ENZYMES

After completing the chapter, students should be

able to:
• Describe the nature, classification and function of
enzymes
• Discuss the mechanism of enzyme reaction
• Explain the factors affecting enzyme reactions
• Discuss the application of important enzymes in
food system
• Describe immobilised enzymes and their
advantages

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 Introduction
• Minor constituents, but play major role in foods

• Cause desirable & undesirable changes in foods

• Used as indicators in analytical methods such

as phosphatase –used in phosphatase test of
pasteurisation of milk

• aid in food manufacturing such as rennin used as

coagulant for milk in production of cheese

• cause hydrolytic rancidity in oils and fats

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 Nature of Enzymes
• Synthesized in cells of plants, animals & micro-
organisms.

• Most enzymes in industrial applications obtained from

microorganisms because microbial enzymes are very
heat stable & have broader pH optimum .

• Holoenzyme is a catalytically active enzyme that

consists of apoenzyme (protein) and non-protein part
including cofactor (non-organic or coenzyme (organic)
such as metals and (vitamin). Apoenzyme is inactive
form of enzyme without cofactor or coenzyme

• holoenzyme - an enzyme complete in both its

apoenzyme and coenzyme components
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 Difference between apoenzyme and coenzyme

apoenzyme coenzyme
1. It is the protein part of the enzyme It is the non-protein organic part of the
composed of only amino acids. enzyme which is attached to the
apoenzyme to form conjugate protein.
2. It is heat sensitive i.e. Thermolabile It is heat stable.
3. It is larger in size. It is smaller in size.
4. It is specific for an enzyme. It can function as a cofactor for a
number of enzymes carrying out specific
functions.
5. It is responsible for catalytic activity of It takes part in the removal of the
the enzyme. products of the reaction.

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 Nature of Enzymes

• the protein part – apoenzyme; & nonprotein part –

cofactor

• Compound being converted in enzymic reaction

is known as substrate

• Substrate(s) enzyme
 products (s)

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 Enzymes-Substrate Reactions
Active Site Theory
“Lock and Key Hypothesis and
Induced Fit”

• The enzyme’s active site has a

shape complementary to the
substrate.

• The substrate locks into the active

site of the enzyme.

• The active site alters its shape

holding the substrate more tightly
and straining it.
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 Enzymes-Substrate Reactions
• An enzyme-substrate
complex is formed.

• The substrate undergoes a

chemical change – a new
substance (product) is
formed.

• The product is released from

the active site.

• The free unaltered active

site is ready to receive fresh
substrate.
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Example of Enzymes-Substrate Reactions8
 Factors Affecting Enzyme Action

• Enzyme action occurs when the enzyme and

substrate collide ‘enzyme-substrate complex’

• During the collision the substrate slots into

the active site of the enzyme.

• Collisions happen because of the rapid

random movement of molecules in liquids.

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 Factors Affecting Enzyme Action
i)Temperature

– at 0°C enzyme action is low because

the movement of molecules is low

– the collision frequency between

enzyme and substrate is therefore low

– increasing the temperature speeds up

the movement of molecules ( > kinetic
energy)

– more ‘enzyme-substrate complexes’

are formed

– therefore enzyme action increases

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 Factors Affecting Enzyme Action
– maximum enzyme action at 40°C - maximum
collision frequency between active enzymes and
substrates

– enzyme action decreases above 40°C because

the enzymes are denaturing more quickly

• when all the enzymes are denatured enzyme action

stops (>60oC)
• Optimum temp. for enzyme function: 30-40°C
- < 0°C - enzyme action slow down;
- > 45°C – enzymes begin denatured & inactivated

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 Factors Affecting Enzyme Action
(ii) pH
• enzyme action is greatest within a
narrow range of pH, because:

• all the enzymes are active

• changing pH changes H-bonds,

thus shape of the active site

• therefore substrate can no longer

bind to the active site

• and so enzyme action decreases

• a major pH change denatures the

enzyme so enzyme action

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 Factors Affecting Enzyme Action
• Optimum pH for enzyme activity: 7-9
- Generally inactive at pH<6.2 & pH >10.8.

iii. Water – important for all chemical reactions,

including enzymatic reactions.

- Serves as a medium in which reactants can be

brought together

- Dehydration keep enzymes and substrates apart,

reducing enzyme activity.

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14
 Classification of Enzymes
• Traditionally, enzymes were simply assigned
names by the investigator who discovered
the enzyme.

• As knowledge expanded, systems of

enzyme classification became more
comprehensive and complex.

• Currently enzymes are grouped into six

functional classes by the International Union
of Biochemists (I.U.B.).
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 Classification of Enzymes

No. Classification Biochemical Properties

Oxidoreductas Act on many chemical groupings to add or remove
1.
es hydrogen atoms.
Transfer functional groups between donor and
acceptor molecules. Kinases are specialized
2. Transferases
transferases that regulate metabolism by
transferring phosphate from ATP to other molecules.
3. Hydrolases Add water across a bond, hydrolyzing it.
Add water, ammonia or carbon dioxide across
4. Lyases double bonds, or remove these elements to produce
double bonds.
Carry out many kinds of isomerization: L to D
5. Isomerases isomerizations, mutase reactions (shifts of chemical
groups) and others.
Catalyze reactions in which two chemical groups are
6. Ligases
joined (or ligated) with the use of energy from ATP.

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 Specificity of Enzymes

• Enzymes are also usually highly selective in terms of

their substrates. However, degrees of specificity
exist within particular classes of enzymes. Eg.
proteolytic enzymes- these are enzymes that cleave
peptide bonds and, thus, function by breaking down
proteins.

• Some of these enzymes, such as subtilisin, will cleave

peptide bonds regardless of the specific amino acids
that are joined by that particular bond.

• Trypsin, on the other hand, only cleaves peptide

bonds on the carboxyl-side of lysine and arginine
residues.
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18
 Some uses of enzymes in food production
Food Enzyme Purpose / function

Dairy Rennet (protease) Coagulant in cheese

production
Lactase Hydrolysis of lactose to
give lactose-free milk
products
Protease Hydrolysis of whey
proteins
Catalases Removal of hydrogen
peroxide
Brewing Cellulases, beta-glucanases, For liquefaction,
alpha amylases, proteases, clarification and to
maltogenic amylases supplement malt enzymes
Alcohol Amyloglucosidase Conversion of starch to
production sugar 19
 Some uses of enzymes in food production
(cont.)
Baking Alpha-amylases Breakdown of starch, maltose production
Amyloglycosidases Saccharification
Maltogen amylase Delays process by which bread becomes
(Novamyl) stale
Protease Breakdown of proteins
Pentosanase Breakdown of pentosan, leading to
reduced gluten production
Glucose oxidase Stability of dough
Wine and Pectinase Increase of yield and juice clarification
fruit
juice Glucose oxidase Oxygen removal
Beta-glucanases
Meat Protease Meat tenderising
Papain
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 Some uses of enzymes in food production
(cont.)
Protein Proteases, trypsin, Breakdown of
aminopeptidases various
components
Starch Alpha amylase, Modification and
glucoamylases, conversion (eg to
hemicellulases, maltogenic dextrose or high
amylases, glucose fructose syrups)
isomerases
dextranases, beta-
glucanases
Inulin Inulinases Production of
fructose syrups21
 Why enzymes are widely used in food
industry?

i. Occur naturally in biological material

ii. Non-toxic
iii. Catalyse specific action without causing
unwanted side reactions
iv. Active at low concentrations
v. Rate of reaction can be controlled easily
by adjusting temp, pH and amount of
enzyme itself
vi. Can be inactivated after reaction has
proceeded to desired extent.
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Some uses of enzymes in food production
• Majority of enzymes used in food industry are
hydrolases, consisting chiefly of carbohydrases,
followed by proteases & lipases (esterases), a few
oxidoreductases & one isomerase.

1. CARBOHYDRASES
 Hydrolytically split glycosidic linkages

 Hydrolysis of poly & oligosaccharides

 Eg. amylases, cellulases, lactase, invertase &

hemicellulase

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a. Amylases (hydrolyze starch)

• Starch degrading enzymes

• 2 types: - amylase & -amylase

• Impt in fruit ripening, potato processing, production

of corn syrup, in brewing & breadmaking

• Hydrolysis of starch by amylases can:

i. Provide sugars for subsequent fermentation &
utilisation by micro-organisms
ii. Provide reducing sugars to participate in non-
enzymic browning
iii. Alter mouthfeel, texture, moistness & sweetness of
foods
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-amylase
• Endo-enzyme also known as liquefying @ dextrinogenic
amylase

• Hydrolyses -1,4 bonds of amylose & amylopectin in

random manner resultin in production of low-molecular
weight dextrins

• Leads in rapid decrease in viscosity & rapid loss in ability to

produce coloured complex with iodine & increase in
reducing power.

• Widely distributed in plant and animal

Note: An endoenzyme,
endoenzyme or intracellular enzyme,
enzyme is an enzyme that functions within the cell in
which it was produced.

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-amylase
• exo-enzyme, also known as saccharifying @
saccharogenic amylase

• Hydrolyses -1,4 bonds by removing maltose units from

nonreducing end of glucosidic chain in an oderly fashion
resulting in production of high-molecular weight dextrins

• Found only in plants such as barley, wheat, sweet

potatoes & soybean

• Impt in baking, brewing & distilling industries.

Note: An exoenzyme,
exoenzyme or extracellular enzyme,
enzyme is an enzyme that is secreted by a cell and
functions outside of that cell

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2. PROTEASES
 Degrade protein by hydrolysing peptide linkages

 Mainly proteolytic enzyme with different degrees of

specificity, no single one capable of hydrolysing all
peptide bonds in protein

Papain
• Most widely used protease in food

• Hydrolyse polypeptides, amides & esters peptides

of low molecular weight

• Source: papaya

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Papain
• Applications:
i. Tenderise meat by hydrolyses one/more muscle
tissue components (esply sarcolemma) without
excessive degradation of muscle fibres.

ii. Chill proof beer (prevent beer haze formation since

polypeptides are major components of chill haze)

iii. Improve oil & protein extraction from animals & plants
iv. Control & modify protein functionalities

v. Improve fish protein processing

vi. Improve hot cereals & marinades

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Bromelain
• Source: pineapple
• Aplications: same as papain

• Rennin/Rennets
• Hydrolyse peptide linkages in casein clotting of milk

• Impt in cheese making as clotting of milk by rennin is

the primary step.

• Aids in development of flavour & texture in ripened

cheese

• Sources: fourth stomach of ruminant animals

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ESTERASES
• Responsible for splitting ester linkages with introduction of
water
RCO-OR’ + H2ORCOOH + R-OH

LIPASES
• Widely distributed in animals, plants & micro-organisms

• Responsible for hydrolysis of oils & fats

• Enzyme action take place at fat-water interface

• Divide into 2 types based on positional specificity for primary

ester eg.:
 1,3 lipases hydrolyses ester linkages at 1 & 3 positions -
obtained from P. roqueforti
 2-lipases attacks ester linkage at the 2 position – obtained
from A. flavus 30
LIPASES
• Sources:
i. Stomach tissue of calves & lambs
ii. Animal pancreatic tissues
iii. A. oryzae
iv. A. niger

• Applications:
i. Animal lipases develop flavour in manufacturing
cheese & in lipolysed butteroil

ii. Microbial lipases catalyses hydrolysis of lipids eg.

in fish oil concentration

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Chlorophyllase
• Catalyses 1st step in degradation of chlorophyll
during senescence @ storage of fruits &
vegetables

• Chlorophyll + H2O phytol + methyl

chlorophyllide

• Widely distributed in plant tissues

• Important to inactivate this enzyme to maintain

desirable green colour in processed vegetables.

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33
Phosphatases

• Present in bacteria (B. subtilis)

• Main application:
i. Production of flavour enhancers: 5’
mononucleotide, guanosine-5-monophosphate
(GMP) & inosine-5’-monophosphate (IMP)

ii. Impt in milk as its inactivation is use to assess

adequacy of HTST pasteurisation as this process
destroys any pathogenic enzymes. Negative
phosphatase test results confirms effectiveness of
pasteurisation.

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 OXIDOREDUCTASES
 Catalyses oxidation-reduction reactions

 application to food is limited

 Main: glucose oxidase; others: catalase & lipoxygenase

Glucosidase
• Catalyses oxidation of -D-glucose with molecular oxygen to
form D-gluconic acid.

• Glucose + H O ------- gluconic acid + H O

2 2 2

• High specificity for -D-glucose & used as an analytical reagent

for specific determination of glucose in biological materials.

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Glucosidase

• Obtained from A. niger which also contains

catalase

• Applications:
i. Removal of glucose from food materials to
minimise Maillard reaction. Added in small
qauntities eg. in egg albumin @ dried egg powder
to hydrolyse aldehyde group of glucose to gluconic
acid; hence suppresses Maillard reaction

ii. Removes oxygen from beverages & salad

dressings to prevent off-flavours & improve storage
stability.

iii. Improves colour & texture of baked goods &

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machinability of bread doughs
Phenolases
• Occur in almost all plants, higher in potatoes, apples,
bananas, avocados, peaches, mushrooms, tea
leaves & coffee beans.

• Involved in browning reaction.

• Beneficial in processing of tea & coffee

• Known as polyphenoloxidase/polyphenolase/
phenolase

• Oxidise phenolic compounds to o-quinones

•
• Detrimental when leads to browning in bruised and
broken plant tissue. 37
Lipoxygenase (Linoleate)
• Formerly known as lipoxidase
• Present in plants
• Major sources: legumes, soybeans, other beans &
peas.
• Catalyses oxidation of unsaturated fats
• Linoleate + H2O -----Hydroperoxides
• Applications: hydroperoxides bleach carotenoids in
dough & oxidise thiol groups in gluten to improve
dough rheology
• Undesirable effects:
i. Destruction of carotene & vitamin A, chlorophyll,
bixin & other pigments
ii. Development of off-flavour in soybean (beany
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flavour) in peas & beans
 IMMOBILISED ENZYMES
• Fixing of enzymes on water-insoluble inert supports

• Fixed enzymes retain their activity & can be easily added to

@ remove from reaction mixture.

• Use of immobilised enzymes permit continuous processing &

increase use of enzyme

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 METHODS OF IMMOBILISING ENZYMES
1. Adsorption on organic polymers, glass, metal oxides &
siliceous materials such as bentonite & silica.

2. Entrapment in natural @ synthetic polymers usually

polyacrylamide

3. Microencapsulation in polymer membranes

4. Ion exchange

5. Crosslinking

6. Adsorption & crosslinking combined

7. Copolymerization

8. Covalent attachment to organic polymers

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 Application of Immobilised Enzymes
1. Main use in food industry: Use of immobilised
glucose isomerase (convert glucose to fructose)
obtained from Streptomyces for production of high
fructose corn syrup

2. Hydrolysis of lactose to glucose & galactose in milk

by lactase obtained from yeast (Saccharomyces
lactis & S. fragilis) or from fungi (Aspergillus oryzae
or A. niger)

3. Production of aspartic acid using aspartase from E.

coli

4. Fumarase from Brevibacterium ammoniagenes used

for L-malic acid production
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