ENZYMES

A protein with catalytic properties due to its

power of specific activation
Enzymes are biological catalysts.

Important ENZYMES
1.
2.
3.
4.

The amylolytic enzymes

Lipolytic enzyme
Proteolytic enzymes
Oxidizing enzymes

Amylolytic ENZYMES
DIASTASE and AMYLASE
Salivary Diastase or Ptylin
Pancreatic diastase or amylopsin
2. Malt diastase starch to maltose
3. Invertase or sucrase sucrose to glucose &
1.

fructose

4. Maltase maltose to glucose

5. Zymase monosaccharides to CO2 and alcohol

6. Emulsin amygdalin to glucose, HCN and
benzaldehyde
7. Myrosin Sinalbin, Sinigrin and other
glycoside

Proteolytic enzymes
Pepsin Proteins into proteoses & Peptons
2. Trypsin proteoses & Peptons to
polpeptides and amino acids
3. Erepsin proteoses & Peptons to amino
acids
4. Rennin Curdles soluble casein of milk
5. Papain meat tenderize proteoses &
Peptons to polpeptides and amino acids r
1.

OXIDIZING Enzymes
Peroxidases Oxidation reaction
2. Thrombin Fibrinogen to Fibrin
3. Zymase Monosaccharide spliting enzyme
but works by oxidation
1.

MALT EXTRACT
BARLEY is dried grain of Hordeum vulgare
Graminae
Cultivated through out the world wherever climate
is favourable
Malt or Malted Barley
Dried, artificially germinated barley grain
Grains kept wet with water in warm room
germinate till caulicle protrudes dried quickly

Enzyme diastase Converts starch to maltose

Stimulate embryo formation which is killed
by drying
Dry malt resembles barley & has agreeable
odour and sweet taste
Contains sugar maltose 50-70%
Dextrins 2-15%
Proteins 8%
Diastase and peptase emzyme
Used in Brewing & alcohol industries

Malt extract

Extracting malt with hot water at 60C.

Concentracted at 60C under pressure
Malt extract is mixed with 10% glycerine
It contains dextrin, maltose, glucose and
amylolytic enzyme
Starch soluble sugars (5times of its own
weight)

Uses

Malt extract is used as easily digested

nutritive and as an aid in starch digestion.
15g
Diastase 50times starch sugars
Lactase converts lactose galactose and
glucose. It is used in pt having lactose
intolerance

Papain
Phytoenzyme
Biological source
Dried and purified latex of the fruit of
Carica papaya family Caricaceae
Geographical source
Indigenous to tropical America
Cultivated in Sri Lanka, Tanzania, Hawaii, Florida

Plant description

Plant attains height of 5-6 meters

Fruit grows to a length of about 12 inches
(30cm)
Weight of fruit is about 5 Kg

Collection procedure

The epicarp of the fruit adheres to the orange

colour, fleshy sarcocarp, which surrounds the
central cavity.
Cavity contains a mass of nearly black seeds
Full grown but unripe fruits are subjected to
shallow incisions on the 4 sides.
Latex flow for few seconds and then soon
coagulates
Latex is then collected

After collection coagulated lumps are

shredded and dried by sun or by artificial heat
Later methods yield better grades of crude
papain
Incisions & collections are made at weekly
intervals as long as the fruit exudes the latex.
Crude papain is purified by dissolving in
distilled water and then precipitating with
alcohol

Papain enzymes

Papain is referred to as Vegetable Pepsin

because it contain enzyme similar to that of
Pepsin. However unlike Pepsin, Papain acts in
acid, neutral and alkaline media.
Papain contain one or more proteolytic
enzymes because a single sample of papain
yield variable result with the type of protein
used

Papain contains one or more proteolytic

enzymes, Peptidase-I, converts proteins
dipeptides and polypeptides
Rennin like coagulating enzyme that act on
milk casein
An Amylolitic enzyme
A Clotting enzyme similar to Pectase Pectin
pectic acid and methyl alcohol
An enzyme that act on fat

Actions and uses of Papain

Papain differ in strength with method of

preparation
It is used as meat tenderizer It can digest
35 times of its own weight of lean meat
It is used extensively in tenderization of beef

Best grade of papain can digest 300 times of

its own weight of egg albumin
It is used as protein digestant as it has same
action on protein as that of Pepsin
Used to relieve the symptoms of episiotomy

Pepsin
Animal enzyme
Proteolytic enzyme
Biological source
Enzyme obtained from the glandular layer of the
fresh stomach of various animals like Pig, sheep
or calf. Commonly from pig,
Sus scrofa family Suidae

Sus is from Greek word Us meaning HOG

Preparation of pepsin

Pepsin is prepared by using stomach linings

The mucous membrane is separated from the
stomach by the process of stripping or it is
scrapped off
Then it is placed in acidified water for
autolysis at 37C for 2 hours
The liquid so obtained consists of both pepsin
and peptone

It is then filtered and sodium or ammonium salts

are added to the liquid till it is half saturated
At this point only the pepsin separates out in the
form of precipitates, and the peptone remains in
solution
The precipitates are collected and subjected to
dialysis for the separation of salts
Remaining amount of pepsin if any in the
aqueous solution is precipitated by the addition
of alcohol into it.

Concentrated solution containing precipitates,

is poured on glass plates to dry forming
Scale pepsin
Or concentrated solution is carefully
evaporated in vacuum forming spongy
pepsin

Description of enzyme

Pepsin is lustrous, transparent or translucent

scales or occurs as granular or spongy masses.
Colour ranges between light yellow to light
brown or as fine white or cream coloured
amorphous powder
It is free from offensive odour and has a
slightly acid or saline taste.
It show optimal activity at a pH 1.8 to 3.5
Reversibly inactive at pH 7 to 8

Standard pepsin

Pepsin digests not less than 3000 and not

more than 3500 times its weight of coagulated
egg albumin.
Commercial pepsin, especially spongy pepsin
is often 4-5 times more active than that of
medicinally used.
A pepsin of higher digestive power may be
reduced to standard pepsin by admixture with
a pepsin of lower grade or with lactose.

Pepsin best act at a temperature 40C and pH

2-4
Pepsin denatured at 70C and in the presence
of alcohol.
Pepsin can be stored at 2-8C for about 1-2
years

Uses

pepsin is used in deficiency of gastric secretion

Pepsin is administered to assist digestive digestion
in combination with pancreatin.
It is proteolytic enzyme is administered after meals
and followed by a dose of hydrochloric acid, its
Usual dose is 500mg
It is used in laboratory analysis of various proteins.
It is used in preparation of cheese and other protein
containing food

Pancreatin

Animal enzyme
Pancreatin is a mixture of several digestive
enzymes. It is composed of
Amylase (hydrolyses starches to oligo &
disaccharides maltose)
Lipase (hydrolyses triglycerides to fatty acids
and glycerols)
Protease (Trypsin hydrolyses proteins to
oligopeptides)

Biological source
Pancreatin is produced by the exocrine cells of
the pancreas of Pig or Hog,
Sus scrofa Family Suidae
or from Pancreas of Ox or calf
Bos taurus Family Bovidae

Preparation method

The Pancreas is a gland that lies directly

inside the posterior wall of the abdomen.
Fresh glands are minced and extracted by
similar methods as employed in the
manufacture of Pepsin

Standard pancreatin

It is cream coloured amorphous powder with a

faint, characteristic but not offensive odour.
Pancreatin contain in each mg,
Not less than 25 USP units of amylase activity
Not less than 2 USP units of lipase activity
Not less than 25USP units of protease activity

USP units

One USP unit of amylase activity is contained

in the amount of pancratin that digests 1mg of
the dry USP potato starch standard
One USP unit of lipase activity is contained in
the amount of pancratin that liberates 1Eq of
acid per minute at a pH 9 and 37C.
One USP unit of protease activity is contained
in the amount of pancratin that digests 1mg of
casein under specific conditions

Properties

It show optimal activity in neutral or faintly

alkaline solution
More than traces of mineral acids or large
amounts of alkali hydroxide render pancreatin
inert, and excess of alkali carbonates inhibits
its action

Pancreatin uses

Pancreatin is a mixture of several digestive

enzymes. This mixture is used to treat
conditions in which pancreatic secretions are
deficient, such as surgical pancreatectomy,
pancreatitis and cystic fibrosis.
As digestive aid for invalids
Enteric coated granules are used to treat infants
with celiac disease and related pancreatic
deficiencies,. Usual dose is 325mg 1g as
tablets, capsules or granules

Routine cancer eradication

As is mixture of enzymes but proteolytic
enzyme play role in cancer eradication
Cancer is often disease of protein metabolism
Pancreatin enzyme cancer defence mechanism
Effected by protein rich foods at inappropriate
time or in excessive amount
Body need 12 hrs time free of protein
consumption for proper working of this
defence system

Pancreatin become inactive in acid or with

alcohol contact
cancer continuously produce acids
Many cosmetic contain acid or
alcoholspecial concern with the skin cancer

Pancrealipase
Is same as Pancreatin but is more concentrated
form of Pancreatin
Pancrealipase contain in each mg,
Not less than 100 USP units of amylase activity
Not less than 24 USP units of lipase activity
Not less than 100USP units of protease activity
Thus the lipase activity is increased by 12folds,
but the amylase and protease activity only 4 folds
when compared to Pancreatin

Biological source
Pancreatin is produced by the exocrine cells of
the pancreas of Pig or Hog,
Sus scrofa Family Suidae
or from Pancreas of Ox or calf
Bos taurus Family Bovidae

Uses

Employed as digestive aid

It increase intestinal absorption of fat used
to treat steatorrhea
Usual dose is 8000 to 24000 USP units
Dose is determined on the basis of clinical
evaluation according to pancreatic
insufficiency

Rennin
Animal enzyme
Protease enzyme
Milk curdling enzyme
Biological source
Rennin is partially purified enzyme obtained
from the glandular layer of the stomach of the
calf, Bos taurus
Family : Bovidae

Rennin (RENNET)
Three main sources,
Bos taurus
animal source
Withania coagulans plant source
Rhizomucor miehei microbial source

Preparation method

Rennin is prepared by macerating the minced

glandular layer of the digestive stomach of the
calf in 0.5% sodium chloride solution.
Filtration is then carried out
Filtrate is acidified with hydrochloric acid
Then saturation is carried out with sodium
chloride
Which precipitates the Rennin
Rennin is then separated and dried and
powdered

Standard Rennin

Rennin occur as a yellowish white powder or

as yellowish grains or scales
It has characteristic and slightly saline taste
It has peculiar odour
Standard Rennin can coagulate approximately
25000 times its own weight of fresh cows
milk

Uses

Rennin is used to coagulate milk, thus

rendering it more digestible for convalescents
It is ingredient in Rennin and pepsin elixir
called pepsin essence
Protease enzyme that curdles the casein in
milk, helping young mammals digest their
mothers' milk.
Its principal used is in the manufacture of
cheese, mainly for coagulation of milk

Used to separate milk into solid curds used for

cheese making and liquid whey

Important enzyme
Trypsin
Proteolytic enzyme from pancreas gland of Ox
Used for debridement of necrotic and pyogenic
surface lesions
Wound and ulcer cleanser
Chymotrypsin
Proteolytic enzyme from pancreas of Ox
Ophthalmic solution

Hyaluronidase (mucolytic enzyme)

The hyaluronidases are a family of enzymes that degrade

hyaluronic acid. It is found in human testes, in bacterial cultures as
by product or in the heads of leeches and snake venoms
By catalysing the hydrolysis of hyaluronic acid, hyaluronidase
lowers the viscosity of hyaluronic acid, thereby increasing
tissue permeability.
It is, therefore, used in medicine in conjunction with other drugs to
speed their dispersion and delivery.
Common applications are ophthalmic surgery, in combination
with local anesthetics.
It also increases the absorption rate of parenteral fluids given
by interstitial or subcutaneous infusions (hypodermoclysis).
It is an adjunct in subcutaneous urography for improving
resorption of radiopaque agents

Streptokinase & streptodornase

Thrombolytic enzyme
Streptokinase breakdown fibrin whereas streptodornase
affects deoxyribonucleic acid and desoxyribo-nucleoprotein, which is chief constituents of pus and necrotic
tissue, therefore these are used wherever, clotted blood or
fibrinous or prulent accumulations appear following injury
to the tissue.
The enzymes are used in 4:1 treptokinase:treptodornase
Usual oral dose is 10000 units of streptokinase in
combination with 2500 units of streptodornase 4times/ day
For IM administration 5000:2500 units twice a day are
used

Streptokinase

Streptokinase is used for its indirect thrombolytic activity

It acts on the endogenous fibrinolytic system by converting
plasminogen to plasmin(proteolytic enzyme) or to fibrinolysin
Fibrinolysin degrades fibrin clots
Used for massive pulmonary emboli and for extensive thrombi
of the deep veins in adults
Streptokinase is administered through continuous IV infusion
@ 10000 units per hour for 24 to 72 hours
It should be used by only the physician who is expert of
managing thrombotic diseases
It should not be used to treat superficial thrombophlebitis

Urokinase

Isolated from human urine

Alternate of the streptokinase for the
treatment of massive pulmonary emboli
Have less chances of allergic reaction due to
human origin
Administered through IV infusion @ 4400
untis/kg body weight per hour for 12 hours

Fibrinolysin

It is present in blood serum as a protease and in plasma

as the inactive precursor plasminogen
It is prepared commercially by activating blood serum
with streptokinase
It act on the protein portions of the dead tissues,exudates
and blood clots found in the wounds, ulcers & burns
It is used to treat the blood clots within the
cardiovascular system
It is used to treat Phlebothrombosis, thrombophlebitis
and pulmonary emboli
It is administered by IV infusion

Sutilains

Proteolytic enzyme
Obtained from Bacillus subtilis
It is cream colour powder
Standard enzyme has not less than 2.5million
USP units of casein proteolytic activity per
gram
It is used in topically in the form of ointment
for wound debridement

Collagenase

It is obtained from the culture of Clostridium

histolyticum
It cleaves collagen
It is used topically to debride the dermal ulcers and
severely burned areas
Enzyme is deactivated by heavy metals so care should
be exercised in this regard
In case of bacteremia, Burrows solution should be
used to stop the action of enzyme
Used in the form of ointment containing 250 units of
collagenase activity per gram

Deoxyribonuclease

Nucleolytic enzyme
Obtained form the pancreas of bovine origin
It is active in dry form
It catalysis cleavages of giant molecules of
DNA (deoxyribonucleic acid) into numerous
small fragments called polynucleotides
Thus it act against the devitalized tissues in
purulent states
It is available in combination with fibrinolysin

L -Asparaginase

Obtained from Escherichia coli (E. coli)

It is used to treat acute leukemia in children
Its anticancer effect is attributed to the fact that this
enzyme catalysis the conversion of Asparagine to
aspartic acid and ammonia, there by depleting the
asparagine, which result in the death of cells that
requires exogenous sources of this amino acids for
their survival (mostly cancer cell or leukemic cell).
Clinical effectiveness of the drug is due to the
requirement for L-asparagine differences of the normal
cells and the cancer/neoplastic cells

It is used in combination with other

anticancers like prednisolone and vincristine
Usual dose IV is 1000 units per kg body
weight daily or IM 6000 units per square
meter of body surface area at 3 day interval
A number of adverse effects have been noted
with L-Asperaginase use, like allergic
reaction, anaphylactic reaction

Properties

of enzymes

Chemical reactions

Chemical reactions need an initial input of energy =

THE ACTIVATION ENERGY
During this part of the reaction the molecules are
said to be in a transition state.

Reaction pathway

Making reactions go faster

Increasing the temperature make molecules move

faster
Biological systems are very sensitive to temperature
changes.
Enzymes can increase the rate of reactions without
increasing the temperature.
They do this by lowering the activation energy.
They create a new reaction pathway a short cut

An enzyme controlled pathway

Enzyme controlled reactions proceed 108 to 1011 times faster

than corresponding non-enzymic reactions.

Enzyme structure

Enzymes are
proteins
They have a
globular shape
A complex 3-D
structure

Human pancreatic amylase

Dr. Anjuman Begum

The active site

H.PELLETIER, M.R.SAWAYA
ProNuC Database

One part of an enzyme,

the active site, is
particularly important
The shape and the
chemical environment
inside the active site
permits a chemical
reaction to proceed
more easily

Cofactors

An additional nonprotein molecule that is

needed by some
enzymes to help the
reaction
Tightly bound cofactors
are called prosthetic
groups
Cofactors that are bound
and released easily are
called coenzymes
Many vitamins are
coenzymes
Nitrogenase enzyme with Fe, Mo and ADP cofactors
Jmol from a RCSB PDB file 2007 Steve Cook
H.SCHINDELIN, C.KISKER, J.L.SCHLESSMAN, J.B.HOWARD, D.C.REES
STRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS
IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997)

The substrate

The substrate of an enzyme are the reactants

that are activated by the enzyme
Enzymes are specific to their substrates
The specificity is determined by the active
site

The Lock and Key Hypothesis

Fit between the substrate and the active site of the enzyme is
exact
Like a key fits into a lock very precisely
The key is analogous to the enzyme and the substrate
analogous to the lock.
Temporary structure called the enzyme-substrate complex
formed
Products have a different shape from the substrate
Once formed, they are released from the active site
Leaving it free to become attached to another substrate

The Lock and Key Hypothesis

S

E
E

Enzymesubstrate
complex

Enzyme may
be used again
P
P

Reaction coordinate

The Lock and Key Hypothesis

This explains enzyme specificity

This explains the loss of activity when
enzymes denature

The Induced Fit Hypothesis

Some proteins can change their shape

(conformation)
When a substrate combines with an enzyme, it
induces a change in the enzymes conformation
The active site is then moulded into a precise
conformation
Making the chemical environment suitable for the
reaction
The bonds of the substrate are stretched to make the
reaction easier (lowers activation energy)

The Induced Fit Hypothesis

Hexokinase (a) without (b) with glucose substrate

http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html

This explains the enzymes that can react with a

range of substrates of similar types

Factors affecting Enzymes

substrate concentration
pH
temperature
inhibitors

Substrate concentration: Non-enzymic reactions

Reaction
velocity

Substrate concentration

The increase in velocity is proportional to the

substrate concentration

Substrate concentration: Enzymic reactions

Vmax
Reaction
velocity

Substrate concentration

Faster reaction but it reaches a saturation point when all the

enzyme molecules are occupied.
If you alter the concentration of the enzyme then Vmax will
change too.

The effect of pH
Optimum pH values

Enzyme
activity

Trypsin

Pepsin
1

7
pH

11

The effect of pH

Extreme pH levels will produce denaturation

The structure of the enzyme is changed
The active site is distorted and the substrate
molecules will no longer fit in it
At pH values slightly different from the enzymes
optimum value, small changes in the charges of the
enzyme and its substrate molecules will occur
This change in ionisation will affect the binding of
the substrate with the active site.

The effect of temperature

Q10 (the temperature coefficient) = the increase in

reaction rate with a 10C rise in temperature.
For chemical reactions the Q10 = 2 to 3
(the rate of the reaction doubles or triples with every
10C rise in temperature)
Enzyme-controlled reactions follow this rule as they
are chemical reactions
BUT at high temperatures proteins denature
The optimum temperature for an enzyme controlled
reaction will be a balance between the Q10 and
denaturation.

The effect of temperature

Q10

Enzyme
activity

10

20
30
40
Temperature / C

Denaturation

50

The effect of temperature

For most enzymes the optimum temperature is about

30C
Many are a lot lower,
cold water fish will die at 30C because their
enzymes denature
A few bacteria have enzymes that can withstand very
high temperatures up to 100C
Most enzymes however are fully denatured at 70C

Inhibitors

Inhibitors are chemicals that reduce the rate of

enzymic reactions.
The are usually specific and they work at low
concentrations.
They block the enzyme but they do not
usually destroy it.
Many drugs and poisons are inhibitors of
enzymes in the nervous system.

The effect of enzyme inhibition

Irreversible inhibitors: Combine with the
functional groups of the amino acids in the
active site, irreversibly.
Examples: nerve gases and pesticides,
containing organophosphorus, combine with
serine residues in the enzyme acetylcholine
esterase.

The effect of enzyme inhibition

Reversible inhibitors: These can be washed
out of the solution of enzyme by dialysis.
There are two categories.

The effect of enzyme inhibition

1. Competitive: These
compete with the
substrate molecules for
the active site.
The inhibitors action is
proportional to its
concentration.
Resembles the substrates
structure closely.

E+I
Reversible
reaction

EI
Enzyme inhibitor
complex

The effect of enzyme inhibition

Fumarate + 2H++ 2e-

Succinate
Succinate dehydrogenase

CH2COOH

COOH

CHCOOH

CH2
CH2COOH

COOH
Malonate

CHCOOH

The effect of enzyme inhibition

2. Non-competitive: These are not influenced by the
concentration of the substrate. It inhibits by binding
irreversibly to the enzyme but not at the active site.
Examples
Cyanide combines with the Iron in the enzymes
cytochrome oxidase.
Heavy metals, Ag or Hg, combine with SH groups.
These can be removed by using a chelating agent such
as EDTA.

Applications of inhibitors

Negative feedback: end point or end product

inhibition
Poisons snake bite, plant alkaloids and nerve
gases.
Medicine antibiotics, sulphonamides,
sedatives and stimulants