile the dairy industry mainly utilizes Catalase to decompose Hydrogen Peroxide and
Lactase to reduce the lactose content of the milk. This directly affects the final product
quality, texture, taste and shelf life. The meat industry utilizes enzymes primarily to improve
product quality (taste, texture, shelf life) and to produce value-added products with
enhanced nutrition. Some enzymes in this regard are, Papain, Bromelain, Transglutaminase,
Protease and Peptin [5].
Similarly, enzymes are used in the fruit and vegetable industry to simplify processes (fruit
peeling, pulping), enhance product quality (clarity, shelf life, easy end-use). Some enzymes
used in this regard are cellulase and amylase. Some industries that utilize enzymes are apple,
lychee, pineapple and tea industry [5].

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8.3 Importance of Determining Optimum Temperature
Enzymes are protein molecules containing “active sites” where the reactant molecule reacts
(See Section 2). The optimum temperature ensures that the reaction happens at the highest
possible rate ensuring maximum product throughput. Deviations from the Optimum
temperature can not only slow down the reaction rate, but can also denaturize the enzyme
structure rendering them ineffective [3]. This can cause financial losses due to substrate
washout, high enzyme purchasing costs and low product throughput.
8.4 Factors affecting Enzymatic Activity
Enzymes catalyze biochemical reactions and this process depends on a number of factors
[6];

• Temperature – Temperature directly affects the reaction rate and is also responsible
for denaturing/decomposing enzymes at extreme values.

• pH – Most enzymes are only active within a narrow pH range of 5 to 7. Extremely

high or low pH can denaturize and deactivate the active sites in the enzyme.

• Enzyme and Substrate Concentration – Only sufficient amounts of enzyme should be

utilized based on the substrate concentration. Adding high amounts of enzyme or
too little substrate can render the process ineffective.

• Activators, Promoters and Poisons

• Light – Photo or UV sensitive enzymes exist and thus, precautions must be taken as
such. Electromagnetic radiation can both enhance reaction rates in some enzymes
whilst decreasing catalytic activity in others.

8.5 Errors and Suggestions

Apart from generic human and experimental errors, it is advisable to ascertain the purity
and source of the Amylase sample. This could have enabled better comparison with data
available in literature.

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References

[1] “Take Online Courses. Earn College Credit. Research Schools, Degrees & Careers.”
Study.com | Take Online Courses. Earn College Credit. Research Schools, Degrees &
Careers, https://study.com/learn/lesson/lock-key-model-vs-induced-fit-model.html.
[2] Gunawardane, Sanja. “Variation of Enzymatic Activity with pH.” Moratuwa, University of
Moratuwa.

[3] Tomasik, Piotr, and Derek Horton. ENZYMATIC CONVERSIONS OF STARCH.

[4] Arendt, Elke K., and Emanuele Zannini. “Millet.” Cereal Grains for the Food and Beverage
Industries, 2013, pp. 312–350., https://doi.org/10.1533/9780857098924.312.

[5] [email protected] (2022) Top 4 application of enzymes in food industry, Infinita

Biotech. Available at: https://infinitabiotech.com/blog/application-of-enzymes-in-the-food-
industry/ (Accessed: November 28, 2022).

[6] Factors affecting enzyme activity- introduction, general features and mechanism (2022)
Embibe Exams. Available at: https://www.embibe.com/exams/factors-affecting-enzyme-
activity/ (Accessed: November 28, 2022).

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