LEARNER GUIDE

Faculty Applied and Computer Sciences

Department Natural Sciences

Course ABMCP3A

Title Microbial Biochemistry 3: Practical

Compiled By Dr. U Terblanche

Year 2024

NQF Level 6

Credits 9
 MODULE INFORMATION

1. Word of Welcome

Welcome to the Department of Natural Sciences.

According to the United Nations Convention on Biological Diversity, Biotechnology is the

use of living systems and organisms to develop or make useful products. Similarly, it
comprises any technological application that uses biological systems, living organisms
or derivatives thereof, to make or modify products or processes for specific use.
(http://www.cbd.int/convention/text/).

The department strives towards integration of existing knowledge with new knowledge
and to afford the learner the ability to:
• Think logically
• Gain knowledge of Biochemical principles in order to make a positive contribution
to the fields of Biotechnology once you have completed your studies.

2. Contact Persons

Title and Surname Office number Telephone number and e-

mail address

Dr. U. Terblanche F 312(h) (016) 950 9614

[email protected]

Mr. G. Thathana F204 (016) 950 9096

[email protected]

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 3. Rationale

Being able to fulfill your role as a well-trained microbial biochemist, it is imperative that
your practical skill set is on par with industry standards and that you are able to apply
your theoretical knowledge in a competent and safe way.
In this course, you will be introduced to basic biochemistry concepts and practical
methodology that spans a wide array of apparatus usage, mathematical calculations,
and scientific writing and reporting.

4. Prerequisites

Chemistry I; Biochemistry II; Biochemistry II: Practical and good comprehension and
usage of English, because the learning medium is English.
Students should be able to communicate about their subject appropriately; cite and
reference work in an appropriate manner. Students should also demonstrate self-
discipline and self-management skills.

5. Assessment Strategy

Assessment in this module occurs continuously through various methods. There will
be one written summative assessment event (exam) during the module,
representing 50% of the final mark. The published assessment date is final. To pass
the semester test, a minimum of 50% is required. The remaining 50% of the final mark
will be based on three to four formal practical reports. Each practical report contributes
to the final mark.

Assessment % Contribution Date

CASS Event 50%
Reports 50%
TOTAL 100%

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 1.5.1 Absent from a Summative Assessment event

In the case of illness, a valid medical certificate should accompany the learner’s
request for a special assessment. The Assessment Office reserves the right to obtain
further information to authenticate such illness.
In the case of death of a first level blood relation member of the family (e.g. father,
mother, child, brother/sister) valid documentation should be submitted.

If you miss a summative assessment event without a valid reason, you will obtain a nil
(0%) mark and will not get a re-assessment opportunity for that subject/module.

1.5.2 Submission of Reports

If you submit an report one day late, 10% of your final mark will be subtracted, two
days late, 20% of your final mark will be subtracted ..…
Very Important: each report is only handed in ONCE. In other words, if you hand in
a sloppy report and get 20% for it, you cannot correct it and hand it in again! Your
first attempt is your final attempt.

1.5.3 Dishonesty

Any dishonesty during the writing of a summative assessment event or a report will be
regarded as a serious offence and can result in a nil mark. It is your own responsibility
to ensure that your answer sheets are submitted at the end of the test period. No late
submission of answers will be accepted.

1.5.4 Plagiarism

It is the responsibility of the leaner that a fellow learner does not copy his/her work. In
the case of plagiarism the assignment mark will be divided between the parties
involved.

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 1.5.5 Re-assessment

A learner, who scores less than 50% for the summative assessment event, may be re-
assessed.

Learners, who failed the reassessment, can reregister for the module the next
semester.

6. Module Plan

Experiment 1 Real-world Titrations Applications

Experiment 2 The presence of key metabolites during Glucose Fermentation

by Yeast
Experiment 3 Enzyme Kinetics of Tyrosinase

Experiment 4 Properties of -amylase

7. Class Attendance

Each practical is compulsory. Everyone should attempt to be punctual in order that the
full time allocate for the practical session.
We do not “give off” classes. It is not allowed to submit a report if you did not conduct
the practical yourself. A practical session is about learning skills in the laboratory. If
you miss a practical session, due to any of the reasons stipulated in 1.5.1, it is your
responsibility to reschedule the session as soon as possible.

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 EXPERIMENT 1: Titration Skills

Introduction
Titrations are crucial in biotechnology, providing essential techniques for analyzing
and quantifying chemical substances. Acid-base titration, iodometric titration, and
back titration are three fundamental types of titrations commonly employed in
biotechnology laboratories. An acid-base titration is used to determine the
concentration of acids or bases in solutions, which is vital for various applications in
biotechnology, such as adjusting pH levels in culture media or assessing the purity of
chemical reagents. Iodometric titration involves redox reactions and measures the
concentration of oxidizing agents, which is essential for studying enzyme kinetics and
oxidative processes in biological systems. Back titration, on the other hand, allows
for determining the concentration of a substance that reacts slowly or incompletely
with the primary titrant, providing valuable insights into reaction kinetics and
equilibrium in biotechnological processes. Understanding these titration techniques is
crucial for biotechnology students as they provide the foundation for accurate analysis
and experimentation in various biotechnological research and applications.

Learning Objectives
1. Understand the principles of acid-base titration and how to perform titrations
accurately.
2. Learn the concept of equivalence point and how to determine it during an acid-
base titration experiment.
3. Gain proficiency in calculating the concentration of an unknown acid or base
solution using titration data.
4. Explore the principles and techniques of iodometric titration.
5. Understand the concept of redox reactions and how they apply to iodometric
titration.
6. Learn how to determine the concentration of an oxidizing agent using
iodometric titration.
7. Develop skills in performing back titrations and understanding their
applications.
8. Gain proficiency in calculating concentrations of substances using back titration
data.
9. Understand the importance of proper experimental techniques and accurate
measurements in titration experiments.
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 10. Analyse and interpret experimental results to draw conclusions about the
concentrations of substances involved in the titration processes.

WEEK 1: Preparation Lab

In this Preparation laboratory session, you are encouraged to work in groups of two
for a collaborative learning experience. During this session, you need to prepare
various chemical solutions. These solutions will be used in the next practical session.
To make the most of your time, please ensure you begin by completing the laboratory
report provided to you. This report will guide you through the necessary calculations
to determine the required masses and volumes to prepare the specified chemicals
accurately. Working together efficiently and following the provided instructions will
contribute to a successful and productive laboratory session.

Prepare 100 ml of the following chemicals:

Sodium Hydroxide 0.1 mol/L
Sodium thiosulfate standard (Na2S2O3) solution, 0.1 mol/L
Sodium hydroxide (NaOH) 0.2 mol/L
Hydrochloric acid (HCl) 0.2 mol/L

8. How to prepare a solution from a solid:

Figure 1: Steps for preparing a solution from a solid (https://quizlet.com/gb/280331984/chemistry-required-practical-1-

make-up-a-volumetric-solution-and-carry-out-a-simple-acid-base-titration-flash-cards/)

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 9. How to prepare a solution from a liquid:

Figure 2: Steps for preparing a solution from a liquid (https://wou.edu/chemistry/courses/online-chemistry-

textbooks/3890-2/ch104-chapter-7-solutions/).

Procedure:

Calculate the weight: Calculate the required mass of the chemical. Write this
calculation in the given laboratory report.
Weigh the solid: Using an analytical balance, accurately weigh the desired
amount of the solid substance (solute) needed to prepare the solution. Record the
mass in grams.
Select the container: Choose an appropriate volumetric flask based on the
volume of the solution you need to prepare. Ensure the container is clean and dry.
Transfer the solid: Carefully transfer the weighed solid substance into the
selected container. To minimize loss, you can use a spatula or funnel.
Add solvent: Using a graduated cylinder or pipette, measure the appropriate
volume of deionized or distilled water (solvent) needed to prepare the desired
concentration. Slowly add the solvent to the container containing the solid. Be
cautious, as some solids may dissolve exothermically (releasing heat). Stir the
mixture continuously with a stirring rod or using a magnetic stirrer to help dissolve
the solid.
Dissolve the solid: Continue stirring the mixture until the solid is completely
dissolved in the solvent. This may take some time, depending on the solubility of
the substance. Ensure no visible solid particles remain.
Adjust the volume: Add more solvent until the desired volume is reached.
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 Mix thoroughly: Stir the solution thoroughly to ensure uniform distribution of the
solute in the solvent. Avoid introducing contaminants or stirring too vigorously,
which may lead to splashing.
Label the container: Clearly label the container with essential information,
including your lab number, the identity of the solute, the concentration of the
solution, the date of preparation, and any hazards associated with the solution.
Storage: Ask the technician to store the solution until it is needed.
Clean up: Dispose of any unused solid material properly and clean all equipment
used to prepare the solution. Follow laboratory waste disposal guidelines.

WEEK 2: Practical work

In this practical session, you must perform a series of different titrations, including an
acid-base titration, an iodometric titration, and a back titration. You need to complete
one of each of the titrations mentioned above. To ensure efficiency and teamwork,
please follow the following instructions carefully:

1. After completing your titrations, please collaborate with two other students to
collect their data for each of the mentioned titrations (you will now have triplicate
data for each).
2. Start working on your laboratory report immediately, including all calculations
and observations. The report should be comprehensive and well-organized.
3. You must submit your completed laboratory report the following day.

A. Acid-base titration

Determination of the free fatty acids in lipids

The acid value of an oil or fat is defined as the weight of sodium hydroxide required
to neutralize the free acidity in 1 g of the sample (Figure 1). The result is often
expresses as the percentage of free fatty acid (FFA). The acid value is a measure of
the extent to which glycerides in the oil has been decomposed by lipase or other
action. The decomposition is accelerated by heat and light. As rancidity is usually
accompanied by free fatty acid formation, the determination is often a general
indication of the condition and edibility of oils. With most oils acidity begins to be palate
when the free fatty acids calculated as oleic acid is about 0.5 – 1.5%. The free fatty
acid is usually calculated as oleic acid (MM = 282 g.mol-1).

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 O O
- +
R1 C OH + NaOH R1 C O Na + H2O

Figure 3: Neutralization of free fatty acid with sodium hydroxide.

Experimental Procedures
Materials
Petroleum ether (Reagent Grade 40-60°C)
Ethanol AR 99.5 % heated.
Phenolphthalein indicator: Dissolve 0.5 g phenolphthalein in 50 ml ethanol and
add 50 ml distilled water.
Sodium Hydroxide 0.1 mol/liter

Method

Set up a burette with 0.1 M NaOH. Remember:

a. the burettes should be clean (no droplets on the inside)
b. no air bubble below the stopcock
c. the stopcock should not be leaking
Weigh 1.00 g extracted oil sample into 250 ml glass Erlenmeyer flask and note the
mass.
Add 50 ml hot ethanol and swirl to dissolve.
Titrate the mixture using the NaOH with phenolphthalein as indicator until a pink
color appears. Shake during the titration and keep the solution warm.
Tabulate your results in table 1.

Table 1: Titration data for the % free fatty acids

Mass of oil (g)

Mass of weighing boat (g)
TITRATION 1 2 3
Final burette reading (ml)
Initial burette reading (ml)
Volume NaOH (ml)
Average volume NaOH (ml)
Mass of free fatty acids (g)
% Yield of free fatty acid

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 B. Iodometric titration

Determination of the peroxide value in lipids

Oils with a high degree of unsaturation (double bonds) are most susceptible to
autoxidation. The best test for autoxidation (oxidative rancidity) is determination of the
peroxide value. Peroxides are intermediates in the autoxidation reaction. During
storage, peroxide formation is slow at first during an induction period, which may vary
form a few weeks to several months according to the particular oil, or fat, the
temperature etc. The peroxide value is determined by the reaction of potassium iodide
in acid solution. Chloroform is normally used as solvent. Titration is done with sodium
thiosulphate in the presence of starch as indicator. The peroxide value is defined as
the amount of peroxide per 1 kilogram of fat or oil. Peroxide values of fresh oils are
less than 10 milliequivalents /kg, when the peroxide value is between 30 and 40
milliequivalents/kg, a rancid taste is noticeable.

Assume: The formation of H2O2 involves the initial stage in oxidizing long-chain fatty
acids and other lipids.

H2O2 + 2H+ + 2I- I 2 + 2H2O

2S2O32- + I2 S4O62- + 2I-

H2O2 + 2H+ + 2S2O32- S4O62- + 2H2O

(thiosulphate ion) (tetrathionate ion)

Figure 4: Reaction between peroxide and thiosulphate

Experimental Procedures
Materials

1. Petroleum ether (Reagent Grade 40-60°C)

2. Glacial acetic acid-chloroform mixture – mix 3 volumes CH3COOH with 2 volumes
CHCl3
3. Iodine Indicator
4. Sodium thiosulfate standard (Na2S2O3) solution, 0.1 mol/l (0.1 N)
5. Saturated Potassium Iodide solution (KI). Store in the dark.

For Na2S203, there is one equivalent of thiosulfate ion (S2O32-) per molecule of sodium
thiosulfate. A 0.1 N solution is the same as a 0.1 M solution.
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 Method

1. Set up a burette with 0.1 N Na2S2O3.

2. Weigh 5.00 g extracted oil sample into 250 ml Erlenmeyer flask and note the mass.
3. Add 30 ml CH3COOH-CHCl3 mixture, and swirl to dissolve.
4. Add 0.5 ml of the saturated KI solution.
5. Let stand with occasional shaking 1 min.
6. Add 30 ml distilled water.
7. Add a spatula tip of iodine indicator.
8. Slowly titrate the oil mixture with 0.1 N Na2S2O3 until the blue just disappears.
9. Conduct blank determination (10 ml chloroform + 15 ml acetic acid + 1.0 ml KI +
30 ml H2O). Add iodine indicator (1 ml) before titrating and titrate drop wise.
10. Subtract form titration.
11. Record your results in table 2.

Table 2: Titration data for the peroxide value

Mass of oil (g)

Mass of weighing boat (g)
TITRATION 1 2 3
Final burette reading (ml)
Initial burette reading (ml)
Volume Na2S2O3 (ml)
Average volume Na2S2O3 (ml)
Peroxide value

(𝑉1 − 𝑉0) 𝑥 𝑁𝑁𝑎2 𝑆2𝑂3 𝑥 1000

𝑃𝑉 =
𝑚𝑎𝑠𝑠 𝑜𝑓 𝑜𝑖𝑙 𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑛 𝑔𝑟𝑎𝑚𝑠

[miliequivalent available oxygen/kg sample ] [meq. / kg]

where:
V1 – volume of thiosulfate solution required to titrate the sample [ml];
V0 – volume of thiosulfate solution required to titrate the blank determination [ml];
N - normality of the sodium thiosulfate solution
m – mass of sample [g]
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 C. Back-titration

Analysis of Antacid Tablets

The stomach has an acidic interior generated by dilute HCl, "stomach acid", which
insures proper digestion. The acidic environment also helps to activate the digestive
enzyme pepsin, by removing a 42-amino acid chain from the inactive form,
pepsinogen.

When the acidity of the stomach becomes high enough to cause discomfort
““heartburn”, brought about by the ingestion of certain types of food, an antacid
preparation can be taken to neutralize the excess stomach acid. The active ingredient
in every antacid is a base, the most common being metal hydroxides, metal
carbonates or a mixture of the two.

The HCl in the gastric juices is neutralize by these active ingredients as the following
reactions show:
NaHCO3 + HCl  NaCl + H2O + CO2
CaCO3 + 2HCl  CaCl2 + H2O + CO2
Al(OH)3 + 3HCl  AlCl3 + 3H2O
Mg(OH)2 + 2HCl  MgCl2 + 2H2O

In this experiment, you will determine the amount of HCl neutralized by two different
commercial antacid tablets. To do so we use a technique called back-titration. An
excess amount of HCl will be added to the antacid tablet. The excess HCl helps to
dissolve the tablet. Some of the HCl will be neutralized by the active ingredient in the
antacid tablet. The remaining HCl is neutralized by sodium hydroxide via titration.

The amount of HCl neutralized by the antacid tablet can then be determined as
followings:

Experimental Procedures
Materials
1. An antacid tablet
2. Sodium hydroxide (NaOH), 0.2 mol/liter
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 3. Hydrochloric acid (HCl), 0.2 mol/liter
4. Thymol blue indicator

Method
1. Set up two burettes: one with 0.2 M NaOH and the other with 0.2 M HCl.
2. Collect two antacid tablets and determine the mass of each tablet.
3. Place each tablet in separate 250 ml Erlenmeyer flasks and add 10 ml water to
each.
4. Add exactly 50 ml 0.2 M HCl to each Erlenmeyer flask from the burette.
5. Dissolve the tablets (some of the inactive ingredients may not the soluble).
6. Add a few drops of thymol blue indictor and stir with a stirring rod. The solution
should be red. If a red colour is not observed, add 10 ml 0.2 M HCl from a refilled
burette and make certain that the red color will persists for more than 30 sec.
7. Record the total volume of 0.2 M HCl added.
8. Slowly titrate the antacid tablet-HCl solution with 0.2 M NaOH until the color
changes to yellow and stay yellow for 30 sec.
9. Record the results in table 3.

Table 3: Titration data for the antacid tablet

Mass of antacid tablet (g)

Mass of weighing boat (g)
Mass of antacid tablet and weighing boat (g)
Total volume of 0.2 M HCl added to antacid tablet (ml)
Final burette reading for 0.2 M NaOH (ml)
Initial burette reading for 0.2 M NaOH (ml)
Total volume 0.2 M NaOH added (ml)
Volume of 0.2 M HCl obtained in back-titration (ml)
= (final – initial burette reading)
Volume of 0.2 M HCl neutralized by the antacid tablet (ml)
= (Total volume HCl – Volume obtained in back-titration)
Gram of HCl neutralized by 1 gram antacid tablet (g)

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 EXPERIMENT 2: The presence of key metabolites during Glucose Fermentation
by Yeast

Introduction:
The glycolysis pathway is a fundamental metabolic process found in nearly all living
organisms, initiating the breakdown of glucose into pyruvate molecules. In aerobic
conditions, pyruvate undergoes oxidative decarboxylation to generate acetyl-CoA,
entering the citric acid cycle for complete oxidation. However, under anaerobic
conditions, alternative pathways are employed, such as fermentation, to extract
energy from glucose. Fermentation involves the conversion of pyruvate into various
end products, including lactic acid and ethanol, depending on the organism and
environmental conditions. In this experiment, we aim to elucidate the fermentation
pathway of glucose by Saccharomyces cerevisiae (yeast) by detecting intermediates
such as pyruvate, acetaldehyde, NADH, and lactic acid. Additionally, we will quantify
the concentration of NADH to gain insights into yeast metabolism under different
physiological conditions.

Objective:
1. To investigate different approaches to demonstrate the presence of
intermediates in a metabolic pathway during glucose fermentation.
2. To grow Saccharomyces cerevisiae under different physiological conditions to
determine the presence of pyruvate, acetaldehyde, and lactic acid during
fermentation.
3. To utilize various chemical tests, including 2,4-dinitrophenylhydrazine and
sodium nitroprusside, to detect the presence of pyruvate and acetaldehyde as
intermediates in the fermentation pathway.
4. To employ a trapping agent, sodium sulfite, to determine the presence of
acetaldehyde during fermentation.
5. To interpret the results obtained from the chemical tests and quantitative assay
to elucidate the metabolic pathways involved in glucose fermentation by yeast.
6. To analyse and compare the metabolic profiles of yeast under different
physiological conditions, providing insights into the regulation of fermentation
pathways and metabolic adaptation.

Experimental Procedures
Materials

1. Disodium hydrogen phosphate Na2HPO4 (0.5 mol/L, pH 10)

2. Potassium dihydrogen phosphate KH2PO4 (0.5 mol/L, pH 4)
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 3. Yeast suspension (100 g/L in Na2HPO4)
4. Yeast suspension (100 g/L in KH2PO4)
5. Yeast suspension (100 g/L in water)
6. 10% glucose (100 g/ L)
7. 5% sodium nitroprusside (50 g/L, prepare just before use)
8. Concentrated ammonia
9. Solid ammonium sulphate
10. Solid sodium sulphite Na2SO3
11. 10% sodium hydroxide NaOH (100 g/L)
12. 2,4-Dinitrophenylhydrazine hydrochloride (saturated solution in 2 M
hydrochloric acid)
13. 3% aqueous piperidine
14. 10% trichloroacetic acid (100 g/L)
15. Phenolphthalein or bromothymol blue

Method

A. Formation of Pyruvate and Acetaldehyde from Glucose:

1. Prepare five test tubes with varying yeast suspensions and glucose solutions
as shown below.

Test tube A B C D E
Glucose solution (ml) 5.0 5.0 5.0 5.0 5.0
Yeast in Na2HPO4 (ml) 5.0 0.0 0.0 0.0 0.0
Yeast in KH2PO4 (ml) 0.0 5.0 0.0 0.0 0.0
Yeast in H2O (ml) 0.0 0.0 5.0 5.0 0.0
Deionized H2O (ml) 0.0 0.0 0.0 0.0 5.0
Solid Na2SO3 (g) 0.0 0.0 0.0 0.5 0.5

2. Cover the test tubes (A – E) with a lid and incubate in a water bath at 37 °C for
1 hour.
3. After the 1 hour incubation, add 2 ml of 10% trichloroacetic acid solution to test
tubes A and B. Mix carefully with a stirring rod.
4. Pour the mixture in test tube A into two centrifuge tubes (equal amounts) and
centrifuge at room temperature for 10 min at 2500 g. Remember to label each
tube clearly.
5. Repeat step 4 for test tubes B – E.
6. Remove the supernatant and do the appropriate tests.

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 B. Test for pyruvate with 2,4-dinitrophenylhydrazine

C. This test should be done with the supernatant of solutions A, B and E only.
D. Add 1 ml of the saturated, 2,4-dinitrophenylhydrazine solution to 2 ml of the
supernatant.
E. Mix thoroughly and place two or three drops of the mixture in a test tube.
F. Add 1 ml of the 10% sodium hydroxide solution and dilute with water to about
5 ml.
G. A red color forms if pyruvate is present.
H. Record the observation in table 1.

C. Test for pyruvate with sodium nitroprusside

1. This test should be done with the supernatant of solutions A, B and E only.
2. Add 2 ml of boiled supernatant to about 1 cm of solid ammonium sulphate in a test
tube.
3. Add two drops of freshly prepared sodium nitroprusside solution (50 g/liter) and
mix thoroughly.
4. Run concentrated ammonia carefully down the side of the tube so as to form two
layers.
5. If pyruvate is present, a green or blue ring form at the junction of the two liquids.
6. A transient pink ring at the junction is due to the presence of thiol groups and this
sometimes appears before the characteristic blue or green color given by pyruvate.
7. Record the observation in table 1.

TABLE 1: Experimental data for identification tests

Identification Tests
Pyruvate
Tube 2,4-Dinitrophenyl Sodium Acetaldehyde Lactic acid
hydrazine nitroprusside

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 E

D. Formation of acetaldehyde form glucose

This test should be done with the supernatant of solutions C, D and E only.
Add 0.5 ml of freshly prepared sodium nitroprusside to 2 ml of the supernatant.
Add 2 ml of aqueous piperidine into each test tube and mix.
If acetaldehyde is present a blue (violet) color is observed. Observations after 2
minutes are invalid.
Record the observation in table 1.

E. Test for Lactic Acid

1. Prepare a set of test tubes, labelling them accordingly for each sample to be tested.
2. Add 2 ml of the sample solution (containing the suspected presence of lactic acid)
into each labelled test tube.
3. Prepare a pH indicator solution by dissolving a suitable pH indicator, such as
phenolphthalein or bromothymol blue, in distilled water according to the
manufacturer's instructions.
4. Add a few drops of the prepared pH indicator solution to each test tube containing
the sample solution.
5. Observe the color change in the sample solution after adding the pH indicator.
Lactic acid is acidic in nature, so it will cause the pH indicator to change colour,
indicating its presence.
6. Compare the observed color change to a pH color chart to determine the
approximate pH of the sample solution, which can indicate the concentration of
lactic acid present.

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 Enzymes

Introduction

An enzyme is a molecule that catalyses or greatly speeds up the rate at which a

chemical reaction occur by lowering the activation energy of the reaction. The
sequence of events for a simple enzymatic reaction involving a single substrate (S)
converted to a single product (P) can be written as:

E + S ES E + P

The speed or rate of the enzyme-catalyzed reaction can be determined by measuring

the amount of product generated or the amount of substrate used up per unit time.

Δ𝑃 −∆𝑆
rate (v) = =
Δ𝑡 Δ𝑡

This is called reaction velocity (v) and is commonly expressed in terms of micromoles
Product/minute (µmole.min-1). Since the rate of enzymatic reactions is of
importance to the overall economy of the cell, the factors that affect the velocity of
enzymatic reactions should be discussed and investigated. These factors include:

1. Enzyme concentration
2. Substrate concentration
3. Presence of inhibitors
4. pH
5. Temperature
6. Presence of cofactors and coenzymes

A. Enzyme concentration

When all the other factors (substrate concentration, temperature, pH, etc.) are held
constant and an enzymatic reaction is done at progressively higher enzyme
concentrations the following graph was observed:

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 Figure 1: Reaction velocity versus enzyme concentration

Increasing enzyme concentration increases the number of collisions between the

enzyme molecules and the substrate molecules. This increases the number of
successful collisions and the number of enzyme-substrate complexes. Therefore the
reaction rate is increased as well and enzyme activity is promoted. Eventually,
increasing the enzyme concentration beyond a certain point has no effect because the
substrate concentration becomes the limiting factor. This is simple to follow because
when most of the substrate has already been acted upon, it leaves only a few to break
down and a lot of enzyme molecules. So the rate of reaction levels out.

B. Substrate concentration

An important factor affecting the rate of an enzymatic reaction is the proportion of the
enzyme molecules that are occupied by substrate molecules at any time. At a low
substrate concentration there are many active sites that are not occupied. This means
that the reaction rate is low. Increasing the substrate concentration increases the rate
of reaction. This is because more substrate molecules will be colliding with enzyme
molecules, so more product will be formed. If only half of the enzymes are occupied,
then the reaction will proceed half as rapidly as when all molecules are saturating the
active sites. Then the enzyme operates at maximum velocity (Vmax). All the active
sites will be saturated so no more enzyme-substrate complexes can be formed and
increasing the substrate concentration further will have no effect on the reaction rate.

The effect of substrate on the reaction velocity is shown by the figure:

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 Figure 2: Effect of substrate concentration on reaction rate, assuming the enzyme concentration is constant.

The proportion of enzymes that are saturated at any fixed substrate concentration will
depend on the affinity of the enzyme for its substrate. An enzyme with a high affinity
will be saturated at lower concentration. The substrate concentration at which only
half of the enzyme molecule are operating, that is, when the velocity (v) of the reaction
Vmax
is half of the maximum velocity ( ) is called the KM (Michaelis constant). A low KM
2
indicates a high attraction between enzyme and substrate and a higher KM is evidence
of a low affinity.

The equation fitting this graph is:

𝑉𝑚𝑎𝑥 +[𝑆]
𝑣= 𝐾𝑀 +[𝑆]

This is the Michaelis-Menten equation.

The values for 𝑉𝑚𝑎𝑥 and KM could be determine graphically from a graph similar to
figure 2, but more precise values may be obtained if the same experimental date are
displayed graphically in a linear plot. The Michaelis-Menten equation has been
manipulated to give the double-reciprocal plot called the Lineweaver-Burk plot.

1 𝐾𝑀 1 1
= x +
𝑣 𝑉𝑚𝑎𝑥 [𝑆] 𝑉𝑚𝑎𝑥
where y= m x + b

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 Figure 3: Lineweaver-Burk plot

C. Presence of inhibitors

An inhibitor is a molecule that interferes with the activity of an enzyme. Enzyme

inhibition can be irreversible or reversible. If enzyme inhibition is irreversible, the
inhibitor forms a strong bond with the enzyme, inactivating it. An irreversible reaction
E + I  EI takes place via a covalent bond, effectively permanently taking the enzyme
out of circulation.

Reversible inhibition involves noncovalent bonding. Three types of reversible

inhibition can be identified, namely competitive, noncompetitive and uncompetitive.
Experimentally, these are distinguished by performing the enzymatic reaction in the
presence of a constant amount of the inhibitor at increasing concentrations of the
substrate. When the inhibited reaction is compared with the normal reaction using the
graphic analyses of the Michaelis-Menten equation the type of inhibition is clearly
indicated.

Figure 4: A Michaelis-Menten plot in the presence of a competitive and noncompetitive inhibitor.

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 o Competitive inhibition

A competitive inhibitor is any compound which closely resembles the chemical

structure and molecular geometry of the substrate. This means that they fit into the
active site, but remain unreacted since they have a different structure to the
substrate. Therefore less substrate molecules can bind to the enzymes so the reaction
rate is decreased. Binding of the inhibitor to the active site on the enzyme prevents
binding of the substrate to the enzyme. In the presence of a competitive inhibitor, it
takes a higher substrate concentration to achieve the same velocities that were
reached in its absence. So while Vmax can still be reached if sufficient substrate is
available, one-half Vmax requires a higher [S] than before and thus Km is larger
(decreased in affinity for substrate).

• Same Vmax
• Larger KM

Figure 5: Michaelic-Menton and Lineweaver-Burk plots in the presence of a competitive inhibitor.

o Noncompetitive inhibition

A noncompetitive inhibitor is not chemically related to the substrate; therefor it does

not prevent the formation of enzyme-substrate complexes. Noncompetitive inhibitors
prevent the formation of enzyme-product complexes so they prevent the substrate
from reacting to form product. A noncompetitive inhibitor binds to a site other than the
active site, called an allosteric site. The net effect of a noncompetitive inhibitor is to
change the shape (3D tertiary structure) of the enzyme and thus the active site, so that
the substrate can no longer interact with the enzyme to give a reaction. Since they do
not compete with substrate molecules, noncompetitive inhibitors are not affected by
substrate concentration. The effect on kinetics is as if the enzyme were less active

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 (Vmax is reduced), but that the affinity for substrate is unaffected (KM remains the same)
since the substrate binding site is not occupied by the noncompetitive inhibitor.

• Reduced Vmax
• Same KM

Figure 6: Lineweaver-Burk plot in the presence of a noncompetitive inhibitor.

D. pH (hydrogen ion concentration)

Most enzymes are active over a limited pH range. Changing the pH alters the enzyme
activity by affecting V, Km or the stability of the enzyme. A plot of activity against pH
usually gives a bell-shaped curve of the type shown in Figure 7.

Figure 7: The effect of pH on and enzyme-catalyzed reaction.

The pH at which an enzyme functions optimally is called the pH optimum. Making the
solution more alkaline or more acidic sharply decreases the rate of the reaction. H+
and OH- ions interfere with hydrogen and ionic bonds that help with the stability of the
tertiary structure of an enzyme. This interference causes a change in shape of the
enzyme, and importantly, its active site. Changes in pH may not only affect the shape

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 of an enzyme but it may also change the shape or charge properties of the substrate
so that either the substrate cannot bind to the active site or it cannot undergo catalysis.
Small changes in pH above or below the optimum do not cause a permanent change
to the enzyme, since the bonds can be reformed. However, extreme changes in pH
can cause enzymes to denature and permanently lose their function.

Thus the effect of pH on the rate of an enzyme-catalyzed reaction is the result of a

combination of factors, namely:

1. the binding of the enzyme to substrate,

2. the catalytic activity of the enzyme,
3. the ionization of the substrate, and
4. the variation in the 3-D shape of the protein.

E. Temperature

Enzymes are also very sensitive to temperature changes as shown in figure 8. At

relative low temperatures, the rate for an enzyme-catalyzed reaction increases
proportionally with increasing temperature. This can be ascribed to an increase in the
kinetic energy that molecules possess. In a fluid, this means that there are more
random collisions between enzyme and substrate molecules, increasing the number
enzyme-substrate complexes which results in more product formation. At the
temperature optimum, the enzyme functions optimally and the rate of the reaction is
maximal.

Figure 8: The effect of temperature of an enzyme-catalyzed reaction.

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 Above the optimum temperature, increasing the temperature also increases the
vibrational energy of the bonds within the enzyme. Bonds such as the weaker
hydrogen and ionic bonds will break as a result of this strain. Breaking bonds within
the enzyme will cause the active site to change shape. Eventually, the enzyme will
become denatured and will no longer function.

In summary, as temperature increases, initially the rate of reaction will increase,

because of increased kinetic energy. However, the effect of bond breaking will become
greater and greater, and the rate of reaction will begin to decrease.

Units of Enzyme Activity

When enzymes catalyse a reaction, we are interested in the amount of product formed
per unit time. This is called the activity.
𝑎𝑚𝑜𝑢𝑡 𝑜𝑓 𝑝𝑟𝑜𝑑𝑢𝑐𝑡
𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 =
𝑡𝑖𝑚𝑒

Usually an enzyme activity if measured in micromole of product per minute. This would
be one unit of activity.
1 𝜇𝑚𝑜𝑙 𝑝𝑟𝑜𝑑𝑢𝑐𝑡
1 𝑢𝑛𝑖𝑡 𝑜𝑓 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (𝑈) =
𝑚𝑖𝑛𝑢𝑡𝑒

Thus when measuring the activity of an enzyme, we are not actually measuring the
number of enzyme molecules, but rather what those enzyme molecules are doing.
The activity is very dependent on reaction conditions such as pH, temperature and the
presence of inhibitors or activators.

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 EXPERIMENT 3: Enzyme Kinetics of Tyrosinase

Introduction

Enzyme kinetics is the study of reaction rates of enzyme-catalyzed reactions. By

observing how reaction rates are affected by the concentration of enzyme, substrate,
inhibitors and activators, we will begin to understand the nature of the reaction.

Tyrosinase, also called polyphenol oxidase, is present in plant and animal cells. In
mammalian cells, it catalyzes two steps in the synthesis of melanin pigments from
tyrosine. It is located in the skin and is activated by UV light from the sun, leading
ultimately to a suntan. Tyrosinase is also involved in the browning of fruits, tubers and
fungi that have been damaged. As shown in figure 9, in the presence of molecular
oxygen (O2), tyrosinase catalyzes the hydroxylation of tyrosine to form 3,4-
dihydroxyphenylalanine (DOPA). Tyrosinase then catalyzes DOPA into dopaquinone
which spontaneously converts to dopachrome. Dopachrome will eventually be turned
into melanin. The conversion of tyrosine to L-DOPA is the rate-limiting step in this
biochemical process. This means the reaction is the slowest. Dopachrome is a
colored compound with a maximum absorbance at 475 nm. Mushroom tyrosinase is
tetrameric, with a molecular weight of 128 000.
-
-
OOC CH NH3+ OOC CH NH3+
CH2 CH2

Tyrosinase Tyrosinase O COO- O

OH NH3+ NH
O2 O O
OH OH
Tyrosine DOPA Dopaquinone Dopachrome
(3,4-dihydroxyphenylalanine) max = 475 nm

Figure 1: The tyrosinase reaction.

Objectives

The objectives of this experiment are:

1. To prepare a tyrosinase-containing mushroom crude extract through
homogenization and filtration of white button mushrooms
2. To determine the optimal amount of enzyme (tyrosinase-containing mushroom
extract) for the tyrosinase reaction.
3. To determine the effect of increasing substrate concentration on the initial
reaction rate of the tyrosinase-catalyzed reaction.
4. To construct different plots of velocity versus [S] and to estimate Km and Vmax
from the graphs.
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 5. To determine the effect of inhibitors on the rate of the tyrosinase-catalyzed
reaction.

Experimental Procedures

Materials
1. White button mushrooms
2. Disodium hydrogen phosphate
3. Potassium dihydrogen phosphate
4. Sodium phosphate buffer, 10 mM, pH 6.0
5. Tyrosinase-containing Mushroom extract. Always keep your enzyme on ice.
6. L- DOPA, 15 mM
7. Benzoic acid (3 mM in sodium phosphate buffer, pH 6.8)
8. Ascorbic acid (3 mM in sodium phosphate buffer, pH 6.8)

Methods
A. Preparation of a crude tyrosinase-containing mushroom extract

In this part of the experiment, you will compare the crude tyrosinase-containing
mushroom extract. Remember to always keep the extract in the dark and on ice.
1. Weigh out 30 g of white button mushrooms.
2. Rinse the mushrooms with distilled water twice.
3. Add 500 ml of a 10 mM phosphate buffer (pH 6.0) to the mushrooms and
homogenize for 1 minute at a high speed.
4. Filter the homogenate through Whatman #1 filter paper using a Buchner funnel.
5. Immediately place the filtrate on ice.
6. Aliquot the filtrate into several tubes and store the aliquots at -15°C until
needed.

B. Determine the optimal amount of enzyme for the tyrosinase reaction

Due to the unique nature of each batch of mushroom extract and the unknown
concentration of tyrosinase in the extract, the amount of extract needed for the assays
must first be determined. For this part of the experiment, the entire practical group
will work as a team. Each student will only run two assays (both in duplicate). The
results will be given to the entire practical group. For the assays, you would need four
disposable cuvettes.

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 1. Assays should be prepared in disposable plastic cuvettes.
2. Set up cuvettes (in duplicate) as follows:

Phosphate DOPA Distilled Crude extract

Cuvette
buffer (µL) 10 mM (µL) water (µL) (µL)
1 600 1000 1350 50 Do not add!
2 600 1000 1300 100
3 600 1000 1250 150
4 600 1000 1200 200
5 600 1000 1150 250
6 600 1000 1100 300
7 600 1000 1000 400
8 600 1000 900 500
9 600 1000 800 600
10 600 1000 600 800

3. Warm up the spectrophotometer and set the wavelength at 475 nm.

4. Cut small squares of Parafilm.
5. Zero the spectrophotometer at 475 nm with the blank.
6. Pipet the appropriate amount of the crude tyrosinase-containing extract into the
first cuvette (e.g., cuvette 1) containing the reaction mixture. Immediately cover
with Parafilm, mix by inversion, and read the change in absorbance at 475 nm
IMMEDIATELY.
7. Note the exact time that the enzyme is added. Read the absorbance value
15 seconds after the enzyme was added. Read the absorbance value for
3 minutes, recording at 15 second intervals.
8. Repeat the step 6 for the remaining cuvettes.
9. Plot a graph of absorbance versus reaction time for volume mushroom extract
used.
10. Calculate the rate of each reaction. Remember that the rate of the enzyme-
catalyzed reaction can be determined by measuring the amount of product
generated per unit time.
𝛥𝑃 ∆𝐴
𝑟𝑎𝑡𝑒 (𝑣) = =
𝛥𝑡 ∆𝑚𝑖𝑛

11. Plot a graph of product concentration (Dopachrome concentration µmol/min)

against the volume of the mushroom extract. The changes in absorbance per
minute must be converted to change in µmol dopachrome (product) using
Beer’s law and the extinction coefficient for dopachrome which is 3600 M-1ml-1
at 475 nm.

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 Table 1: Absorbencies at 15 second intervals for varying tyrosinase-containing
mushroom extract.

Mushroom Absorbance
extract 0s 15s 30s 45s 60s 75s 90s 105s 120s 135s 150s 165s 180s
(µl)
50
75
100
125
150
175
200
250
300
500
600
800

Table 2: Reaction rates for different volumes of tyrosinase-containing mushroom

extract
Reaction rate in:
Mushroom extract
Cuvette ∆𝑨 µ𝐦𝐨𝐥 𝐩𝐫𝐨𝐝𝐮𝐜𝐭
(µL)
∆𝒎𝒊𝒏 𝒎𝒊𝒏
1 50
2 75
3 100
4 125
5 150
6 175
7 200
8 250
9 300
10 500
11 600
12 800
∆𝑨
= “units of enzyme activity”
∆𝒎𝒊𝒏

C. Effect of increasing substrate concentration on initial reaction rate

After determining the appropriate volume of the tyrosinase-containing mushroom
extract, the kinetic parameters Km, and Vmax of the mushroom tyrosinase enzyme can

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 now be determined. In this part, the concentration of substrate will vary, but the
enzyme concentration will be constant.
1. Set up a protocol as before by preparing cuvettes as set out below.

Phosphate DOPA Distilled water Crude extract

Cuvette
buffer (µL) 10 mM (µL) (µL) (µL)
1 600 100 2050 250 Do not add
2 600 200 1950 250
3 600 300 1850 250
4 600 400 1750 250
5 600 500 1650 250
6 600 750 1400 250
7 600 1000 1150 250
8 600 1300 850 250
9 600 1500 650 250

2. Pipet the L-DOPA and phosphate buffer in each test tube, but DO NOT ADD
THE TYROSINASE.
3. Mix by gentle inversion using Parafilm to cover the top of the test tube.
4. When you are ready to assay your first reaction, transfer the reaction mixture,
without the enzyme, to a cuvette (this is the blank).
5. Zero the spectrophotometer at 475 nm with the blank.
6. Pipet the appropriate amount of the crude tyrosinase-containing extract into the
first cuvette (e.g., cuvette 1) containing the reaction mixture. Immediately cover
with Parafilm, mix by inversion, and read the change in absorbance at 475 nm
IMMEDIATELY.
7. Note the exact time that the enzyme is added. Read the absorbance value 15
seconds after the enzyme is added. Read the absorbance value for 3 minutes,
recording at 15 second intervals. (Tabulate the results in table 3).

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 Table 3: Absorbencies at 15 second intervals for varying substrate concentrations.

Cuvette Absorbance at 475 nm

Start 15s 30s 45s 60s 75s 90s 105s 120s 135s 150s 165s 180s
1
2
3
4
5
6
7
8
9

8. Repeat steps 2-7 for the other substrate concentrations. Remember to add the
enzyme immediately before reading each cuvette!
9. Calculate the number of units of enzyme activity for the varying amount of
substrate. (Tabulate the results in table 6).
10. Calculate the substrate concentration in the cuvettes at start of the reaction.
(Tabulate the results in table 4).

Table 4: Reaction rates and different substrate concentrations for the tyrosinase
reactions.

DOPA Reaction rate in:

L-DOPA
Cuvette 10 mM ∆𝑨 µ𝐦𝐨𝐥 𝐩𝐫𝐨𝐝𝐮𝐜𝐭
(mM)
(µL) ∆𝒎𝒊𝒏 𝒎𝒊𝒏
1 100
2 200
3 300
4 400
5 500
6 750
7 1000
8 1300
9 1500

11. Plot a Michaelis-Menten graph. Plot v (µmol/min) vs. [S].

12. Plot a Lineweaver-Burk plot to determine Km and Vmax.

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 D. Effect of inhibitors, anisic acid and benzoic acid, on the rate of reaction
of tyrosinase

In this part, the effect of two inhibitors, ascorbic acid and benzoic acid on the kinetic
parameters Km and Vmax of tyrosinase will be determined. You should do the
experiment only with one inhibitor. Select a partner in the laboratory that will do this
experiment using the other inhibitor. Thereafter, collect the results and use the data in
your own report.

1. Set up a protocol as before by preparing six test tubes as set out below.
DOPA Crude
Phosphate Inhibitor Distilled water
Cuvette 10 mM extract
buffer (µL) (µl) (µL)
(µL) (µL)
1 600 100 300 1750 250
2 600 200 300 1650 250
3 600 300 300 1550 250
4 600 400 300 1450 250
5 600 500 300 1350 250
6 600 750 300 1100 250
7 600 1000 300 850 250
8 600 1300 300 550 250
9 600 1500 300 350 250

2. Cut six small squares of Parafilm.

3. Pipet the L-DOPA, phosphate buffer and inhibitor in each test tube, but DO
NOT ADD THE TYROSINASE.
4. Mix by gentle inversion using Parafilm to cover the top of the test tube.
5. When you are ready to assay your first reaction, transfer the reaction mixture,
without the enzyme, to a cuvette (this is the blank).
6. Zero the spectrophotometer at 475 nm with the blank.
7. v into one cuvette (e.g. cuvette 1) containing the reaction mixture. Immediately
cover with Parafilm, mix by inversion and read the change in absorbance at 475
nm IMMEDIATELY.
8. Note the exact time that the enzyme is added. Read the absorbance value 15
seconds after the enzyme is added. Read the absorbance value for 3 minutes,
recording at 15 second intervals. (Tabulate the results in table 5).

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 Table 5: Absorbencies at 15 second intervals in the presence of an inhibitor.

Cuvette Absorbance at 475 nm

Start 15s 30s 45s 60s 75s 90s 105s 120s 135s 150s 165s 180s
1
2
3
4
5
6
7
8
9

9. Repeat steps 4-8 for the other substrate concentrations. Remember to add the
enzyme immediately before reading each cuvette!
10. Calculate the number of units of enzyme activity for the varying amount of
substrate. (Tabulate the results in table 6).
11. Calculate the substrate concentration in the cuvettes at start of the reaction.
(Tabulate the results in table 6).

Table 6: Reaction rates and amount of enzyme for different tyrosinase reactions in the
presence of an inhibitor.

DOPA Reaction rate in:

L-DOPA
Cuvette 10 mM ∆𝑨 µ𝐦𝐨𝐥 𝐩𝐫𝐨𝐝𝐮𝐜𝐭
(mM)
(µL) ∆𝒎𝒊𝒏 𝒎𝒊𝒏
1 100
2 200
3 300
4 400
5 500
6 750
7 1000
8 1300
9 1500

Plot a Michaelis-Menten graph on the same “Michaelis-Menten graph” from part C.

Estimate Km and Vmax in the presence of an inhibitor from the graph.

Km from Michaelis-Menten graph _________

Vmax from Michaelis-Menten graph _________
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 Plot a Lineweaver-Burk plot on the same “Lineweaver-Burk plot” from part C and
determine Km and Vmax in the presence of an inhibitor.

Km from Lineweaver-Burk plot _________

Vmax from Lineweaver-Burk plot _________

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 EXPERIMENT 4: Properties of -amylase

Introduction

Amylases are enzymes that catalysis the hydrolysis of α(14)glycosidic bonds in

starch or glycogen. Amylases are produced by a variety of living organisms, ranging
from bacteria to plants and humans. Bacteria and fungi secrete amylases to the
outside of their cells to carry out extracellular digestion. When they have broken down
the insoluble starch, the soluble end products such as (glucose or maltose) are
absorbed into their cells.

Maltose is a reducing sugar because it has a free aldehyde group that can reduce
Fehling's and Benedict's reagents. Maltose can be used as a standard for estimating
reducing sugar in unknown samples, such as for the breakdown of starch by -
amylase. Constructing a standard curve / graph for maltose helps us to estimate
concentration of reducing sugars present in an unknown sample and for determining
the activity of amylase enzyme in forthcoming experiments. The standard curve for
maltose is usually constructed using 3,5-dinitrosalicylic acid (DNS) as the reagent.
Maltose reduces the pale yellow coloured alkaline 3,5-dinitrosalicylic acid (DNS) to the
orange-red coloured, 3-amino-5-nitrosalicylic acid.

COO- COO-
OH OH
+ Reducing sugar + Oxidised sugar
O2N NO2 O2N NH2

3,5-Dinitrosalicylic acid 3-Amino-5-nitrosalicylate

max = 540 nm
Figure 2: The DNS method

The intensity of the colour is proportional to the concentration of maltose present in

the solution. This intensity change in colour is measured using a spectrophotometer
at 540 nm.

Change the pH alters the enzyme activity by affecting V, Km., or the stability of the
enzyme protein. If the enzyme is unstable at certain pH values, then the optimum pH
is no longer a characteristic of the enzyme.

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 Objectives

The objectives of this experiment are:

1. To construct a standard curve (calibration curve) for maltose.
2. To determine the amount of maltose molecules produced from the hydrolysis
of starch at various pH values
3. To explain the effect of pH on -amylase.
4. To determine the effect of temperature of the activity of -amylase.

Experimental Procedures

Materials
1. Sodium phosphate buffer, 0.1 M, pH 6.7
2. Buffered starch substrate (5 g/liter in phosphate buffer): Mix 5 g of soluble
starch to a smooth paste with about 50 ml of buffer solution. Add this
quantitatively to 500 ml of boiling phosphate buffer solution, continue to boil for
1 min, the cool to room temperature, and dilute to 1 liter with buffer solution.
3. Sodium chloride (10 g/liter)
4. α-Amylase (0.6 mg/ml)
5. Sodium hydroxide (2 mol/liter)
6. Sodium potassium tartrate (Dissolve 300 g of this salt in about 500 ml of water)
7. 3,5-Dinitrosalicylate acid (Dissolve 10 g of this reagent in 200 ml of 2 M sodium
hydroxide)
8. DNS (dinitrosalicylate) reagent (Prepare this fresh by mixing solutions 6 and 7
and making it up to 1 liter with water.
9. Maltose (4 µmol/ml)

Methods
A: Construct a standard curve

In this part of the experiment, you will construct a standard curve (calibration curve)
for maltose. This calibration curve can be used to determine the concentration of a
maltose sample with an unknown concentration.

1. Prepare duplicate maltose solutions containing 0; 0.2; 0.4; 0.8; 1.2; 1.8; 2.8; 3.2
and 4.0 µmol/ml by appropriate dilution of the standard maltose solution.
2. Tabulate your dilution procedure in table 1.

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 Table 1: Absorbencies for varying maltose concentrations.
Maltose Maltose Absorbance
Water Average
solution concentration
(ml) (1) (2) absorbance
(ml) (µmol/ml)
0.0 5.0 0.0 blank
0.2
0.4
0.8
1.2
1.8
2.8
3.2
5.0 0.0 4.0

3. Warm up the spectrophotometer and set the wavelength at 540 nm.

4. Pipette 1 ml of each of the prepared maltose solutions into a test tube.
5. Pipette 1 ml DNS reagent to all the tubes.
6. Incubate all the test tubes in a boiling water bath for 5 minutes and cool to room
temperature.
7. Add 10 ml distilled water to each tube and mix well.
8. Zero the spectrophotometer at 540 nm with the blank.
9. Read the absorbance at 540 nm and record the data in table 1.
10. Calculate the average absorbance for each maltose concentration (µmol/ml).
11. Plot a standard curve (calibration cure) for maltose. Plot absorbance vs.
[maltose] (µmol/ml.

B: The effect of pH on -amylase

In this part of the experiment, you will work in a group of two. The amount of product
(maltose) produced after incubating starch at various pH values with -amylase will
be determined. The amount of product can be determined from the calibration curve
of maltose.

1. Prepare buffered starch solutions with a pH of: 3.0; 4.0; 5.0; 5.5; 6.0; 6.5; 7.0;
7.5; 8.0; 9.0 and 11.0. Each student in the laboratory can prepare enough on
one buffer starch solution with a given pH value.
2. Each group will prepare (for each pH value) three test tubes, as set out below.

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 Test Tube Blank A B
Buffered Starch at pH 3.0 (ml) 0.5 0.5 0.5

Water (ml) 0.5 0.0 0.0

α-Amylase 0.0 0.5 0.5 Do not add

Total volume (ml) 1.00 1.00 1.00

3. Mark the test tubes clearly. Remember to write the pH value and the number
on each test tube.
4. Pipet the buffered starch and water in the test tubes, but DO NOT ADD THE
α-AMYLASE!!!
5. Equilibrate a test tube with 13 ml α-amylase and the test tubes containing the
reaction mixtures for 5 minutes in a water bath at 25C.
6. When you are ready to add the α-AMYLASE, one member of your group should
handle the pipet and the other should note the time that you add α-amylase to
each test tube. Remember α-amylase is added only to test tube A and B!!!
7. Add α-amylase to the test tubes labeled A serially. This is a crucial part in the
experiment so you need to pay attention. When you pipet 0.5 ml α-amylase to
the first test tube, your partner should start the timer. Thereafter, he/she should
tell you at timed intervals (e.g. every 15 seconds) to pipet 0.5 ml α-amylase to
the next test tube. DO NOT STOP THE TIMER!!!

Start 15s 30s 45s 60s 75s 90s 105s 120s 135s 150s 165s 180s

8. After exactly 5 minutes, add 1 ml DNS reagent at the same time interval (e.g.
every 15 seconds) to the first test tube labeled A.

5:00 5:15s 5:30s 5:45s 6:00s 6:15s 6:30s etc.

9. Repeat steps 3-8 for the test tubes labeled B.

10. Incubate all the test tubes in a boiling water bath for 5 minutes and cool to room
temperature.
11. Add 10 ml distilled water to each tube and mix well.
12. Zero the spectrophotometer at 540 nm with the blank. Remember each pH
value has its own blank.
13. Read the absorbance at 540 nm and record the data in table 2.

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 Table 2: Absorbencies at various pH values for α-amylase.

Absorbance Product Number of

Average Enzyme
pH concentration moles of
absorbance activity
(1) (2) (µmol/ml) product
3.0
4.0
5.0
5.5
6.0
6.5
7.0
7.5
8.0
9.0
11.0

14. Calculate the average absorbance at each pH value and determine the amount
of product (µmol/ml) produced from the calibration curve of maltose.
15. Calculate the enzyme activity for α-amylase at each pH value and plot a graph
of enzyme activity vs pH.

𝜇𝑚𝑜𝑙 𝑚𝑎𝑙𝑡𝑜𝑠𝑒 𝑟𝑒𝑙𝑒𝑎𝑠𝑒𝑑

𝐸𝑛𝑧𝑦𝑚𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (𝑈) =
5 𝑚𝑖𝑛
= 𝜇𝑚𝑜𝑙/𝑚𝑖𝑛

Questions

1. What is the optimum pH for -amylase?

2. How would you calculate specific activity for α-amylase at a pH of 6.5?

C: Determination of the effect of temperature on -amylase

In this part of the experiment you will work in a group of two. Each group will do the
experiment at only one given temperature, but in duplicate. To complete the report,
data for other temperatures should be collected from the reset of the class.

1. Each group will prepare, in duplicate, eight three test tubes, as set out below.

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 Test Tube Blank A B C D E F G

Buffered starch (ml) 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5

Phosphate buffer (ml) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Sodium chloride (ml) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

Time incubated (min) - 0 5 10 15 20 30 40

2. Mark the test tubes clearly.

3. Pipet the buffered starch, buffer and sodium chloride solution into the test tubes,
but DO NOT ADD THE α-AMYLASE!!!
4. Equilibrate a test tube with 6 ml α-amylase in a water bath at 37C for 10
minutes.
5. Place the test tubes (A – G) containing the reaction mixture at the given
temperature for 10 minutes.
6. Pipet 0.5 ml enzyme into test tubes A – G and 0.5 ml water into the blank. Note
the time that the enzyme is added and start the timer.
7. Immediately add 0.5 ml of a 2 M sodium hydroxide solution to test tube A.
8. Incubate the remaining test tubes for 5, 10, 15, 20, 30 and 40 minutes at the
given temperature.
9. After 5 minutes stop the reaction by adding 0.5 ml of a 2 M sodium hydroxide
solution.
10. Repeat the step 9 after 10, 15, 20, 30 and 40 minutes.
11. Pipette 1 ml DNS reagent to all the tubes.
12. Incubate all the test tubes in a boiling water bath for 5 minutes and cool to room
temperature.
13. Add 10 ml distilled water to each tube and mix well.
14. Zero the spectrophotometer at 540 nm with the blank.
15. Read the absorbance at 540 nm and record the data in table 3.

Table 3: Absorbencies at various temperatures for α-amylase.

Temp Absorbance
(C) 0 min 5 min 10 min 15 min 20 min 30 min 40 min
15
25
35
45
55
65
75

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 16. Plot the absorbance of each solution against the time of incubation and prepare
a progress curve of the reaction.
17. Collect the data from the other groups and plot a graph of absorbance against
temperature at a specific time (say 5 minutes).
18. Determine the initial velocity of α-amylase from the progress curve for each
temperature.

Table 4: Reaction rates of α-amylase at different temperatures.

Temp ∆𝑨
(C) ∆𝒎𝒊𝒏
15
25
35
45
55
65
75

19. Plot a graph of initial velocity against temperature.

Questions

1. What is the optimum temperature of α-amylase?

2. Does your optimum temperature compare favorably with those found in the
literature?
3. Explain why enzymes are denaturated at high temperatures.

References
1. FARRELL, S.O. & RANALLO, R.T. 2000. Experiments in Biochemistry: A
Hands-on Approach. Australia: Brooks/Cole Publishing Company.
2. PLUMMER, D.T. 1987. An Introduction to Practical Biochemistry. 3rd ed.
London: McGraw-Hill Book Company
3. Unknown 2012a. Amylase, Alpha Assay. [Online]. Available at:
<http://www.worthington-biochem.com/aa/assay.html> Accessed: 28/06/2012.

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 4. Unknown 2012b. Amylase, Alpha Assay. [Online]. Available at:
<http://prr.hec.gov.pk/Chapters/131S-3.pdf> Accessed: 28/06/2012.

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