SUBMITTED TO MISS DR HIRA MAAZ

SUBMITTED BY

AYESHA SHAMIM 1141-2023

FIZA 678-2023
RUTABA KAUSER 474-2022
SAMI NADEEM 692-2023
MIRZA MAAZ 719-2023

ENZYME
&
PROTEIN

ASSIGNEMENT OF PHARMACOGNOSY

11-11-2024
 PROTEIN

INTRODUCTION:
Proteins are large, complex molecules that play many critical roles in the body. They do
most of the work in cells and are required for the structure, function, and regulation of the
body’s tissues and organs.The importance of proteins was recognized by chemists in the
early 19th century, including Swedish chemist Jöns Jacob Berzelius, who in 1838 coined
the term protein, a word derived from the Greek prōteios, meaning “holding first place.”
Proteins are species-specific; that is, the proteins of one species differ from those of
another species. They are also organ-specific; for instance, within a single
organism, muscle proteins differ from those of the brain and liver.

Proteins are made up of hundreds or thousands of smaller units called amino acids,they
contain carbon, hydrogen, oxygen, nitrogen, and sulphur molecules,which are attached to
one another in long chains. There are 20 different types of amino acids that can be
combined to make a protein. The sequence of amino acids determines each protein’s
unique 3-dimensional structure and its specific function. Amino acids are coded by
combinations of three DNA building blocks (nucleotides), determined by the sequence of
genes.

PROTEIN STRUCTURE:

Nucleic Acids:
The two forms of nucleic acid are deoxyribonucleic acid and ribonucleic acid. The basic
building blocks of nucleic acids are called nucleotides. Each nucleotide consists of a
phosphate unit, a sugar and a nitrogen base. DNA nucleotide bases include adenine,
thymine, guanine and cytosine. RNA uses the same set of bases, except for the
substitution of unit cells for thymine.

Nucleotides bind to one another to form strands or other structures. In the DNA molecule,
nucleotides are arranged and twisted, and a double strand called a double helix. The
sequence of different nucleotides along the DNA double helix is the “master code” for
assembling proteins and other nucleic acids.

Protein structure is defined as a polymer of amino acids joined by peptide bond. A

peptide bond is established from the following reaction:
 Formation of Peptide Bond

the peptide bond (-CO-NH) is formed between the amine group of one molecule and the
carboxyl group of the adjacent molecule followed by the elimination of a water molecule.
This bond is otherwise an amide linkage. When peptide bonds are established among
more than ten amino acids, they together form a polypeptide chain. Very often, when a
polypeptide chain has a mass exceeding 10000u and the number of amino acids in the
chain exceeding 100, we get a protein.

CLASSIFICATION OF PROTEIN STRUCTURES:

Classification of proteins is done on the basis of the following:

 Shape
 Constitution
 Nature of molecules

On The Basis Of Shape:

1. Fibrous Proteins:(Scleroprotein)
When the polypeptide chains run parallel and are held together by hydrogen and
disulfide bonds, then the fiber-like structure is formed. Such proteins are generally
insoluble in water. These are water-insoluble proteins.Fibrous proteins are resistant to
proteolytic enzymes and are coiled and exist in threadlike structures to form fibres.

Example – keratin (present in hair, wool, and silk) and myosin (present in
muscles),collagen, actin, etc.

2. Globular Proteins:
This structure results when the chains of polypeptides coil around to give a spherical
shape. These are usually soluble in water.

Example – Insulin and albumins and hormones like oxytocin, are common examples of
globular proteins.

On The Basis Of Constitution:

1. Simple proteins: These proteins are made up of amino acids only. e.g. albumins,
globulins, prolamins, etc.
2. Conjugated proteins: These are complex proteins that are combined with the
characteristic of non–amino acid substance called as a prosthetic group. These are of
following types:–

 Nucleoproteins: Combination of protein and nucleic acid

 Mucoproteins: Combination of proteins and carbohydrates (>4%)
 Glycoproteins: Combination of proteins and carbohydrates(<4%)
 Chromoproteins: Combination of proteins and coloured pigments.
 Lipoproteins: Combination of proteins and lipids.
 Metalloprotein: Combination of proteins and metal ions.
 Phosphoprotein: Combination of proteins and phosphate group.


3. Derived proteins: When proteins are hydrolyzed by acids, alkalies or enzymes, the
degradation products obtained from them are called derived proteins.

On The Basis Of Nature Of Molecules:

1. Acidic proteins: They exist as anion and contain acidic amino acids. e.g. blood groups.

2. Basic proteins: They exist as cations and are rich in basic amino acids e.g. lysine,
arginine etc.

FURTHER CLASSIFICATION:
While there are hundreds of amino acids in nature, humans use only 20 of them. One way
to further classify them is by defining which ones healthy bodies can and cannot make.

The three classes of proteins are:

1. Non-Essential
2. Conditionally Essential
3. Essential Amino Acids

1. Non Essential: There are five amino acids termed non-essential because they can be
obtained from foods and also generated within the body. The non-essential amino acids
are: Alanine; Asparagine; Aspartic acid; Glutamic acid; Serine

2. Conditionally-Essential Amino Acids: There are six amino acids termed

conditionally-essential because healthy bodies can generate them under normal
physiologic conditions. They become essential under certain conditions lIke starvation or
inborn errors of metabolism. The conditionally essential amino acids are: Arginine;
Cysteine ; Glutamine; Glycine; Proline; Tyrosine

3. Essential Amino Acids: There are nine amino acids termed essential because they
cannot be generated within the body. Dietary protein, therefore, provides these amino
acids, which are needed to make certain hormones and other important molecules. The
essential amino acids are: Histidine; Isoleucine; Leucine; Lysine; Methionine;
Phenylalanine; Threonine; Tryptophan; Valine.
LEVELS OF PROTEIN STRUCTURE:
Protein molecules are large, complex molecules formed by one or more twisted and
folded strands of amino acids. Each amino acid is connected to the next amino acid by
covalent bonds (peptide bond). Linderstrom-Lang (1952) in particular first suggested a
hierarchy of protein structure with four levels: central, secondary, tertiary , and
quaternary.
1. Primary (first level) – Protein structure is a sequence of amino acids in a chain.
2. Secondary (secondary level) – Protein structure is formed by folding and twisting of
the amino acid chain.
3. Tertiary (third level) – Protein structure is formed when the twists and folds of the
secondary structure fold again to form a larger three dimensional structure.
4. Quaternary (fourth level) – Protein structure is a protein consisting of more than
one folded amino acid chain.
Proteins can bond with other organic compounds and form “mixed” molecules. For
example, glycoproteins embedded in cell membranes are proteins with sugars attached.
Lipoproteins are lipid-protein combinations.

1. Primary Structure of Protein

 The Primary structure of proteins is the exact ordering of amino acids forming their
chains.
 The exact sequence of the proteins is very important as it determines the final fold
and therefore the function of the protein.
 The number of polypeptide chains together form proteins. These chains have amino
acids arranged in a particular sequence which is characteristic of the specific
protein. Any change in the sequence changes the entire protein.
The following picture represents the primary protein structure (an amino acid chain). As
you might expect, the amino acid sequence within the polypeptide chain is crucial for the
protein’s proper functioning. This sequence is encrypted in the DNA genetic code. If
mutation is present in the DNA and the amino acid sequence is changed, the protein
function may be affected.
The protein ‘s primary structure is the amino acid sequence in its polypeptide chain. If
proteins were popcorn stringers designed to decorate a Christmas tree, a protein ‘s
primary structure is the sequence in which various shapes and varieties of popped maize
are strung together.

Covalent, peptide bonds which connect the amino acids together maintain the primary
structure of a protein.

All documented genetic disorders, such as cystic fibrosis, sickle cell anemia, albinism,
etc., are caused by mutations resulting in alterations in the primary protein structures,
which in turn lead to alterations in the secondary , tertiary and probably quarterly
structure.

Amino acids are small organic molecules consisting of a chiral carbon with four
substituents. Of those only the fourth the side chain is different among amino acids.

2. Secondary Structure of Protein

Secondary structure of protein refers to local folded structures that form within a
polypeptide due to interactions between atoms of the backbone.

 The proteins do not exist in just simple chains of polypeptides.

 These polypeptide chains usually fold due to the interaction between the amine and
carboxyl group of the peptide link.
 The structure refers to the shape in which a long polypeptide chain can exist.
 They are found to exist in two different types of structures α – helix and β – pleated
sheet structures.
  This structure arises due to the regular folding of the backbone of the polypeptide
chain due to hydrogen bonding between -CO group and -NH groups of the peptide
bond.
 However, segments of the protein chain may acquire their own local fold, which is
much simpler and usually takes the shape of a spiral an extended shape or a loop.
These local folds are termed secondary elements and form the proteins secondary
structure.

(a) α – Helix:
α – Helix is one of the most common ways in which a polypeptide chain forms all
possible hydrogen bonds by twisting into a right-handed screw with the -NH group of
each amino acid residue hydrogen-bonded to the -CO of the adjacent turn of the helix.
The polypeptide chains twisted into a right-handed screw.

(b) β – pleated sheet:

In this arrangement, the polypeptide chains are stretched out beside one another and then
bonded by intermolecular H-bonds. In this structure, all peptide chains are stretched out
to nearly maximum extension and then laid side by side which is held together by
intermolecular hydrogen bonds. The structure resembles the pleated folds of drapery and
therefore is known as β – pleated sheet

3. Tertiary Structure of Protein

 This structure arises from further folding of the secondary structure of the protein.
 H-bonds, electrostatic forces, disulphide linkages, and Vander Waals forces
stabilize this structure.
  The tertiary structure of proteins represents overall folding of the polypeptide
chains, further folding of the secondary structure.
 It gives rise to two major molecular shapes called fibrous and globular.
 The main forces which stabilize the secondary and tertiary structures of proteins
are hydrogen bonds, disulphide linkages, Vander Waals and electrostatic forces of
attraction.


4. Quaternary Structure of Protein

The spatial arrangement of various tertiary structures gives rise to the quaternary
structure. Some of the proteins are composed of two or more polypeptide chains referred
to as sub-units. The spatial arrangement of these subunits with respect to each other is
known as quaternary structure.

The exact amino acid sequence of each protein drives it to fold into its own unique and
biologically active three-dimensional fold also known as the tertiary structure. Proteins
consist of different combinations of secondary elements some of which are simple
whereas others are more complex. Parts of the protein chain, which have their own three-
dimensional fold and can be attributed to some function are called “domains”. These are
considered today as the evolutionary and functional building blocks of proteins.

Many proteins, most of which are enzymes contain organic or elemental components
needed for their activity and stability. Thus the study of protein evolution not only gives
structural insight but also connects proteins of quite different parts of the metabolism.

Rules of Protein Structure

 The type determines the function of a protein.

 A protein’s shape is determined by its primary structure (the amino acid sequence).
 The amino acid sequence within a protein is determined by the encoding sequence
of nucleotides in the gene (DNA).

.FUNCTIONS OF PROTEINS:

 Structural functions: Proteins are called as the building blocks of the body. They are
an essential component of various structures in the cell and tissues. We also find these
proteins in the outer membrane of all cells in the human body. We can also find
structural proteins in hair, skin, and muscles. Proteins often act to strengthen these
structures. Proteins working together can allow movement within the body, such as
contraction of muscles and movement of food through the digestive system etc. They
are needed for the growth, development, healing, and repair of tissues.
 Protective: Proteins are the main constituent of antibodies that protect our body
against antigens and pathogens thus preventing infections.
 Hormonal regulation: Hormones are majorly composed of proteins. Hormones play a
vital role in regulating muscle mass, sex hormones, and growth and development.
 Enzymes: Proteins are called as biological buffers because they, as enzymes, regulate
many different biochemical reactions that are occurring in the body.

 Proteins are synthesised in the cytoplasm in a process termed translation.The typical

protein is constructed from a single set of amino acids. Every protein is specially
equipped for its function. Any protein in the human body can be created from
permutations of only 20 amino acids.

 Protein is a vital part of the human diet.

 The protein content of eg. Muscles contain about 30 percent protein, the liver 20 to 30
percent, and red blood cells 30 percent. Higher percentages of protein are found in
hair, bones, and other organs and tissues with a low water content

 Positive negative attractions between different atoms in the long amino acid strand
cause it to coil on itself again and again to form its highly complex shape. Folded
 proteins may combine with other folded proteins to form even larger more
complicated shapes.
 The folded shape of a protein molecule determines its role in body chemistry.
Structural proteins are shaped in ways that allow them to form essential structures of
the body. Collagen, a protein with a fibre shape, holds most of the body tissues
together. Keratin, another structural protein forms a network of waterproof fibres in
the outer layer of the skin.
 Functional proteins have shapes that enable them to participate in chemical processes
of the body. Functional proteins include some of hormones, growth factors, cell
membrane receptors, and enzymes.
There are seven types of functional proteins: antibodies, contractile
proteins, enzymes, hormonal proteins, structural proteins, storage proteins, and
transport proteins.

TYPES OF FUNCTIONAL PROTEINS:

1. Antibodies: Antibodies are specialized proteins that defend the body against antigens
or foreign invaders. Their ability to travel through the bloodstream enables them to be
utilized by the immune system to identify and defend against bacteria, viruses, and
other foreign intruders in blood. One way antibodies counteract antigens is by
immobilizing them so that they can be destroyed by white blood cells.

2. Contractile Proteins: Contractile proteins are responsible for muscle contraction and
movement. The cytoplasm of cells is a colloidal network of contractile proteins. Actin
filaments are the major components of this network Muscle Cells (Myocyte).
Eukaryotes tend to possess copious amounts of actin, which controls muscle
contraction as well as cellular movement and division processes. Myosin powers the
tasks carried out by actin by supplying it with energy.

3. Enzymes: All enzymes identified thus far are proteins.Enzymes, which are the
catalysts of all metabolic reactions, enable an organism to build up the chemical
substances necessary for life—proteins, nucleic acids, carbohydrates, and lipids—to
convert them into other substances, and to degrade them. Life without enzymes is not
possible.

4. Hormonal Proteins: Hormonal proteins are messenger proteins that help coordinate
certain bodily functions.eg:Growth factors are highly specific proteins, a subdivision
of cytokines. Growth factors stimulate the division and differentiation of a particular
type of cell. In skeletal muscle hypertrophy, growth factors include insulin-like
growth factor (IGF). IGF is secreted by skeletal muscle. It regulates insulin
metabolism and stimulates protein synthesis.Testosterone is an androgen, or a male
sex hormone. The primary physiological role of androgens are to promote the growth
 and development of male organs and characteristics. Testosterone affects the nervous
system, skeletal muscle, bone marrow, skin, hair and the sex organs.Cortisol is a
steroid hormone (hormones which have a steroid nucleus that can pass through a cell
membrane without a receptor) which is produced in the adrenal cortex of the kidney.
It is a stress hormone.

5. Structural Proteins: A large group of structural proteins maintains and protects the
structure of the animal body. The most common example of a structural protein
is collagen which is found in the bones, cells and skin. Structural proteins are also
found in cells. They are used to provide an internal structure to the cell
(the cytoskeleton) and are sometimes involved in cell movement. Structural proteins
are especially important in larger cells.

6. Storage Proteins: Storage proteins reserve amino acids for the body until ready for
use. Examples of storage proteins include Ferritin a storage protein that stores iron.

7. Transport Proteins: Transport proteins are carrier proteins that move molecules
from one place to another in the body. The respiratory protein hemoglobin acts as
oxygen carrier in the blood, transporting oxygen from the lung to body organs and
tissues. Cytochromes, another type of transport protein, operate in the electron
transport chain as electron carrier proteins, Adenosine triphosphate (ATP)

Examples of protein functions

Function Description Example

Antibody Antibodies bind to specific foreign particles, Immunoglobulin

such as viruses and bacteria, to help protect G (IgG)
the body.

Enzyme Enzymes carry out almost all of the Phenylalanine

thousands of chemical reactions that take hydroxylase
place in cells. They also assist with the
formation of new molecules by reading the
genetic information stored in DNA.

Messenger Messenger proteins, such as some types of Growth hormone

hormones, transmit signals to coordinate
biological processes between different cells,
Function Description Example

tissues, and organs.

Structural These proteins provide structure and support Actin

component for cells. On a larger scale, they also allow
the body to move.

Transport/storage These proteins bind and carry atoms and Ferritin

small molecules within cells and throughout
the body.

DIET

Protein is a vital part of the human diet and is present in various foods eg eggs, meats,
dairy, seafood legumes, nuts, and seeds. Irrespective of the source of the protein
consumed, it gets broken down in reformed into new proteins in our bodies.

Most animal proteins are referred to as complete proteins as they contain all nine
essential amino acids whereas most plant proteins are considered to be incomplete as
they are missing at least one of the essential amino acids. Soy products, quinoa and
the seed of a leafy green called amaranth are a few of the plant-based proteins that
contain all nine essential amino acids.
 Protein powder

Human beings aren't able to store protein, for the most part. The human body can
break down its muscle tissue to get certain amino acids, or the building blocks of
protein, but it has no specialized cells to store protein efficiently, as it does fat and
carbohydrate. For this reason, eating protein regularly is of paramount importance.

 The amount of protein we need changes throughout our lifetime and elderly
people will actually need more protein than younger adults.
 Protein-rich foods tend to make people feel fuller for longer compared to fats or
carbohydrates. Proteins do this by increasing thermogenesis and through the
direct effect of its constituent amino acids (notably leucine) on the brain.
 Millions of people take sports supplements hoping for a range of health
benefits, from weight loss to muscle building. But some supplements are being
sold illegally and can be very harmful..

RELATED MEDICAL CONDITIONS:

Proteins serve crucial roles in human biochemistry. The major role is to provide the
body's building blocks. They are the precursors of several biologically relevant
molecules. Therefore either the excess or deficiency of protein can lead to disease
result in nervous system defects, metabolic problems, organ failure, and even death.
eg the most severe form of protein deficiency is called kwashiorkor, Anorexia
Nervosa.

 A dysfunctional protein can lead to a variety of medical conditions and, often,

death.
 Dysfunctional proteins can lead to childhood obesity, breakdown of the retina
leading to blindness, hearing loss, and type 2 diabetes.
Eg the protein cilia and how its dysfunction manifests.

 Inadequate cilia in flagella lead to sperm dysmotility.

 Defective cilia in the respiratory tract lead to chronic lung infections.
 Dysfunctional cilia in Fallopian tubes cause infertility.

Girl with kwashiorkor

 ENZYMES

Introduction to Enzymes:
Enzymes are the organic catalysts produced by living organisms. They are
biological catalyst and increase the rate of chemical reaction. They make possible the
many complex chemical reactions that make up life processes. Although produced by
living organisms, enzymes are lifeless. When isolated, they still exert their
characteristic catalytic effect.

Properties of Enzymes:
1. Enzyme are colloids and are soluble in water and dilute alcohol, but are
precipitated by concentrated alcohol.
2. Most enzyme act best at temperature between 35 - 40 0C, temperature above
65oC, especially in the presence of moisture, usually completely destroy them,
where as their activity is negligible at 0oC.
3. Enzymes are sensitive to heat and are denatured by excess heat or cold, i.e.
their active site becomes permanently warped, thus the enzyme is unable to
form an enzyme substrate complex. This is what happens when you fry an egg,
the egg white (Augmentin, a type of protein, not an enzyme), is denatured.
4. Certain heavy metals, formaldehydes and free iodine retard the enzyme
activity. There activity is markedly affected by the pH of the medium in which
they act or by the presence of other substances in this medium.
5. They are highly selective in their action.
6. The enzymes are proteins that range in molecular weight from about 13,000 to
as much as 840,000 Dalton.
7. Minute quantities of enzymes are required to complete a chemical reaction.
8. Enzymes are created in cells but are capable of functioning outside of the cell.
This allows the enzymes to be immobilized, without killing them.
9. Enzymes are reusable and some enzymes are capable of catalyzing many
hundreds of thousands of reactions, for example, catalase working on hydrogen
peroxide, try putting some liver into hydrogen peroxide.
10. Enzymes will only catalyze one reaction, for example, invertase will only
produce glucose and fructose, when a glucose solution is passed over beads of
enzyme.
 11. Enzymes are capable of working in reverse, this act as a cut off point for the
amount of product being produced. If there are excess reactants, the reaction
will keep going and be reversed, so that there is no overload or buildup of
product.
12. Enzymes are lifeless and when isolated, they still exert their characteristic
catalytic effect.
13. Their chemical composition varies, and
they do show several common
properties.

Classification of Enzymes:
There are several methods for classification of enzymes. Few are:

 Classification on the basis of reaction they control

 Classification on the basis of site of action
 Classification on the basis of nature of substrate

CLASSIFICATION ON THE BASIS OF REACTION THEY

CONTROL:
Enzymes are presently classified according to their action by Commission on
Enzymes of the International Union of Biochemistry. Under this heading,
enzymes can be classified in six major classes; each class has 4 to 13 subclasses
and each enzyme is assigned with a systematic code number (E.C.) comprised of
four digits.

1. Oxidoreductase
2. Transferase
3. Hydrolases
4. Lyases
5. Isomerases
6. Ligases

i. Oxidoreductases:
These enzymes are also called as redox enzymes. This class comprises the
enzymes which were earlier called dehydrogenases, oxidases, peroxidases,
hydroxylases, oxygenase’s etc. The group, in fact, includes those enzymes which
bring about oxidation-reduction reactions between two substrates (substances), S and
S′.

Sreduced + S′oxidized → Soxidized + S

′reduced
 More precisely, they catalyze electron transfer reactions. In this class are included
the enzymes catalyzing oxidoreductions of CH—OH, C=O, CH—CH, CH—
NH2 and CH=NH groups.

Examples are:

 Alcohol: NAD oxidoreductase [Common or trivial name,

Alcohol dehydrogenase]

Alcohol + NAD → Aldehyde or Ketone + NADH + H+

 Glucose 6-phosphate: NADP+

𝑁𝐴𝐷𝑃+→𝑁𝐴𝐷𝑃𝐻 +𝐻+
Glucose 6 – phosphate −−−−−−−−−−−−−−−→ 6 - Phosphoglucolactone

Other Oxidoreductases are:

 O2 oxidoreductase
 H2O2 oxidoreductase

ii. Transferases:
These enzymes bring about a transfer of functional groups such as phosphate,
amino, acyl and methyl from one molecule to another molecule called donor and
acceptor molecules respectively.

S—G + S′ → S + S′—G

In these are included the enzymes catalyzing the transfer of one-carbon groups,
aldehydic or ketonic residues and acyl, glycosyl, alkyl, phosphorus or sulfur-
containing groups.

 Acyltransferases. For example: Acetyl-CoA : choline O-acetyltransferase

[Choline acetyltransferase]

Acetyl-CoA + choline → CoA + O-acetylcholine

Other Examples are:

 Glycosyltransferases
 Orthophosphate glycosyl transferase [Phosphorylase]
 D-hexose-6-phosphotransferase [Hexokinase]
 Methyl transferase
 Acyl transferase

iii. Hydrolases:
These catalyze the hydrolysis of their substrates by adding constituents of
water across the bond they split. The substrates include ester, glycosyl, ether, peptide,
acid-anhydride, C—C, halide and P—N bonds. For example:

 Enzymes acting on ester bonds e.g. Glycerol ester hydrolase [Lipase]

 A triglyceride + H2O → A diglyceride + a fatty acid

 Enzymes acting on glycosyl compounds e.g. β-D-galactosidase galactohydrolase

[β-galactosidase]

A β-D-galactosidase + H2O → An alcohol + D-galactose

 Enzymes acting on peptide bonds

Here the classical trivial names (pepsin, trypsin, thrombin, plasmin etc.) have been
largely retained due to their consistent long usage and also due to dubious specificities
which make systematic nomenclature almost impractical at this time.

 Enzymes acting on C—N bonds, other than peptide bonds E.g. L-arginine Ure
hydrolase [Ariginase]

L-arginine + H2O → L-ornithine + urea

iv. Lyases (Demolases)

These are those enzymes which catalyze the removal of groups from substrates
by mechanisms other than hydrolysis, leaving double bonds.

In these are included the enzymes acting on C—C, C—O, C—N, C—S and C—
halide bonds.

 Carbon-carbon lyases e.g. Ketose-1-phosphate aldehyde-lyase [Aldolase]

A ketose-1-phosphate → Dihydroxyacetone phosphate + an
aldehyde

 Carbon-oxygen lyases e.g. L-malate hydro-lyase [Fumarase]

𝐹𝑢𝑚𝑎𝑟𝑎𝑠𝑒
L-malate −−−−−−−→ Fumarate + H2O

 Carbon-nitrogen lyases e.g. L-histidine ammonia-lyase [Histidase]

L-histidine → Urocanate + NH3

v. Isomerases:
These catalyze inter-conversions of optical, geometric or positional isomers by
intramolecular rearrangement of atoms or groups. For Example:
 Racemases and epimerases e.g. Alanine racemase

L-alanine → D-alanine

 Cis-trans Isomerases e.g. All trans-retinene 11-cis-trans isomerase [Retinene

isomerase]

All trans-retinene → 11-cis-retinene

 Intramolecular oxidoreductases e.g. D-glucose-6-phosphate keto-

isomerase [Glucosephosphate isomerase]

D-glucose-6-phosphate → D-
fructose-6-phosphate

vi. Ligases (Synthetase):

These are the enzymes
catalyzing the linking together of two
compounds utilizing the energy made
available due to simultaneous
breaking of a pyrophosphate bond in
ATP or a similar compound. This
category includes enzymes catalyzing
reactions forming C—O, C—S, C—N
and C—C bonds.

 Enzymes catalyzing formation of C—S bonds For example: Acetate : CoA

ligase (AMP) [Acetyl-CoA synthetase]

ATP + acetate + CoA → AMP + pyrophosphate + acetyl-CoA

 Enzymes catalyzing formation of C—N bonds For example : L-glutamate :

ammonia ligase (ADP) [Glutamine synthetase]

ATP + L-glutamate + NH3 → ADP + orthophosphate + L-glutamine

 Enzymes catalyzing formation of C—C bonds. For example : Acetyl-CoA : CO2

ligase (ADP) [Acetyl-CoA carboxylase]

ATP + acetyl-CoA + CO2 + H2O → ADP + orthophosphate + malonyl-

CoA

To date, over 2,000 different enzymes are known, of which

the oxidoreductases, transferases and hydrolases predominate. Because
official names are
 often lengthy, the trivial names of enzymes are generally used after initial
identification.

CLASSIFICATION ON THE BASIS

OF SITE OF ACTION.
Under this heading, enzymes are classified into two types:

 Endoenzymes
 Expenzymes

1. Endoenzymes:
 Most of the enzymes usually act within the cells in which they are produced
and hence are called intracellular enzymes or endoenzymes.
 E.g. most of the plant enzymes. As these enzymes catalyze the metabolic
reactions of the cell, they are also referred to as metabolic enzymes.
 Examples are synthetase, isomerase and phosphorylase.

2. Exoenzymes:
 Certain enzymes which are liberated by living cells catalyze useful reactions
outside the cell in its environment and hence are known as extracellular
enzymes or exoenzymes, e.g., enzymes found in bacteria, fungi and some
insectivores like Drosera and Nepenthes.
 They act chiefly as digestive enzymes, catalyzing the breakdown of complex
substances to simpler ones which can readily be absorbed by the cell. Such
enzymes are digestive in their function i.e. breakdown complex molecule into
small units
 E.g. proteases, lipases, amylases.

CLASSIFICATION ON THE BASIS OF CHEMICAL NATURE

 Many enzymes are inactive when first produced. Such enzymes in inactive form
are called pro-enzymes or zymogens. They are inactive because the active site is
not exposed and unable to bind with substrate.
 Later by the action of essential coenzymes (organic in nature, loosely attached
with molecule e.g. NAD, FAD, NADP) or activator they made active.
  Enzymes often occur in combination with inorganic and organic substances that
have an important part in the catalytic action. If these are non protein organic
compounds known as coenzymes. If they are inorganic ions, they are referred as
activator e.g. Hexokinase (Mg2+), Arginiosuccinate (Cu2+), Carbonic anhydrase
(Zn2+), Phosphofructokinase (Fe2+).
 Coenzymes are integral part of enzyme system. Several vitamins, thiamine
chloride, riboflavin, nicotinic acid are recognized as having a coenzymatic
function. Both are collectively called cofactors. The cofactors bound to the protein
part of enzyme tightly. In this case cofactor is termed as prosthetic cofactor or
prosthetic group. It may be organic or inorganic.
 In globular structure of enzymes, one or more polypeptide chains twist and fold,
bringing together a small number of amino acids to form active site. Active site is
that location on the enzyme where the substrate binds. Enzyme and substrate fails
to bind if there shape does not match exactly.

CLASSIFICATION ON THE BASIS OF SUBSTRATE

On the basis of substrate type enzymes are classified into following types:

1. Esterases:
An esterase is a Hydrolases enzyme that splits esters into an acid and an
alcohol in a chemical reaction with water called hydrolysis. A wide range of
different Esterases exist that differ in their substrate specificity, their protein structure,
and their biological function. E.g. lipase, phospholipase and acetylcholinesterases etc.

2. Carbohydratases:
E.g. diastase, lactase, maltase, Invertase, cellulose, lysozyme and others

3. Nucleases:
 A nuclease is an enzyme capable of cleaving the phosphodiester bonds between
monomers of nucleic acids. E.g. Ribonuclease, deoxyribonuclease, and others.

4. Nuclein deaminases:
Includes adenase, adenosine deaminase, and others

5. Amidases:
Amidase e.g. deaminase, fatty acylamidase, N-acetylaminohydrolase
(ambiguous)) is an enzyme that catalyzes the hydrolysis of an amide.

6. Proteolytic Enzymes
Proteolytic enzymes (or proteases) refer to the various enzymes that digest
(break down into smaller units) protein. These enzymes include the
pancreatic proteases chymotrypsin and trypsin, bromelain (pineapple enzyme), papain
(papaya enzyme), fungal proteases, and Serratia peptidase (the ―silk worm‖ enzyme).

Enzyme and Catalyst:

Similarities:

 Both catalyst and enzyme increase the rate of a chemical reaction by

lowering the activation energy.
 Both catalyst and enzyme are not changed by the reaction.
 Both catalyst and enzyme temporary bind to their substrates.
 The rate of both forward and backward reactions are increased by catalysts
and enzymes.
 Both catalyst and enzyme have no effect on the equilibrium constant of the
reaction
Chemical Nature Enzymes:

All enzymes are proteinaceous in nature (Sumner, 1926) with the exception of
recently discovered RNA enzymes. Some enzymes may additionally contain a non-
protein group.

On the basis of differences in chemical nature, the enzymes may be described

as follows:

(i) Simple Enzymes:

Some enzymes are simple proteins, i.e., on hydrolysis, they yield amino acids
only. Digestive enzymes such as pepsin, trypsin and chymotrypsin are of this nature.

(ii) Conjugate Enzymes:

It is an enzyme which is formed of two parts – a protein part called apoenzyme
(e.g., flavoprotein) and a non-protein part named cofactor. The complete conjugate
enzyme, consisting of an apoenzyme and a cofactor, is called holoenzyme.

There can be an enzymatic activity only when both components (apoenzyme

and cofactor) are present together. The cofactor is sometimes a simple divalent
metallic ion (e.£.,Ca, Mg, Zn, Co, etc), and sometimes a nonprotein organic com-
pound. However, some enzymes require both kinds of cofactors. If the cofactor is
firmly bound to the apoenzyme, it is called prosthetic group.

For example, cytochromes are the enzymes that possess porphyrins as their
prosthetic groups. If, instead of being more or less permanently bound to the
apoenzyme the cofactor attaches itself to the apoenzyme only at the time of reaction, it
is called a coenzyme.

(iii) Metallo-enzymes:

The metal cofactors involved in enzymic reactions are both monovalent (K+)
and divalent cations (Mg++, Mn++, Cu++). These may be loosely held by the
enzyme, or as in some cases, go into the composition of the molecule itself. If the
metal forms part of the molecule, as iron of haemoglobin or cytochrome is, the
enzymes are called metallo-enzymes.

(iv) Isoenzymes (Isozymes):

At one time it was believed that an organism has only a single enzyme for a
given step of a metabolic reaction. It was later discovered that a substrate may be
acted upon by a number of variants of an enzyme producing the same product.
 The multiple molecular forms of an enzyme occurring in the same organism
and having a similar substrate activity are called isoenzymes or isozymes. Over 100
enzymes are known to have isoenzymes. Thus a-amylase of wheat endosperm has 16
isozymes, lactic dehydrogenase has 5 isoenzymes in man, while alcohol
dehydrogenase has 4 isozymes in maize. Isoenzymes differ in activity optima and
inhibition.

The most thoroughly studied isozyme is lactic dehydrogenase (LDH) which

occurs in five possible forms in organs of most vertebrates as observed by starch gel
electrophoretic separation. Two basically different types of LDH occur. One type,
which is inhibited strongly by relatively low concentrations of pyruvate, predominates
in the heart and is called heart LDH.

The other type, less easily inhibited by pyruvate, occurs in many skeletal
muscles and is thus called muscle LDH. The heart LDH consists of 4 identical
subunits, which are called H subunits. The muscle LDH consists of 4 identical M
subunits. The two types of subunits, H and M, have different amino acid
compositions, enzyme kinetics and immunological properties. These subunits in
different combinations produce 5 isoenzymes.

They are thus useful to organism in adapting to varied environmental conditions.

Factors Affecting Enzyme Activity:

1- Effect of Substrate Concentration (Single-Substrate Reactions)
2- Effect of Inhibitors
3- Effect of pH
4- Effect of
Temperature 5- Effect of
Pressure
6- Effect of Water
 Enzymes in food can be detected only indirectly by measuring their catalytic
activity and, in this way, differentiated from other enzymes.
 This is the rationale for acquiring knowledge needed to analyze the parameters
which influence or determine the rate of an enzyme-catalyzed reaction.
 The reaction rate is dependent on the concentrations of the components involved
in the reaction.
 Here, it means primarily the substrate and the enzyme. Also, the reaction can be
influenced by the presence of activators and inhibitors.
 Finally, the pH, the ionic strength of the reaction medium, the dielectric
constant of the solvent (usually water) and the temperature exert an effect.
1. Effect of Substrate Concentration (Single-Substrate Reactions):
 Let us consider a single-substrate reaction.
 Enzyme E reacts with substrate A to form an intermediary enzyme-substrate
complex, EA.
 The complex then forms the product P and releases the free enzyme:

 In order to determine the catalytic activity of the enzyme, the decrease in substrate
concentration or the increase in product concentration as a function of time can be
measured.
 The activity curve obtained has the following regions:
o The maximum activity which occurs for a few msec until equilibrium is
reached between the rate of enzyme-substrate formation and rate of breakdown
of this complex.
 Measurements in this pre-steady state region which provide an insight into
the reaction steps and mechanism of catalysis are difficult and time
consuming.
 Hence, further analysis of the pre-steady state should be ignored.

(Progress of an enzyme-catalyzed reaction)

o The usual procedure is to measure the enzyme activity when a steady state has
been reached. In the steady state, the intermediary complex concentration
remains constant while the concentration of the substrate and end product are
changing.
o The reaction rate decreases in this region in spite of an excess of substrate. The
decrease in the reaction rate can be considered to be result of:
 Enzyme denaturation which can readily occur
 Decreasing the enzyme concentration in the reaction system, or the product
formed increasingly inhibits enzyme activity or, after the concentration of
the product increases,
  The reverse reaction takes place, converting the product back into the
initial reactant.

2. Effects of Inhibitors:
 The catalytic activity of enzymes, in addition to substrate concentration, is affected
by the type and concentration of inhibitors, i.e. compounds which decrease the rate
of catalysis, and activators, which have the opposite effect.
 Activitors include
 metal ions and
 Compounds which are active as prosthetic groups or which provide stabilization
of the enzyme‘s conformation or of the enzyme-substrate complex.
 Inhibitors are found among food constituents.
 Based on kinetic considerations, inhibitors are divided into two groups:
o inhibitors bound irreversibly to enzyme and
o inhibitors bound reversibly to enzyme .
 Proteins which specifically inhibit the activity of certain peptidases, amylases or
β- fructofuranosidase are examples.
 Furthermore, food contains substances which non-selectively inhibit a wide
spectrum of enzymes.
 Phenolic compounds of food and mustard oil belong to this group.
 Food might be contaminated with pesticides, heavy metal ions and other chemicals
from a polluted environment which can become inhibitors under some
circumstances.
 These possibilities should be taken into account when enzymatic food analysis is
performed.
 Food is usually heat treated to suppress undesired enzymatic reactions.
 As a rule, no inhibitors are used in food processing.
 An exception is the addition of, for example, SO2 to inhibit the activity of
phenolase.
 Much data concerning the mechanism of action of enzyme inhibitors have been
published in recent biochemical research.
 These data cover:
o the elucidation of the effect of inhibitors on functional groups of an enzyme
o the effect of inhibitors on the active site and
o the clarification of the general mechanism involved in an enzyme catalyzed
reaction.

3. Effect of pH on Enzyme Activity:

 Each enzyme is active only in a narrow pH range and each has a pH optimum
which is often between pH 5.5 and 7.5.
 The optimum pH is affected by the type and ionic strength of the buffer used in
the assay.

 The reasons for the sensitivity of the enzyme to changes in pH are:

o Sensitivity is associated with a change in protein structure leading to
irreversible denaturation
o The catalytic activity depends on the quantity of electrostatic charges on the
enzyme‘s active site generated by the prototropic groups of the enzyme.
 In addition the ionization of dissociable substrates as affected by pH can be of
importance to the reaction rate.
 However, such effects should be determined separately.

4. Influence of Temperature:
 Thermal processes are important factors in the processing and storage of food
because they allow the control of chemical, enzymatic and microbial changes.
 Undesired changes can be delayed or stopped by refrigerated storage. Heat
treatment may either accelerate desirable chemical or enzymatic reactions or
inhibit undesirable changes by inactivation of enzymes or microorganisms.
 Temperature and time are two parameters responsible for the effects of a thermal
treatment.
 They should be selected carefully to make sure that all necessary changes, e. g.,
killing of pathogens, are guaranteed, but still all undesired changes such as
degradation of vitamins are kept as low as possible.
 Enzyme-catalyzed reactions and the growth of microorganisms show a so-called
temperature optimum, which is a temperature-dependent maximum resulting from
the overlapping of two counter effects with significantly different activation
energies:
 o increase in reaction or growth rate
o increase in inactivation or killing rate

5. Influence of Pressure:
 The application of high pressures can inhibit the growth of microorganisms and
the activity of enzymes.
 This allows the protection of sensitive nutrients and aroma substances in foods.
 Some products preserved in this gentle way are now in the market.
 Microorganisms are relatively sensitive to high pressure because their growth is
inhibited at pressures of 300–600 MPa and lower pH values increase this effect.
 However, bacterial spores withstand pressures of >1200 MPa.
 In contrast to thermal treatment, high pressure does not attack the primary
structure of proteins at room temperature.
 Only H-bridges, ionic bonds and hydrophobic interactions are disrupted.
Quaternary structures are dissociated into subunits by comparatively low pressures
(<150 MPa).
 Higher pressures (>1200 MPa) change the tertiary structure and very high
pressures disrupt the H-bridges which stabilize the secondary structure.
 The hydration of proteins is also changed by high pressure because water
molecules are pressed into cavities which can exist in the hydrophobic interior of
proteins.
 In general, proteins are irreversibly denatured at room temperature by the
application of pressures above 300 MPa while lower pressures cause only
reversible changes in the protein structure.

6. Influence of Water:
 Up to certain extent, enzymes need to be hydrated in order to develop activity.
 Hydration of e.g. lysozyme was determined by IR and NMR spectroscopy.
 Enzymatic activity starts at a water content of 0.2 g/g protein, which means
even before a monomolecular layer of the polar groups with water has taken
place.
 Increase in hydration resulting in a monomolecular layer of the whole available
enzyme surface at 0.4 g/g protein raises the activity to a limiting value reached at a
water content of 0.9 g/g protein. Here the diffusion of the substrate to the enzyme‘s
active site seems to be
completely guaranteed.
 For preservation of food it is
mandatory to inhibit enzymatic
activity completely if the
storage temperature is below
the phase transition
temperature.

7. Effect of
Product
Concentration:
The accumulation of
reaction products generally
decreases the enzyme velocity.
For certain‘ enzymes, the products
combine with the active site of
enzyme and form a loose complex
and, thus, inhibit the enzyme activity. In the living system, this type of inhibition is
generally prevented by a quick removal of products formed.

8. Effect of Activators:
Some of the enzymes require certain inorganic metallic cations like Mg2+,
Mn2+, Zn2+, Ca2+, Co2+, Cu2+, Na+, K+ etc. for their optimum activity. Rarely, anions are
also needed for enzyme activity e.g. chloride ion (CI–) for amylase.

Some Important Enzymes:

1. The amylolytic Enzyme or carbohydrates:
 Diastase and amylase: Term applied to 2 well known amylolytic enzymes
salivary diastase or ptyalin and pancreatic diastase or amylopsin are found in
digestive tract of animals
 Malt diastase: It is formed during the germination of barley grains and
converts starch into lactose.
 Invertase or sucrase: Found in yeast and intestinal juices. It brings hydrolysis
of sucrose into glucose and fructose.
  Maltase: cause conversion of maltose into glucose.
 Zymase: Fermentation enzyme cause conversion of monosaccharides into
alcohol and carbon dioxide.
 Emulsin: This enzyme is found in almonds. It causes hydrolysis of beta-
glucosides; thus, amygdalin is hydrolyzed into to glucose, HCN and
benzaldehyde.
 Myrosin: It is found in white and black mustard; it hydrolyzes Sinalbin,
Sinigrin and other glycoside.

2. The Esterases:
 Lipase: it is a lipolytic enzyme widely distributed in animal and vegetable
kingdoms. Found in pencreatic juice of animals and oily seeds.
 Pectase: splits pectin into pectic acid and methyl alcohol.
 Steapsin: it is lipolytic enzyme capable of methyl alcohol.
 Urease: obtained from soybeans and used in laboratory reagent for
converting urea to ammonia.

3. The Proteolytic Enzyme:

 Pepsin: is protolytic enzyme found in the gastric juice. And active at pH 1.8.
 Trypsin: it is formed when the proenzyme trypsinogen is acted on by the
enterokinase of the intestinal juice. It is more active than pepsin and converting
proteoses and peptones into polypeptides and amino acids. It is active at pH 8.
 Erepsin: it is found in intestinal juices. It converts proteoses and peptones into
amino acids.
 Rennin: it is a coagulating enzyme present in the mucous membrane of the
stomach of mammals. It curdles the soluble casein of milk.
 Papain: it is a mixture of active proteolytic enzymes found in the unripe fruit
of the papaya tree.

4. The Oxidizing Enzymes:

 Peroxides: are widely distributed in plants, they bring about the oxidation
reactions that cause the discoloration of bruised fruits.
 Thrombin: converts the fibrinogen of the circulating blood into the
insoluble fibrin of the blood clot.
 Zymase: splitting of monosaccharides by oxidation.
 Enzymes Obtained From Plant Source
(Phytoenzymes):
Enzymes that have natural plant origin are called as Phytoenzymes e.g.

I. Malt Extract
II. Papain
III. Bromelain

MALT EXTRACT.
Introduction:
 Malt extract is a type of phyto-enzyme.
 It is obtained by extracting malt or malted barley, which is partially and
artificially germinated grain of one or more verities of barley grain.

Synonyms:
 Diastase
 Malt Extract
 Malt or Malted Barley

Biological Source:
Hordeum vulgare

Family:
Germinae

Part Used:
Dried Grain

Geographical Source:
 Barley is widely cultivated throughout the world wherever climate is suitable.
 The major producers are United States, Russia, Canada, India, and Turkey.
 It is also cultivated in highlands of China and Tibet.
 Cultivation and Collection:

 Barley is one of the oldest cultivated cereals.

 It is an annual erect stout herb resembling wheat.
 The crop becomes ready for harvest in about four months after sowing.
 The grains are threshed out by beating with sticks or trampling by oxen.
 Dried barley grains are artificially germinated by keeping their heaps wet with
water in a warm room.
 When the caulicle (projection during germination) of the grains starts protruding
out, the germinated grams are dried quickly.
 The enzyme diastase in the moist warm grains converts the starch to maltose,
thereby stimulating the embryo to growth. The embryo is killed when the grain is
dried.
Preparation:
 Dry germinated barley or dry malt is subjected to extraction.
 The malt is infused with water at 60°C.
 An infusion is concentrated below 60°C under reduced pressure and then dried.
 Malt extract is mixed with 10% glycerin.
 Less purified malt extract contains sugars (dextrin, maltose, glucose), and
amylolytic enzymes.
 Its further purification affords diastase.
 It can convert not less than 5 times its weight of starch into water soluble
sugars.

Description:
 It occurs as yellowish-brown or amber colored viscous liquid.
 Dry malt resembles barley.
 It has agreeable odour and sweet taste.

Chemical Characteristics:
 Malt extract contains enzymes, which are most active in neutral solution.
 The acidic conditions destroy the activity.
 It converts starch into disaccharide maltose.
 The enzyme is destroyed by heat.
 Many heat sterilized malt extracts do not contain diastase.
 It is completely soluble in cold water, more readily in warm water.
 The aqueous solution shows flocculant precipitate on standing.
 Limit for arsenic should not exceed one part per million.
Chemical Constituents:
Malt extract contains:

 Sugar maltose.....................................…. 50-70%

 Dextrins................................................... 2-15%
 Proteins.....................................................8%
 Amylolytic enzyme Diastase...................8%
 Traces of glucose and Peptase Emzyme

Classes:
1. Standard Malt:

It can be thought of as the original starch or grain based sweetener.

2. Specialty and Black Malt Extract:

It can be used for yeast food, brewing or flavoring agent.

3. Co-Extracts of Malt:

Un-malted grains or starch sources can be converted into extracts using malted
barley as a natural enzyme source in extraction process. This is done mostly for
economy and in some cases, to make lighter flavored syrup.

Dose:
Usually 15g

Note
:
Many commercial extracts of malt do not contain diastase, which is destroyed
by the heat used for their sterilization. Such extracts should not be confused with this
product.

Uses:
 Malt is used in brewing & alcohol industries.
 Malt extract and purified diastase, both are used as amylolytic enzymes.
 It is used as an aid in digesting starch.
 They are used as bulk producing laxatives.
 Malt extract is used as easily digested nutritive.
 Diastase i.e. in starch sugars is 50 times than its own weight.
  Lactase converts lactose galactose and glucose. It is used in patients having
lactose intolerance.
 It is used as flavoring agent to cover bitter taste.
 It is used as a vehicle for preparation of cod liver oil and halibut liver oil.

Commercial Product:
Maltsupex

2. PAPAIN
Introduction:
Papain is a Phytoenzymes which contains a mixture of proteolytic enzymes
found in the unripe fruit of papaya tree.

Synonyms:
 Papayotin
 Vegetable pepsin
 Tromasin
 Arbuz
 Papaya proteinase

Biological Source:
Carica papaya

Family:
Caricaceae

Part Used:
Latex of unripe fruit

Geographical Source:
The papaya tree is indigenous to tropical America and is cultivated in Sri
Lanka, Tanzania, Hawaii, and Florida.

Plant Description:
  Plant attains a height of about 5 or 6 meters.
 The fruit grows to a length of about 30 cm (12 inches) and a weight of 5 kg.

Cultivation and Collection:

 Papaya is planted during spring (February – March), monsoon (June-July)
and autumn (October-November).
 The epicarp adheres to the orange-colored, fleshy sarcocarp, which
surrounds the central Cavity.
 This cavity contains a mass of nearly black seeds.
 Papain is distributed throughout the plant, but mostly concentrated in the
latex of the fruit.
 The full grown but unripe fruit is subjected to shallow incisions on the 4 sides.
 Incisions are about 1/8 inch deep.
 The incisions are made early in the morning, at intervals of three to seven days.
 The latex flows freely for a few seconds, but soon coagulates which is
collected with help of scrapper.

Preparation:
 After collection, the coagulated lumps are shredded and dried by the sun or by
the use of artificial heat.
 Incisions and collections are made at weekly intervals as long as the fruit
exudes the latex.
 The crude papin is purified by dissolving in the water and precipitating with
alcohol.

Spray Drying Method:

 Latex of these fruits is collected in aluminium trays.
 To the collected latex, potassium metabisulphaite is added.
 The extraneous matter is cleared out by passing through sieves and latex is
dried in vacuum shelf drier at 55 – 60.
 This latex is called papain.

Note:
 Rapid drying or exposure to sun or higher temperature above 38°C produce
dark colour product with weak in proteolytic activity.
 The use of artificial heat yields the better grade of crude papain.
 The final product should be creamy white and friable.
  It is sealed in air-tight containers to prevent loss of activity.
 If 10% common salt or 1% solution of formaldehyde is added before drying,
the product retains its activity for many months.
 Fully grown fruits give more latex of high enzyme potency than smaller or
immature fruits. The yield of Papain varies from 20 to 250 g per tree. The yield
of commercial papain from latex is about 20%.

Description:
 Papain is available as light brown or grayish white colored amorphous with
typical odor and taste.
 It is a hygroscopic powder.
 It shows maximum proteolytic activity between pH 5-6.
 It is insoluble in water and glycerin.
 A temperature range of 60–90°C is favourable for the digestive process with
65° the optimum point.
 Crystalline papain is most stable in the pH range 5–7 and is rapidly destroyed
at 30°C below pH 2.5 and above pH 12.
 It is resistant to heat, inactivated by metal ions, oxidants and reagents which
react with thiols, and is an endopeptidase activated by thiols and reducing
moieties, for example, cysteine, thiosulphate, and glutathione

Chemical Nature:
Mixture of enzymes which acts upon polypeptide and amides

Chemical Constitutes:
Papain is referred to as ―Vegetable Pepsin‖ because it contain enzyme similar
to that of Pepsin. Papain; however, unlike pepsin, papain acts in acid, neutral, or
alkaline media. Papain contains one or more proteolytic enzymes because a single
sample of papain yield variable result with the type of protein used.

 One or more proteolytic enzymes, among which is peptidase I, capable of

converting proteins into dipeptides and polypeptides
 A rennin like, coagulating enzyme that acts on the casein of milk
 An amylolytic enzyme; a clotting enzyme similar to pectase and an enzyme
that has a feeble activity on fats.
 Although differing in strength in accordance with the method of
manufacture, papain can digest about 35 times its own weight of meat.

Identification:
  It decolorizes aq. Potassium permanganate solution
 It causes curdling of milk (prototypic activity)

Test:
Papain is reacted with a gelatin solution at 80°C in the presence of an activating
cysteine chloral hydrate solution for an hour. The solution is cooled to 4°C for long
time. The treated solution must not regel in comparison to a blank solution under
identical conditions.

Uses:
 Papin is anti-inflammatory.
 It is used in clarification of beverages.
 Papain is used to prevent adhesions; in infected wounds
 Internally it is used as protein digestant, as anathematic (nematode)
 Papain is used as a digestant for proteins because it has an action much like
that of pepsin.
 The best grade of papain digests 300 times its own weight of egg albumin.
 It is employed to relieve the symptoms of episiotomy (surgical incision of
the vulva for obstetric purposes).
 It is used to tenderize meats.
 In the meat packing industry, papain is used extensively for tenderizing beef.
 It is used in cheese manufacturing and acts as substitute of renin.
 It causes degumming of silk fiber
 It is used in digestive mixtures, liver tonics, for reducing enlarged tonsils, in
prevention of postoperative adhesions, curbuncles, and eschar burns.
 It is an allergic agent causing severe paroxysmal cough, vasomotor rhinitis and
dyspnea.
 It is a powerful poison when injected intravenously in surgery to reduce
incidence of blood clots where thromboplasma is undesirable and for local
treatment of buccal, pharyngeal, and laryngeal disorders.

Adulteration:
Commercial papain is often adulterated with arrowroot starch, dried milk of
cactus, gutta percha, rice flour, and pepsin.

Dose:
Oral Therapy
  For herpes zoster (shingles): An enzyme combination containing papain
for 14 days
 For pharyngitis: Lozenges containing 2 mg papain, 5 mg lysozyme, and 200
I.U. bacitracin for 4 days

Podiatry Product:
Caroid and Papase

2.BROMELAIN.
Synonyms:
 Bromelin
 Bromelain

Introduction:
 Bromelain or bromelin is a protein digesting and milk-clotting enzyme
 It is obtained from the juice of the pineapple plant.

Biological Source:
Ananas comosus

Family:
Bromeliaceae

Part Used:
 Although this enzyme can appear in the juice of the fruit, it can also occur in
the stem of the plant.
 It differs from papain because it is obtained from both the ripe and unripe
fruits.

Geographical Source:
Pineapple is a native of tropical America. It is grown in almost all parts of the
world including India, China, Thailand, United States, Brazil, Philippines, Mexico,
Hawaii, and Taiwan.
Cultivation and Collection:
 Bromelin is found in pineapple fruit juice and stem.
 Pineapple is perennial, and it does not have a natural period of dormancy.
 It is propagated through suckers, slips, and crowns.
 In Pakistan it is planted in August, the plant generally flowers in February–
March, and the fruit ripens during July–October.
 The fruits must be left on the plant to ripen for the full flavour to develop.
 Dark green unripe fruits gradually change to yellow and finally to deep orange.

Preparation:
 The fruits are cut off.
 The enzyme bromelin does not disappear as the fruit ripens.
 The enzyme from fruit and stem are known as fruit bromelin and stem
bromelin, respectively.
 It is isolated from pineapple juice by precipitation with acetone and also
with ammonium sulphide

Description:
 It is odorless to slightly putrid buff color powder with irritating taste.
 It is soluble in water, insoluble in organic solvents like ether, chloroform
and alcohol.
 The optimum pH of bromelain is 5.0–8.0.
 In solution pH below 3.0 and above 9.5 inactivates the enzyme.
 The optimum temperature is between 50 and 60°C, still it is effective
between 20 and 65°C too.
 The moisture content should not exceed 6%.

Chemical Constituents:
 Bromelain is not a single substance, but rather a collection of enzymes and
other compounds.
 It is a mixture of sulphur-containing protein-digesting enzymes, called
proteolytic enzymes or proteases.
 It also contains several other substances in smaller quantities, including
peroxidase, acid phosphatase, protease inhibitors, and calcium.

Uses:
  Bromelain is used as adjunctive therapy to reduce inflammation and edema
and to accelerate tissue repair, especially following episiotomy.
 Its effectiveness in such conditions is apparently owing to depolymerization
and permeability modifications that it induces following oral administration.
 Bromelain is also employed in the production of protein hydrolysates, in
tenderizing meats, and in the leather industry.

Dose
:
Oral Dose

 For osteoarthritis: a combination product (Phlogenzym), which contains rutin

100 mg, trypsin 48 mg, and bromelain 90 mg, given as 2 tablets 3 times daily
has been used.

Prescription Product:
Ananase

Enzymes Obtained From Animal Source:

3. PEPSIN
Other Names:
 Peptide hydrolyser
 Protein digesting enzyme

Introduction:
 This enzyme has animal source.
 It is a type of proteolytic enzyme.
 This enzyme is obtained from the glandular layer of the fresh stomach of
various animals like pig, sheep or calf but commonly from pig.

Biological Source:
Sus scrofa

Family:
Suidae
Part Used:
Glandular layer of fresh stomach

Note
:
The generic name ‗Sus‘ is from the Greek, meaning HOG; ‗Scrofa‘ is latin and
means BREADING; and ‗Domesticus‘ is latin and means THE HOUSE HOLD.

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 37ºC 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.

Types of Pepsin Based on Concentration:

 Concentrated solution containing precipitates is poured on glass plates to
dry; forming the Scale pepsin.
 Or concentrated solution is carefully evaporated in vacuum; forming the
spongy pepsin.

Description of Enzyme:
 Pepsin is lustrous, transparent or translucent scales or occurs as granular or
spongy masses.
 Its colour ranges between light yellow to light brown or as fine white or
cream colored amorphous powder.
 It is free from offensive odour and has a slightly acid or saline taste.
 It shows optimal activity at a pH 1.8 to 3.5.
 It is reversibly inactive at pH 7 to 8.
 It is soluble in dilute acids, water, and physiological salt (NaCl) solution.
  It is best active at a temperature of 40°C.

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.
 Pepsin of higher digestive power may be reduced to standard pepsin by
admixture with pepsin of lower grade or with lactose.
 Pepsin best act at a temperature 40ºC and pH 2-4
 Pepsin denatured at 70ºC and in the presence of alcohol.

Storage:
Pepsin can be stored at 2-8ºC 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 converts protein into peptone and proteose.
 It is used in laboratory analysis of various proteins.
 It is used in preparation of cheese and other protein containing food.

Dose:
It is proteolytic enzyme is administered after meals and followed by a dose of
hydrochloric acid; its usual dose is 500mg. It is often combined with pancreatin in
product formulations.

Note:
Pepsin has a long history of use in medicine, but its actual beneficial
contribution is poorly documented.

4.PANCREATIN
Other Names:
  Pancreatinum
 Pancreatic enzymes.

Introduction:
 It is an animal enzyme.
 Pancreatin is a mixture of several digestive enzymes.

Biological Source:
 Pancreatin is produced by the exocrine cells of the pancreas of Pig or Hog,

Sus scrofa

 Or from Pancreas of Ox or calf,

Bos taurus

Family:

 Sus scrofa Family Suidae

 Bos taurus Family Bovidae

Part Used:
Exocrine cells of pancreas of different animals

Preparation Method:
 The Pancreas is a gland that lies directly inside the posterior wall of the
abdomen.
 Fresh glands are minced and extracted.
 The mucous membrane is separated from the pancreas by the process of
stripping or it is scrapped off.
 Then it is placed in acidified water for autolysis at 37ºC for 2 hours.
 The liquid so obtained consists of both pancreatin 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 pancreatin 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 pancreatin if any in the aqueous solution is
precipitated by the addition of alcohol into it.

Constituents:
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)

Standard Pancreatin:
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

Note:
Pancreatin of a higher digestive power may be labeled to indicate its strength
in whole-number multiples of the 3 minimum activities or may be diluted by
appropriate admixture to conform to aforementioned specifications.

USP Units:
 One USP unit of amylase activity is contained in the amount of pancreatin
that digests 1mg of the dry USP potato starch standard
 One USP unit of lipase activity is contained in the amount of pancreatin that
liberates 1μEq of acid per minute at a pH 9 and 37ºC.
 One USP unit of protease activity is contained in the amount of pancreatin
that digests 1mg of casein under specific conditions

Description / Properties:
 It is cream coloured amorphous powder with a faint, characteristic but not
offensive odour.
 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

Uses:
  Pancreatin is a mixture of several digestive enzymes. This mixture is used to
treat conditions in which pancreatic secretions are deficient, such as:
o Surgical pancreatectomy (surgical removal of the pancreas)
o Pancreatitis (inflammation of the pancreas)
o Cystic fibrosis (Progressive, genetic disease that causes persistent
lung infections and limits the ability to breathe over time)
 As digestive aid for invalids
 Enteric coated granules are used to treat infants with celiac disease and
related pancreatic deficiencies.

Dose:
 Usual dose is 325mg –1g as tablets, capsules or granules

Prodietary Products:
 Elzyme
 Panteric ®
 Viokase
 Products containing both pepsin and pancreatin in combination with bile salts

PANCREALIPASE
Other Names:
 Pancreatinum
 Concentrated pancreatic enzymes

Introduction:
 It is an animal enzyme.
 Pancrealipase is a mixture of several digestive enzymes.
 It is same as Pancreatin but is more concentrated form of Pancreatin.

Biological Source:
 Pancreatin is produced by the exocrine cells of the pancreas of Pig or Hog,

Sus scrofa

 Or from Pancreas of Ox or calf,

Bos taurus

Family:
 Sus scrofa Family Suidae
 Bos taurus Family Bovidae

Part Used:
Exocrine cells of pancreas of different animals

Preparation Method:
 The Pancreas is a gland that lies directly inside the posterior wall of the
abdomen.
 Fresh glands are minced and extracted.
 The mucous membrane is separated from the pancreas by the process of
stripping or it is scrapped off.
 Then it is placed in acidified water for autolysis at 37ºC for 2 hours.
 The liquid so obtained consists of both pancreatin 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 pancreatin 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 pancreatin if any in the aqueous solution is
precipitated by the addition of alcohol into it.

Constituents:
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)

Standard Pancrealipase:
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.

USP Units:
 One USP unit of amylase activity is contained in the amount of pancreatin
that digests 1mg of the dry USP potato starch standard
 One USP unit of lipase activity is contained in the amount of pancreatin that
liberates 1μEq of acid per minute at a pH 9 and 37ºC.
 One USP unit of protease activity is contained in the amount of pancreatin
that digests 1mg of casein under specific conditions

Description / Properties:
 It is cream coloured amorphous powder with a faint, characteristic but not
offensive odour.
 It show optimal activity in neutral or faintly alkaline solution
 More than traces of mineral acids or large amounts of alkali hydroxide
render pancrealipase inert, and excess of alkali carbonates inhibits its action

Uses:
 It is employed as digestive aid.
 It increase intestinal absorption of fats used to treat steatorrhea.

Dose:
 Usual dose is 8000 to 24000 USP units
 Dose is determined on the basis of clinical evaluation according to
pancreatic insufficiency.

Commercial Products:
 Ultresa
 Viokace
 Zenpep

4. RENIN
Introduction:
 It is an animal enzyme.
 It is a protease enzyme.

Other Names:
 Milk curdling enzyme
 Rennet
 Chymosin

Biological Source:
Rennin is partially purified enzyme obtained from the glandular layer of the
stomach of the calf, Bos taurus

Family:
Bovidae

Main Sources:
There are three main sources:

 Bos taurus i.e. an animal source

 Withania coagulans i.e. a plant source
 Rhizomucor miehei i.e. a microbial source

Part Used:
Glandular layer of the stomach

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
  Standard Rennin can coagulate approximately 25000 times its own weight
of fresh cow‘s milk.
 If it coagulates milk more then 25,000 times of its own weight than lactose
or NaCl is mixed to bring its required strength.
 Rennin curdles milk by converting milk protein casein into para-casein by
partial hydrolysis.

Properties:
 Rennin occur as a yellowish white powder or as yellowish grains or scales
 It has characteristic and slightly saline taste.
 It has peculiar odour.

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
digests their mothers' milk.
 Its principal used is in the manufacture of cheese, mainly for coagulation of
milk.
 It is used to separate milk into solid curds used for cheese making and liquid
whey.

Dose
:
7-day therapy of drug with dose 1.5 mg/kg/day

Commercial Products:
Aliskiren
REFERENCE:

https://www.physio-pedia.com/Proteins

https://medlineplus.gov/genetics/understanding/howgeneswork/protein/

https://byjus.com/chemistry/enzym-definition/#:~:text=Proteins%20are%20organic%20molecules
%20that,collagen%2C%20insulin%2C%20and%20anticorps.

https://byjus.com/chemistry/protein-structure-and-levels-of-protein/

https://www.britannica.com/science/protein/Physicochemical-properties-of-the-amino-acids

Enzyme | Definition, Mechanisms, & Nomenclature | Britannica

Enzymes: Function, definition, and examples

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Enzyme: Definition, classification, modification, mode of action,sub classs &

Diagram

www.com.shareslide.enzymes