AG69010 Food Analysis Laboratory

Experiment 5- Enzymatic Activity in Food

Submitted by

Prashant Kumar (21AG63R26)

MTech. FPE 2021-23

Submitted
To

Prof. P Srinivasa Rao

Department of Agricultural and Food Engineering

Indian Institute of Technology Kharagpur

Experiment – 5
 Enzymatic Activity in Food
Aim: To determine the enzymatic activity of food
Objective: Spectrophotometric Assay of PPO and POD Activity Determination
Theory
Enzymes are special class of proteins that act
as biological catalysts (biocatalysts). Catalysts accelerate chemical reaction.
Enzymes speed up the reaction by lowering down the activation energy of the
reaction. The molecules upon which enzymes may act are called substrate, and
form enzyme-substrate complex. The enzyme converts the substrates into
different molecules known as products.
Particular type of Enzymes binds the particular type of substrate (reactant) on
its active site and hold them in particular orientation that maximizes the
chance of particular reaction to occur and form products.

Fig- mechanism of enzyme activity

• 1000s of amino acid are arrange in specific ways to form different type
of enzyme.
• Non- protein enzyme is Ribozymes.
• Enzymes without co-factor is known as Apoenzymes.
• Enzymes with co-factor is called Holoenzymes.
• Co-factors of enzyme are
I. Prosthetic groups- always with the enzyme (means before, at and
after the reaction).
II. Coenzymes- comes at the time of reaction and make holoenzyme,
then reaction take place.
III. Metal ions- 𝑍𝑛2+ ….
Basic theory of enzyme action.
1. Key and lock fit- This suggests that the substrate is completely
complementary in shape to the active site, so that it fits in 'perfectly' -
i.e. the way a key (the substrate) fits into a lock (the enzyme). There is
no change in shape of the active site when the substrate binds.
2. Induced fit model- The induced fit model maintains that enzyme
substrates are not shaped perfectly to the active sites of their respective
enzymes before binding occurs (the opposite was the lock and key
model, proposed by a man named Emil Fisher; this model was widely
accepted for quite a while, but did not explain the changes that occurred
during the process of catalysis).
According to the model, the interactions between the substrates and
binding sites are relatively weak at first. The initial binding of the
substrate causes change in the conformation of the binding site, causing
it to become the correct shape to bind with the rest of the substrate.
This is also sometimes called the hand-in-glove model, due to the
analogy of a hand changing the shape of a glove as it is put on,
progressively making it easier for the hand to fit inside.

Factors affecting enzyme activity in the cell

▪ Concentration of Enzymes and Substrates: The rate of reaction increases
with increasing substrate concentration up to a point, beyond which any
further increase in substrate concentration produces no significant
change in reaction rate. This occurs because after a certain
concentration of the substrate, all the active sites on the enzyme are full
and no further reaction can occur.
 ▪ Temperature: With the increase in temperature, the enzyme activity
increases because of the increase in kinetic energy of the molecules.
There is an optimum level when the enzymes work at the best and
maximum. This temperature is often the normal body temperature of
the body. When the temperature increases beyond a certain limit,
enzymes, which are actually made up of proteins, begin to disintegrate
and the rate of reaction slows down.
▪ pH: Enzymes are very sensitive to changes in the pH and work in a very
small window of permissible pH levels. Below or above the optimum pH
level, there is a risk of the enzymes disintegrating and thereby the
reaction slows down.
▪ Inhibitors: Presence of certain substances that inhibit the action of a
particular enzyme. This occurs when the inhibiting substance attaches
itself to the active site of the enzyme thereby preventing the substrate
attachment and slows down the process
Different types of enzymes.
1. Oxidoreductases- These enzymes bring about oxidation and reduction
reactions and hence are called oxidoreductases. In these reactions,
electrons in the form of hydride ions or hydrogen atoms are transferred.
When a substrate is being oxidized, these enzymes act as the hydrogen
donor. These enzymes are called dehydrogenases or reductases. When
the oxygen atom is the acceptor, these enzymes are called oxidases.
Examples - Glucose oxidase and Ascorbic oxidase
2. Transferases- These enzymes are responsible for transferring functional
groups from one molecule to another. Example: alanine
aminotransferase which shuffles the alpha‐amino group between
alanine and aspartate etc. Some transferases also transfer phosphate
groups between ATP and other compounds, sugar residues to form
disaccharides such as hexokinase in glycolysis.
3. Hydrolases- These enzymes catalyse reactions that involve the process
of hydrolysis. They break single bonds by adding water. Some hydrolases
function as digestive enzymes because they break the peptide bonds in
proteins. Hydrolases can also be a type of transferases as they transfer
the water molecule from one compound to another.
Example: Glucose-6-phosphatase that removes the phosphate group
from glucose-6-phosphate, leaving glucose and H3PO4
4. Lyases- These enzymes catalyze reactions where functional groups are
added to break double bonds in molecules or where double bonds are
formed by the removal of functional groups. Example: Pyruvate
decarboxylase is a lyase that removes CO2 from pyruvate.
 5. Isomerases- These enzymes catalyze the reactions where a functional
group is moved to another position within the same molecule such that
the resulting molecule is actually an isomer of the earlier molecule.
Example: triosephosphate isomerase and phosphoglucose isomerase for
converting glucose 6-phosphate to fructose 6-phosphate
6. Ligases- These enzymes perform a function that is opposite to that of the
hydrolases. Where hydrolases break bonds by adding water, ligases form
bonds by removal of the water component. There are different
subclasses of ligases which involve the synthesis of ATP.

Spectrophotometric Assay of PPO and POD Activity Determination

Polyphenol oxidase (PPO)- PPO is a copper-containing enzyme which is
probably present in all plants. It is involved in the biosynthesis of melanins in
animals and in the browning of plants. The enzyme catalyzes the oxidation of
phenolic compounds to form corresponding quinone intermediates which
polymerize to form undesirable pigment.
It catalyzes two types of the oxidative reaction involving molecular oxygen
I. The hydroxylation of monophenols to o-diphenols
II. The oxidation of o-diphenols to o-quinones, which lead to the formation
of black or brown pigments
Peroxidase (POD)- It is an oxidoreductase that is directly involved in many
plant functions such as hormone regulation, defense mechanisms, indolacetic
degradation and lignin biosynthesis.
It catalyzes a reaction in which hydrogen peroxide acts as the acceptor and
another compound acts as the donor of hydrogen atom.
POD involved in enzymatic browning since diphenols may function as reducing
substrate in this reaction.
The involvement of POD in browning is limited by the availability of electron
acceptor compounds such as superoxide radicals, hydrogen peroxide and lipid
peroxidase.
Principle:
Peroxidase (POD) activity is assayed using Catechol as hydrogen donor and
𝐻2 𝑂2 as electron acceptor. The rate of formation of brown colored oxidised
catechol product is a measure of the POD activity and can be assayed
spectrophotometrically at 420 nm.

Polyphenol oxidase (PPO) catalyzes the oxidation of ortho-phenols to ortho-

chinons. The chinons polymerize and build colored complexes, whose increase
in concentration can be determined by measuring the absorbance of the
sample at wavelength of 420 nm.
Here catechol (pyrocatechol) will be used as o-diphenol and the oxidized
product will be the quinone.

Material required-
• Food Sample
 • Weighing balance
• Cooling centrifuge
• Spectrophotometer
• Lab accessories (eppendorf, spatula, tissue paper, cuvette, etc.)

Chemicals and Reagents required-

❖ 0.1 M catechol solution. (Prepare by adding 1.1 g in 100 ml phosphate
buffer pH 7).
❖ 5% catechol solution. (Freshly prepared and filtered using whatman
paper no. 1).
❖ 0.5% 𝐻2 𝑂2 solution. (Freshly prepared).
❖ Buffer - 0.2 M Na-K phosphate buffer pH 6.8.
❖ 1% Polyvinyl Pyrrolidone (PVP).
❖ 0.1% triton X (non-ionic surfactant).
Method
Enzyme extraction
• Take Banana flesh and banana peel and make it homogenous paste.
• Take 1g of paste in flask (20ml) with 10 ml of 0.2M phosphate buffer,
containing 1% PVP and 0.1% triton X.
• Vortex the content of microtube & centrifuge at 14000 rpm for 30 min at
4°C and collect the supernatant for assay.
Enzyme assay
• We pipette out the corresponding amount of reagent solution into
macro cuvette according to Table and measure its absorbance at 420 nm
in spectrophotometer against a blank for 15 min at regular interval of 10
sec.
Table- Assay conditions for measuring PPO and POD activities
Calculation- Draw the Absorbance vs Time curve and calculate the enzyme
activity using the slope of the linear portion of the curve. Express the enzyme
activity as change in absorbance per unit time-per g of sample.
• Draw the plot of data.
• Select the straight-line portion.
• Plot the data with initial straight-line curve.
• Fit the data into straight line.
• Get the equation of straight line.
• Report the slope of line as enzyme activity.
• Compare the enzyme activities of different samples.
Data table
Min S1 s2
0 0.01 0.01
0.167 0.021 0.056
0.333 0.049 0.072
0.5 0.083 0.09
0.667 0.118 0.108
0.833 0.156 0.127
1 0.194 0.147
1.167 0.231 0.167
1.333 0.266 0.187
1.5 0.301 0.207
1.667 0.334 0.224
1.833 0.368 0.238
2 0.4 0.251
2.167 0.429 0.265
2.333 0.458 0.28
2.5 0.486 0.294
2.667 0.512 0.308
2.833 0.537 0.323
3 0.562 0.337
 3.167 0.586 0.35
3.333 0.609 0.364
3.5 0.632 0.376
3.667 0.652 0.389
3.833 0.673 0.401
4 0.692 0.413
4.167 0.711 0.424
4.333 0.728 0.434
4.5 0.745 0.445
4.667 0.761 0.456
4.833 0.776 0.469
5 0.79 0.48
5.167 0.804 0.491
5.333 0.816 0.501
5.5 0.827 0.511
5.667 0.838 0.521
5.833 0.848 0.531
6 0.857 0.541

chart

absorbance vs time
1
y = 0.147x + 0.0686
0.8
Time (min)

0.6 y = 0.0839x + 0.0669

0.4

0.2

0
0 1 2 3 4 5 6 7
Absorbance

S1 s2 Linear (S1) Linear (s2)

 absorbance vs time (S1)
0.7

0.6 y = 0.1948x - 0.002

0.5

0.4
time (min)

0.3

0.2

0.1

0
0 0.5 1 1.5 2 2.5 3 3.5
-0.1
absorbance

Slope of straight-line part of sample 1= 0.1948. so, enzyme activity of sample 1

(s1) is 0.1948.

absorbance vs time (s2)

0.4
0.35 y = 0.1038x + 0.0386
0.3
time (min)

0.25
0.2
0.15
0.1
0.05
0
0 0.5 1 1.5 2 2.5 3 3.5
absorbance

Slope of straight-line part of sample 2= 0.1038. so, enzyme activity of sample 1

(s2) is 0.1038.
Conclusion- slope of s1 is higher than slope of s2. Therefore, enzyme activity of
s1 is higher than s2.