4/5/2020 Assignment-1

KBT-403
Enzyme Engineering

Abhay Rawat (1800454001)

Btech Biotechnology 2nd yr
Q.1Name three major properties of enzyme active sites?
Ans-
The part of the enzyme where the substrate binds is called the active site.
Enzymes bind substrates at their active site to form the enzyme-substrate complex.
These active sites have several important properties which are following-
1. Active sites contain special residues that are responsible for binding of
substrate molecule and holding it while the chemical reaction takes place.
2. The active site of enzymes lowers the energy of the transition state and
stabilizes the structure. It also contains residues called catalytic groups that are
responsible for catalyzing the reaction.
3. The shape of the active site is complementary to the substrate. This ensures a
proper fit of substrate.

Q.2 What is the lock and key model of enzyme substrate

binding?
Ans-
The concept of lock and key model of enzyme substrate binding was suggested by
the 19th-century chemist Emil Fischer. Where the lock represents an enzyme and
the key represents a substrate. It is assumed that both the enzyme and substrate
have fixed conformations that lead to an easy fit. Because the enzyme and the
substrate are at a close distance with weak attraction, the substrate must need a
matching shape and fit to join together. At the active sites, the enzyme has a
specific geometric shape and orientation that a complementary substrate fits into
perfectly.
The theory behind the Lock and Key model involves the complementarity
between the shapes of the enzyme and the substrate. Their complementary
shapes make them fit perfectly into each other like a lock and a key. According to
this theory, the enzyme and substrate shape do not influence each other because
they are already in a predetermined perfectly complementary shape.
Q.3 List and define four classes of enzyme specifications.
Ans-
The ability of enzyme to bind with specific substrate or catalyse a specific set of
chemical reactions, is called "Enzyme Specificity". In general, there are four
distinct types of specificity :-
1. Absolute specificity - the enzyme will catalyse only one reaction.
2. Group specificity - the enzyme will act only on molecules that have specific
functional groups, such as amino, phosphate and methyl groups.
3. Linkage specificity - the enzyme will act on a particular type of chemical bond
regardless of the rest of the molecular structure.
4. Stereochemical specificity - the enzyme will act on a particular steric or optical
isomer.

Q.4 What is the difference between the enzyme

specificity and enzyme activity?
 Ans-
Enzyme activity and specific activity are two enzyme units which measure the
enzymatic activity. The measurement of enzymatic activity by enzyme assays is
important for the study of enzyme kinetics as well as enzyme inhibition.
The main difference between enzyme activity and specific activity is that enzyme
activity is the moles of substrate converted by the enzyme per unit time whereas
specific activity is the activity of enzyme per milligram of total enzyme.
Furthermore, enzyme activity measures the amount of active enzymes present
under a given condition while specific activity measures the enzyme purity in the
mixture.

Q.5 What is the role of co-factor in enzyme activity?

Ans-
A cofactor is a non-protein chemical compound that is required for the protein's
biological activity. They help enzymes by increasing the rate of reaction or allowing
them to function. There are two types of cofactors- Coenzymes which are organic
molecules that bind to enzymes and help them function,
Prosthetic groups, which are cofactors made of inorganic substances that bind
tightly to the enzyme, such as iron or zinc.
If the cofactor is removed, the enzyme will not be able to do its job and will no
longer work as a catalyst.
The precise role that a cofactor plays in an enzymatic reaction depends on the
enzyme. Each enzyme has its own reaction mechanism, a sequence of chemical
steps through which the reaction it catalyzes takes place, and the role of the
cofactor is specific to that mechanism.
Your blood, for example, contains an enzyme called carbonic anhydrase which
catalyzes the reaction between water and carbon dioxide to form carbonic acid.
Carbonic anhydrase requires a zinc ion as a cofactor. If no zinc is present, the
enzyme will not work.
Q.6 What is the allosteric enzyme?
Ans-
An allosteric enzyme is an enzyme that contains a region to which small,
regulatory molecules ("effectors") may bind in addition to and separate from
the substrate binding site and thereby affect the catalytic activity.
On binding the effector, the catalytic activity of the enzyme towards the
substrate may be enhanced, in which case the effector is an activator, or
reduced, in which case it is a de-activator or inhibitor.

Q.7 Describe the function of enzymes in biological

system.
Ans-
Enzyme plays two roles in biological system either they help to break down large
nutrient molecules, such as proteins, fats, and carbohydrates, into smaller
molecules (occurs during the digestion of foodstuffs) or they promote the
formation of large, complex molecules from the small, simple ones to produce
cellular constituents.
Enzymes are also responsible for numerous other functions, which include the
storage and release of energy, the course of reproduction, the processes of
respiration, and vision. They are indispensable to life.
Some common examples are-
Lipases – a group of enzymes that help digest fats in the gut.
Amylase – helps change starches into sugars. Amylase is found in saliva.
Maltase – also found in saliva; breaks the sugar maltose into glucose. Maltose is
found in foods such as potatoes, pasta, and beer.
Trypsin – found in the small intestine, breaks proteins down into amino acids.
Lactase – also found in the small intestine, breaks lactose, the sugar in milk, into
glucose and galactose.
Acetylcholinesterase – breaks down the neurotransmitter acetylcholine in nerves
and muscles.
DNA polymerase – synthesize DNA from deoxyribonucleotides.

Q.8 Describe the industrial uses of enzymes in various

sectors.
Ans-
Enzymes are used in the food, agricultural, cosmetic, and pharmaceutical
industries to control and speed up reactions in order to quickly and accurately
obtain a valuable final product. Enzymes are crucial to making cheese, brewing
beer, baking bread, extracting fruit juice, tanning leather, and much more.
Enzymes are also used in forensic science as a means of detecting body fluids and
as markers of genetic individuality. Detection of occurrence of saliva in forensic
samples has relied upon the presence of α-amylase, an enzyme which catalyses
the hydrolysis of starch and glycogen. Alcohol dehydrogenase has been used to
monitor ethanol levels in forensic samples.
The traditional use of yeast (e.g., Saccharomyces cerevisiae) in the baking and
brewing industries arose because they contain enzymes for alcohol fermentation.
Immobilized enzymes may be components of artificial kidney machines, which
are used to remove urea and other waste products from body.
The clarification of cider, wines and fruit juices is usually achieved by treatment
with fungal pectinases.

Q.9 What are intracellular enzymes? How are they

recovered?
Ans-
Enzymes which occur inside a cell are called intracellular enzymes. They may be
in cytoplasm, in the nucleus or in an organelle like chloroplast or mitochondria.
Membrane bound enzymes, like Na/K ATPase are also considered intracellular.
They are recovered by disrupting live cells.

Q.10 Define immobilized enzymes. What are the merits

of enzyme immobilization by entrapment. Describe any
one method widely used for entrapment of enzymes.
Ans-
Immobilized enzymes are those enzymes which are physically confined or
localized in a certain defined region of space with retention of their catalytic
activities, which can be used repeatedly and continuously.
Merits of enzyme immobilization by entrapment are –
 Product is not contaminated with enzyme.
 Easy separation of enzyme from product.
 Allow development of multi-enzyme reaction system.
 Reduce effluent disposal problem.
 Multiple or repetitive use of single enzyme.
 Fastest method of immobilization.
 It is cheap.
 Easy to practice at small scale.
 It can be used for sensing application.

Occlusion within a cross linked gel is widely used for entrapment of enzymes in
this method a highly cross-linked gel is formed as a result of the polymerization
which has a fine "wire mesh" structure and can more effectively hold smaller
enzymes in its cages. Amounts in excess of 1 g of enzyme per gram of gel or fibre
may be entrapped.
Some synthetic polymers such as polyarylamide, polyvinylalcohol, etc... and
natural polymer (starch) have been used to immobilize enzymes using this
technique.

Q.11 Describe purification of enzymes using

chromatography.
Ans-
Chromatographic techniques are used for the separation of mixture into their
compound in order to analyse purify, analyse, identify & quantify the mixture.

There are three principle types of chromatography used for purification of

enzyme based on different properties of enzyme-
 Ion exchange chromatography (ionic property of enzyme)
 Affinity chromatography (ionic property of enzyme)
 Gel exclusion chromatography (molecular weight & size)
Ion exchange chromatography is used most commonly used because it is cheaper,
faster & can handle large quantities of crude enzyme. In this, the charged
functional groups are covalently bound to the solid surface of the matrix
according to their charge & then elute enzymes.
In affinity chromatography enzymes are bound to the solid surface of matrix
according to their affinity towards the matrix & then elute enzymes.
In gel exclusion chromatography enzymes enzymes are embedded in the matrix
according to their molecular weight and size & then elute enzymes.

Q.12 Name microbial protease with their source and

application.
Ans-
Some commercially used microbial proteases are as follows:
Product name Microbial source Applications
Alcalase Bacillus licheniformis Detergent, silk degumming
Savinase Alkalophilic Bacillus sp. Detergent, textile
Esperase B. lentus Detergent, food,silk degumming
Biofeed proteases B. licheniformis Feed
Neutrase Bacillus amyloliquefaciens Upgrade proteins of animal
and vegetable origin
Novozyme B. licheniformis Denature cleaner
Proleather Alkalophilic Bacillus sp. Food
Q.13 Differences between biocatalyst and chemical
biocatalyst.
Ans-
Biocatalyst Chemical biocatalyst
High molecular weight globular Simple inorganic molecules with low
proteins molecular weight
Composition may change at the Remain unchanged at the end of
end of reaction. reaction.
Catalyzing effect is very high Slower than enzyme
Reaction specific Not reaction specific
Intolerant to temperature and pH Function within wide range of
changes temperatures, pH or pressure

Q.14 Explain multi substrate kinetics.

Ans-
Involve more than one substrate often resulting in multiple products.
Also known as bi-bi reaction (2 substrate, 2 products and one enzyme)
The following equation represents a bi-bi reaction in which A & B are the
substrate, E is the enzyme and P & Q are the products (considering forward
direction)
A+B↔P+Q
The multi-substrate reaction follows a complex equation that describes how the
substrate molecule binds and in which sequence.
Sequential mechanism:
In this mechanism, substrate binds to the enzyme and at the same time to the
product and an EAB ternary complex is formed. This form of an enzyme, exists
in solution in the absence of any substrate often small molecule can bind to it, is
called the free enzyme. An intermediate formed from the free enzyme by binding
to the substrate molecule is called an enzyme-substrate complex. A complex
derived from the free enzyme and one other molecule is called a binary complex;
one derived from one free enzyme and two other molecules is called a ternary
complex.
Random ordered: In random ordered sequential mechanism, the substrate and
product are bound and then released in no preferred order or random order.
Compulsory order mechanism: In the multi reactant enzyme reaction, the
reactant must bind to the enzyme’s active site in a specific order. Then the
reaction type is called compulsory order mechanism.

Non-sequential mechanism/ Ping-Pong mechanism:

It is characterized by the change of the enzyme into its intermediate form. When
the first substrate to product reaction occurs it is important to note that the term
intermediate indicates that this form is only temporary. At the end of the reaction.
The enzyme must be found in its original form.
e.g., chymotrypsin

Q.15 Briefly discuss the industrial application of

immobilized enzymes.
Ans-
Industrial applications of immobilized enzymes are as follows:
Biomedical: Immobilized enzymes are widely used for diagnosis and treatment of
many diseases such as inborn disorder.
Food industry: Enzymes like pectinases and celluloses immobilized on suitable
carriers are successfully used in the production of jams, jelly’s and syrup from
fruits and vegetables.
Research: The use of immobilized enzyme allow researcher to increase the
efficiency of different enzymes such as different pro-teases for cell and organelle
lysis.
Biodiesel production from vegetable oils.
Textile industry: Scouring, bio polishing and desizing of fabrics.
Waste water management: Treatment of sewage and industrial effluents.
Detergent industry: Immobilization of lipase enzyme for effective dirt removal
from cloths.

Q.16 Discuss the various methods to immobilized

enzymes.
Ans-
There are various methods to immobilized enzyme-
1) Adsorption:-
This method is based on the physical adsorption of enzyme protein on the
surface of water-insoluble carriers. Examples of suitable adsorbents are ion-
exchange matrices, porous carbon, clay, hydrous metal oxides, glasses and
polymeric aromatic resins.
The bond between the enzyme and carrier molecule may be ionic, covalent,
hydrogen, coordinated covalent or even combination of any of these.
Immobilization can be brought about by coupling an enzyme either to external or
internal surface of the carrier.
The external surface binding method is advantageous as it does not involve
conditions like pore diffusion. The disadvantages, however, include exposure of
enzymes to microbial attack, physical abrasion of enzyme due to turbulence
associated with the bulk solution.
The major disadvantage of the internal immobilization method is the pore
diffusion.
2) Covalent bonding:-
Covalent binding is the most widely used method for immobilizing enzymes. The
covalent bond between enzyme and a support matrix forms a stable complex.
The functional group present on enzyme, through which a covalent bond with
support could be established, should be non essential for enzymatic activity.
The most common technique is to activate a cellulose-based support with
cyanogen bromide, which is then mixed with the enzyme.
3) Cross linking:-
This method is based on the formation of covalent bonds between the enzyme
molecules, by means of multifunctional reagents, leading to three dimensional
cross linked aggregates.The most common reagent used for cross-linking is
glutaraldehyde.
4) Entrapment:-
In entrapment, the enzymes or cells are not directly attached to the support
surface, but simply trapped inside the polymer matrix. Entrapment is carried out
by mixing the biocatalyst into a monomer solution, followed by polymerization
initiated by a change in temperature or by a chemical reaction.
Polymers like polyacrylamide, collagen, cellulose acetate, calcium alginate or
carrageenan etc are used as the matrices.

Q.17 What is an active site? Discuss the key features of

an active site.
Ans-
The part of the enzyme where the substrate binds is called the active site.
Enzymes bind substrates at their active site to form the enzyme-substrate complex.
 It contains both the binding and the catalytic site, termed as active site or active
centre, of the enzyme.
 It consists of residues that form temporary bonds with the substrate and the
residues that catalysis the reaction with that of substrate.
 It comprises only small portion of the total volume of enzyme and is usually at
or near the surface, since it must be accessible to the substrate molecule.
Q.18 What do you understand by enzyme inhibition?
Explain reversible enzyme inhibition.
Ans-
Inhibitors are substances which tend to decrease the rate of an enzyme catalyzed
reaction when these inhibitors bind to enzymes & reduces enzyme activity it is
called enzyme inhibition. It is of two types-
1) Reversible inhibition
2) Irreversible inhibition

Reversible inhibition involve those inhibitors that bind to an enzyme in a

reversible fashion and can be removed by dialysis (or simple dilution) to restore
full enzymatic activity.
Further reversible inhibition divided in 3 parts that are follows:

Competitive inhibitors: These inhibitors often resemble substrate whose

reactions they inhibit and because of this structural similarity they may compete
for the same binding site on the enzyme. Therefore the value of km increases
however Vmax re-mains constant.
The plot of the enzyme activity against substrate concentration shifts towards right
due to increase in the km value.

Uncompetitive inhibition: In uncompetitive inhibition, inhibitors bind only to

enzyme-substrate complex and not to free enzyme. Substrate binding could cause
a conformational change to take place in the enzyme and reveal an inhibitor
binding site or the inhibitor binds directly to the enzyme-substrate complex. In
neither case, does the inhibitor competes with the substrate for the same binding
site, so inhibition cannot be overcome by increasing the substrate concentration.
Both the km and Vmax decreases.
Non-competitive inhibition: In non-competitive inhibition, inhibitor combines
with an enzyme molecule to produce a dead-end complex, regardless of whether
the substrate molecule is bound or not. Hence the inhibitor binds to the site
other than the substrate however it doesn’t affect substrate binding. This
decreases Vmax and km remains unchanged.

Q.19 Discuss the properties of immobilized enzymes.

Ans-
Properties of immobilized enzymes are-
 They are insoluble in water.
 They have a high capacity to bind enzyme and the enzyme binding capacity is
determined by the available surface area, both internal (pore size) and external
(bead size or tube diameter), the ease with which the support can be activated
and the resultant density of enzyme binding sites.
 They are chemically inert, the inertness refers to the degree of non-specific
adsorption and the pH, pressure and temperature stability.
 Must be mechanically stable.

Q.20 What do you understand by enzyme

immobilization? What are the various applications of
immobilized enzymes?
Ans-
It is the process whereby the movement of enzymes, cells, organelles, etc. in
space is completely or severely restricted usually resulting in a water-insoluble
form of the enzyme.

Various applications of immobilized enzymes are following-

Biomedical: Immobilized enzymes are widely used for diagnosis and treatment of
many diseases such as inborn disorder.
Food industry: Enzymes like pectinases and celluloses immobilized on suitable
carriers are successfully used in the production of jams, jellies and syrup from
fruits and vegetables.
Research: The use of immobilized enzyme allow researcher to increase the
efficiency of different enzymes such as different pro-teases for cell and organelle
lysis.
Biodiesel production from vegetable oils.
Textile industry: Scouring, bio polishing and desizing of fabrics.
Waste water management: Treatment of sewage and industrial effluents.
Detergent industry: Immobilization of lipase enzyme for effective dirt removal
from cloths.