The document discusses enzymatic activity in foods. It defines enzymes as proteins that act as biological catalysts, speeding up reactions in living organisms. It describes how enzymes are made of amino acids linked into specific shapes that determine their functions. The document then discusses several theories of enzymatic action and factors that can affect enzymatic activity, such as temperature, pH, and substrate concentration. It also categorizes the six main types of enzymes and provides examples of how enzymes like polyphenol oxidase and peroxidase cause browning in fruits and vegetables. The document concludes with an overview of how to assay enzymatic activity, using polyphenol oxidase activity and catechol as a specific example.

1. Date: 29.10.2021
Enzymatic Activity in Foods
By Amit Kumar
Agricultural and Food Engineering Department
Indian Institute of Technology Kharagpur
West Bengal, India

2. Enzymes
• Enzymes are basically proteins that are produced
by living organisms to bring about certain
metabolic and biochemical reactions in the body.
• They are biological catalysts that speed
up reactions inside the body.
• Enzymes are actually made up of 1000s of amino
acids that are linked in a specific way to form
different enzymes. The enzyme chains fold over
to form unique shapes and it is these shapes that
provide the enzyme with its characteristic
chemical potential.
• Most enzymes also contain a non-protein
component known as the co-factor.

3. • Key and lock fit Hypothesis
• Induced fit theory
Basic theory of enzymatic action

4. Enzyme kinetics Michaelis-Menten kinetics

5. Factors affecting enzymatic activity
• Temperature
• pH of surrounding medium
• Ionic strength of medium
• Hindering elements
• Substrate competitors
• Substrate concentration

6. Effect of factors

7. Types of Enzymes
1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyases
5. Isomerases
6. Ligases
The biochemical reactions occurring in the body are basically due to following 6 types of enzymes.

8. Oxidation in fruits and vegetables
• Browning of vegetables, fruits and flowers alter their appearances, flavors,
textures, and lower their marketing values.
• The browning process can be caused by both enzymatic and non-enzymatic
biochemical reactions.
• Polyphenol oxidase (PPO) and peroxidase (POD) are two well known enzymes
involved in the browning process.
• Fruits and vegetables have its protective system developed by nature.
• Fruits and vegetables have small amount of catechol which turn into
benzoquinone by the action of some enzyme called catecholase or polyphenol-
oxidase enzyme.
• Benzoquinone is an antimicrobial compound which prevent any microbial damage
to fruits and vegetables.
• Upon polymerization benzoquinone forms melanoids, brown reddish color
pigment.

9. Polyphenol oxidase
• PPO catalyzes the conversion of phenolic compounds to quinones and assists their
products’ polymerization.
• Its catalysis, in the presence of oxygen, leads to the formation of undesirable brown
pigments and off-flavored products.
• The browning of injured, peeled or diseased fruit tissues can causes undesirable
quality changes during handling, processing and storage.
• PPO is a dicopper-containing enzyme.
• PPO involve in the oxidation of the polyphenols from plants.
• PPO activity can be monitored by oxygen consumption or spectrophotometrically
using a variety of substrates such as pyrogallol, pryocatechol, 4-methylcatechol.
• PPO shows high activity with diphenols.

10. Polyphenol oxidase action

11. Peroxidase
• POD can be found in plants, animals and microbes.
• It is one of the most thermo-stable enzymes responsible for performing single
electron oxidation on a wide variety of compounds, in the presence of hydrogen
peroxide.
• POD reduces H2O2 to water while oxidizing a variety of substrates.
• Furthermore, the related activity of PPO and POD is due to the generation of
hydrogen peroxide during the oxidation of phenolic compounds in PPO-catalyzed
reactions.
• The catalytic process of POD occurs in a multistep reaction.
S stands for substrate and S• represents the corresponding radical.
AH2 and AH• represent a reducing substrate and its radical product, respectively.

12. Peroxidase action

13. Enzyme Assay
• The assay is the act of measuring how fast a given (unknown) amount
of enzyme will convert substrate to product (the act of measuring a
velocity/reaction rate).
• An assay requires to determine the concentration of a product or
substrate at a given time after starting the reaction.

14. Enzyme Assay Principle
Enzyme/ Enzyme extract + Substrate = ES = P
Estimate
PPO Activity estimation
Enzyme extract + Catechol = ES = Quinone
(Brown Colour complex/420nm )

15. Today’s class:
• Food sample: Banana flesh and peel
• Enzyme: PPO enzyme
• Materials required:
1. Food Sample
2. Weighing balance
3. Cooling centrifuge
4. Spectrophotometer
5. Lab accessories (eppendorf, spatula, tissue paper, cuvette, etc.)
6. Chemicals (PVP, Triton X, Catechol, buffer)
• Polyvinylpyrrolidone (PVP): bind with phenolics and oxygen
• Triton X: non-ionic surfactant
• Catechol: Substrate for the enzyme

16. Triton-X 100
• Triton X-100 (C14H22O(C2H4O)n) is a non-ionic surfactant that
has a hydrophilic polyethylene oxide chain (on average it has
9.5 ethylene oxide units) and an aromatic hydrocarbon
lipophilic or hydrophobic group.

17. Poly-vinyl-pyrrolidone (PVP)
• Polyvinylpyrrolidone (PVP), also commonly called polyvidone
or povidone, is a water-soluble polymer made from the
monomer N-vinylpyrrolidone.
Polyvinylpyrrolidone
Vinylpyrrolidone

18. Catechol
• Catechol is an organic compound with the molecular formula
C6H4(OH)2
• It serve as substrate for the polyphenol-oxidase enzyme
(Catecholase).
• Catechol occurs as feathery white crystals that are very rapidly
soluble in water.

19. Catechol, Why?

20. Steps
1. Preparation of required solution
2. Enzyme extraction: Homogenized sample (1 g) in 50 ml of extraction solution. Shake for 30-60 min.
3. Centrifugation: 12000 rpm for 20 min at 4°C.
4. Reaction: 1) Time lapse method, 2) continuous observation method
5. Spectrophotometry: Set the spectrophotometer at 420 nm and perform kinetics
6. Kinetic analysis: Determine the slope of the curve as the enzymatic activity of the sample.
7. Report preparation: Report the observations and analysis.
a) Buffer: Sodium-Potassium-Phosphate buffer of ph 6.8 with 0.2 M ionic strength.
b) Extraction solution: 10 ml Buffer + 0.1 g PVPP + 0.1% triton-X
c) Substrate solution: 0.01 M catechol (0.011 g in 10 ml buffer)
d) Hydrogen peroxide: 0.3 % solution

21. Calculations
min S1
0 0.085
0.167 0.121
0.333 0.18
0.5 0.241
0.667 0.303
0.833 0.361
1 0.415
1.167 0.467
1.333 0.517
1.5 0.561
1.667 0.6
1.833 0.634
2 0.658
2.167 0.678
2.333 0.692
2.5 0.701
2.667 0.705
2.833 0.706
3 0.705
3.167 0.701
3.333 0.697
3.5 0.69
3.667 0.683
3.833 0.676
4 0.668
4.167 0.66
4.333 0.651
4.5 0.643
4.667 0.635
4.833 0.628
5 0.621
5.167 0.614
5.333 0.608
5.5 0.602
5.667 0.596
5.833 0.59
6 0.584

22. Thank you.
For any query and suggestion please contact me at
amitchauhan9091@gmail.com

23. Disclaimer
This presentation is prepared by the authors and does not
represent the institute and organization.
All the information are to provide an idea about enzymes and
theirs actions in foods.
All the images are representative and taken from open source
google search platform.
In case any ambiguity in the concepts, please feel free to
communicate the author.