Introduction
Enzyme: Asubstance that acts as a catalyst in living organisms, regulating the rate at
which chemical reactions proceed without itself being altered in the process.
Chemical nature
All enzymes were once thought to be proteins, but since the 1980s the
catalytic ability of certain nucleic acids, called ribozymes (or catalytic RNAs), has
been demonstrated, refuting this axiom. Because so little is yet known about the
enzymatic
functioning
of RNA,
this
discussion
will
focus
primarily
on protein enzymes.
A large protein enzyme molecule is composed of one or more amino
acid chains called polypeptide chains. The amino acid sequence determines the
characteristic folding patterns of the protein’s structure, which is essential to enzyme
specificity. If the enzyme is subjected to changes, such as fluctuations
in temperature or pH, the protein structure may lose its integrity (denature) and its
enzymatic ability. Denaturation is sometimes, but not always, reversible.
Bound to some enzymes is an additional chemical component called
a cofactor, which is a direct participant in the catalytic event and thus is required for
enzymatic activity. A cofactor may be either a coenzyme—an organic molecule, such
as a vitamin—or an inorganic metal ion; some enzymes require both. A cofactor may
be either tightly or loosely bound to the enzyme. If tightly connected, the cofactor is
referred to as a prosthetic group.(1)
Nomenclature
An enzyme will interact with only one type of substance or group of
substances, called the substrate, to catalyze a certain kind of reaction. Because of this
specificity, enzymes often have been named by adding the suffix “-ase” to the
substrate’s name (as in urease, which catalyzes the breakdown of urea). Not all
enzymes have been named in this manner, however, and to ease the confusion
surrounding enzyme nomenclature, a classification system has been developed based
on the type of reaction the enzyme catalyzes.
There are
six
principal
categories
-1-
and
their
reactions:
(1) oxidoreductases:
which
are
involved
in
electron
transfer;
(2) transferases: which transfer a chemical group from one substance to another;
(3) hydrolases: which cleave the substrate by uptake of a water molecule (hydrolysis);
(4) lyases: which form double bonds by adding or removing a chemical group;
(5) isomerases: which transfer a group within a molecule to form an isomer;
-2-
(6) ligases, or synthetases: which couple the formation of various chemical bonds to
the
breakdown
of
a
pyrophosphate
bond
in adenosine
triphosphate or
a
similar nucleotide.
Mechanism Of Enzyme Action
In most chemical reactions, an energy barrier exists that must be overcome for
the reaction to occur. This barrier prevents complex molecules such as proteins and
nucleic acids from spontaneously degrading, and so is necessary for the preservation
of life. When metabolic changes are required in a cell, however, certain of these
complex molecules must be broken down, and this energy barrier must be
surmounted. Heat could provide the additional needed energy (called activation
energy), but the rise in temperature would kill the cell. The alternative is to lower the
activation energy level through the use of a catalyst. This is the role that enzymes
play.
Key and lock
They react with the substrate to form an intermediate complex—a “transition
state”—that requires less energy for the reaction to proceed. The unstable
intermediate compound quickly breaks down to form reaction products, and the
unchanged enzyme is free to react with other substrate molecules.
Only a certain region of the enzyme, called the active site, binds to the
substrate. The active site is a groove or pocket formed by the folding pattern of the
-3-
protein.(2)
This three-dimensional structure, together with the chemical and electrical
properties of the amino acids and cofactors within the active site, permits only a
particular substrate to bind to the site, thus determining the enzyme’s specificity.
(figure1)
enzyme; active site: The active site of an enzyme is a groove or pocket that binds a
specific substrate.Encyclopædia Britannica, Inc.
Enzyme synthesis and activity also are influenced by genetic control and
distribution in a cell. Some enzymes are not produced by certain cells, and others are
formed only when required. Enzymes are not always found uniformly within a cell;
often they are compartmentalized in the nucleus, on the cell membrane, or in
subcellular structures. The rates of enzyme synthesis and activity are further
influenced by hormones, neurosecretions, and other chemicals that affect the cell’s
internal environment.
2.Induced fit model
In 1958, Daniel Koshland suggested a modification to the lock and key model: since
enzymes are rather flexible structures, the active site is continuously reshaped by
interactions with the substrate as the substrate interacts with the enzyme. As a result,
the substrate does not simply bind to a rigid active site; the amino acid side-chains
that make up the active site are molded into the precise positions that enable the
-4-
enzyme to perform its catalytic function. In some cases, such as glycosidases, the
substrate molecule also changes shape slightly as it enters the active site. The active
site continues to change until the substrate is completely bound, at which point the
final shape and charge distribution is determined. Induced fit may enhance the fidelity
of molecular recognition in the presence of competition and noise via the
conformational proofreading mechanism.(3)
Cofactors
Some enzymes do not need additional components to show full activity. Others
require non-protein molecules called cofactors to be bound for activity. Cofactors can
be either inorganic (e.g., metal ions and iron-sulfur clusters) or organic compounds
(e.g., flavin and heme). These cofactors serve many purposes; for instance, metal ions
can help in stabilizing nucleophilic species within the active site. Organic cofactors
can be either coenzymes, which are released from the enzyme's active site during the
reaction, or prosthetic groups, which are tightly bound to an enzyme. Organic
prosthetic groups can be covalently bound (e.g., biotin in enzymes such as pyruvate
carboxylase).(4)
Coenzymes are small organic molecules that can be loosely or tightly bound to an
enzyme. Coenzymes transport chemical groups from one enzyme to another.
Factors Affecting Enzyme Activity
Because enzymes are not consumed in the reactions they catalyze and can be
used over and over again, only a very small quantity of an enzyme is needed to
catalyze a reaction. A typical enzyme molecule can convert 1,000 substrate molecules
per second. The rate of an enzymatic reaction increases with increased substrate
concentration, reaching maximum velocity when all active sites of the enzyme
molecules are engaged. The enzyme is then said to be saturated, the rate of the
reaction being determined by the speed at which the active sites can convert substrate
to product.
Enzyme activity can be inhibited in various ways. Competitive inhibitionoccurs when
molecules very similar to the substrate molecules bind to the active site and prevent
-5-
binding
of
the
actual
substrate.
Penicillin, for example, is a competitive inhibitor that blocks the active site of an
enzyme that many bacteria use to construct their cell walls.
Noncompetitive inhibition occurs when an inhibitor binds to the enzyme at a
location other than the active site. In some cases of noncompetitive inhibition, the
inhibitor is thought to bind to the enzyme in such a way as to physically block the
normal active site. In other instances, the binding of the inhibitor is believed to
change the shape of the enzyme molecule, thereby deforming its active site and
preventing it from reacting with its substrate. This latter type of noncompetitive
inhibition is called allosteric inhibition; the place where the inhibitor binds to the
enzyme is called the allosteric site. Frequently, an end-product of a metabolic
pathway serves as an allosteric inhibitor on an earlier enzyme of the pathway. This
inhibition of an enzyme by a product of its pathway is a form of negative feedback.(5)
Allosteric control can involve stimulation of enzyme action as well as
inhibition. An activator molecule can be bound to an allosteric site and induce a
reaction at the active site by changing its shape to fit a substrate that could not induce
the change by itself.
Common activators include hormones and the products of
earlier enzymatic reactions.
Allosteric stimulation and inhibition allow production
of energy and materials by the cell when they are needed and inhibit production when
the supply is adequate.
Factors affecting Enzyme Activity
The activity of an Enzyme is affected by its environmental conditions.
Changing these alter the rate of reaction caused by the enzyme. In nature,
organisms adjust the conditions of their enzymes to produce an Optimum rate of
reaction,
where necessary,
or
they
may
have
enzymes
which
are adapted to function well in extreme conditions where they live.
Temperature
Increasing temperature increases the Kinetic Enery that moleculespossess.
In a fluid, this means that there are more random collisionsbetween molecules per
unit time.
-6-
Since
enzymes
catalyse
reactions
by randomly
colliding with Substrate
molecules, increasing temperature increases the rate of reaction, forming more
product.
However, increasing
temperature also increases the Vibrational
Energythat
molecules have, specifically in this case enzyme molecules, which puts strain on
the bonds that hold them together.
As
temperature
increases, more
bonds,
especially
the weaker Hydrogenand Ionic bonds, will break as a result of this strain.
Breaking bonds within the enzyme will cause the Active Site to change shape.
This change in shape means that the Active Site is less Complementaryto
the shape of the Substrate, so that it is less likely to catalyse the reaction.
Eventually, the enzyme will become Denatured and will no longer function.
As temperature increases, more enzymes' molecules' Active Sites' shapes will
be less Complementary to the shape of their Substrate, and more enzymes will
be Denatured. This will decrease the rate of reaction.(6)
In
summary,
as temperature
increases, initially the rate of
reaction
will increase, because of increased Kinetic Energy. However, the effect of bond
breaking will become greater and greater, and the rate of reaction will begin
to decrease.
-7-
The temperature at which the maximum rate of reaction occurs is called the
enzyme's Optimum Temperature. This is different for different enzymes. Most
enzymes in the human body have an Optimum Temperature of around 37.0 °C.
pH - Acidity and Basicity
pH measures the Acidity and Basicity of a solution. It is a measure of
the Hydrogen Ion (H+) concentration, and therefore a good indicator of
theHydroxide Ion (OH-) concentration. It ranges from pH1 to pH14. Lower
pH values mean higher H+ concentrations and lower OH- concentrations.
Acid solutions have pH values below 7, and Basic solutions (alkalis are bases)
have pH values above 7. Deionised water is pH7, which is termed 'neutral'.
H+
and
OH- Ions(9)
are charged and
therefore interfere with Hydrogen and Ionic bonds
that hold
together an enzyme, since they will be attracted or repelled by the charges created
by the bonds. This interference causes a change in shape of the enzyme, and
importantly, its Active Site.
Different enzymes have different Optimum pH values. This is the pH value
at which the bonds within them are influenced by H+ and OH- Ions in such a way
that the shape of their Active Site is the most Complementary to the shape of
their Substrate. At the Optimum pH, the rate of reaction is at an optimum.
Any change in pH above or below the Optimum will quickly cause
a decrease in
the rate of reaction, since more of the enzyme molecules will have Active
Sites whose shapes are not (or at least are less) Complementary to the shape of
their Substrate.
pH Indicators
-8-
Small changes
a permanent
in pH above
change to
the
or
below
enzyme,
However, extreme changes in pH can
since
the Optimum do not cause
the bonds can
be reformed.
cause enzymes
to Denature and permanently lose their function.
Enzymes in different locations have different Optimum pH values since
their environmental conditions may be different. For example, the enzyme
Pepsin functions best at around pH2 and is found in the stomach, which
contains Hydrochloric Acid (pH2).
Concentration
Changing
the Enzyme and Substrate concentrations affect
the rate of
reaction of an enzyme-catalysed reaction. Controlling these factors in a cell is
one way that an organism regulates its enzyme activity and so its Metabolism.
Changing the concentration of a substance only affects the rate of
reaction if it is the limiting factor: that is, it the factor that is stopping a
reaction from preceding at a higher rate. (15)
If
it
is the
limiting
factor, increasing
concentration will increase the rate of reaction up to a point, after which
any increase will not affect the rate of reaction. This is because it will no
-9-
longer be the limiting factorand another factor will be limiting the maximum
rate of reaction.
As
a reaction
proceeds,
the rate
of
reaction will decrease,
since
the Substrate will get used up. The highest rate of reaction, known as the Initial
Reaction Rate is the maximum reaction rate for an enzyme in an experimental
situation.
Substrate Concentration
Increasing Substrate Concentration increases the rate of reaction. This is
because more substrate molecules will be colliding with enzyme molecules,
so more product will be formed.
However, after a certain concentration, any increase will have no effecton
the rate of reaction, since Substrate Concentration will no longer be the limiting
factor. The enzymes will effectively become saturated, and will be working at
their maximum possible rate.
Enzyme Concentration
Increasing
Enzyme
Concentration will increase the rate of
reaction,
as more enzymes will be colliding with substrate molecules.
However, this too will only have an effect up to a certain concentration,
where the Enzyme Concentration is no longer the limiting factor.
- 10 -
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. a chloride ion (CI–) for
amylase.
Source of enzymes
Since the late 19th century scientists have been studying animal enzyme
sources. Their focus was on cancerous tumors and the effect of enzymes on them.
This was because cancer cells are surrounded by protein to protect them from the
immune system. They studied how trypsin, chymotrypsin and pancreatin break down
protein to expose the cancer cells to the immune system. Other areas of study with
enzymes include heart disease, and strokeand inflammation. Fibrin, which is a
protein, contributes to these diseases by causing blood clots. Protease breaks down the
fibrin and can reduce inflammation. Most people don’t know that inflammation can
cause
a
heart
attack.
What are the disadvantages of animal source enzymes? If you were a vegetarian you
would not want to consume them. Also, animals can be exposed to antibiotics and
steroids, which wouldn’t be healthy. Thirdly, they’re weakcompared to microbial
enzymes. Animal enzymes are unstable at a low pH or acidic environment. Since the
stomach is acidic much of the enzyme is destroyed before it can do its job. One way
to get around this would be to put the enzymes in an enteric-coated tablet. This
coating doesn't dissolve in acid. Thus it dissolves in the intestine, which isn't acidic.
One
popular
brand
that's
enteric-coated
- 11 -
is Wobenzym
N.
They are also temperature sensitive and since we don’t have the same body
temperature as the animal source this can affect the enzyme. (20)
What are the advantages of animal source enzymes? The advantage is what is called
the law of similar. The law of similar is the basis of homeopathy. The theory is
although the source is not human your body recognizes it as similar and therefore is
able to use it more effectively.
The two types of plant-based enzymes are bromelain, which comes from
pineapples and papain, which comes from papayas. They are quite stable in acidic
pH and are not affected by temperature. One disadvantage is they are only beneficial
as digestive enzymes. They can’t be used as systemic enzymes. Another is they could
contain harmful substances such as phenolic compounds.
Most enzyme sources come from fungi or yeast. Some consider this plant-based but
fungi are actually not plants. This is because the study of fungi has historically been a
branch of botany. The most popular fungus used is called aspergillus. Over half of
enzymes come from fungi and yeast, over a third from bacteria. About 8% are from
animal sources and 4% from plant sources
What are the advantages of microbial enzymes? They are usually cheaper to produce.
They are extracted from fermented fungus or bacteria. Their enzyme contents are
more controllable and predictable. Manufacturers can manipulate aspergillus fungus
in order to make different types of enzymes. This is done by changing the type of
aspergillus that’s used. They’re the most potent enzymes and can be up to a hundred
times more effective digesting proteins, carbohydrates and fats. One doesn’t have to
worry about contamination with antibiotics or steroids. The pH range is broad which
makes them active in stomach acid and throughout our body. Last but not least, there
is a
reliable
supply
of
raw
material to
make
microbial
enzymes.
Microbial Enzymes
Enzymes are biocatalysts produced by living cells to bring about specific biochemical
reactions generally forming parts of the metabolic processes of the cells. Enzymes are
highly specific in their action on substrates and often many different enzymes are
required to bring about, by concerted action, the sequence of metabolic reactions
- 12 -
performed by the living cell. All enzymes which have been purified are protein in
nature, and may or may not possess a nonprotein prosthetic group. The practical
application and industrial use of enzymes to accomplish certain reactions apart from
the cell dates back many centuries and was practiced long before the nature or
function of enzymes was understood. Use of barley malt for starch conversion in
brewing, and of dung for bating of hides in leather making, are examples of ancient
use of enzymes. It was not until nearly the turn of this century that the causative
agents or enzymes responsible for bringing about such biochemical reactions became
known. Then crude preparations from certain animal tissues such as pancreas and
stomach mucosa, or from plant tissues such as malt and papaya fruit, were prepared
which found technical applications in the textile, leather, brewing, and other
industries. Once the favorable results of employing such enzyme preparations were
established, a search began for better, less expensive, and more readily available
sources of such enzymes. It was found that certain microorganisms produce enzymes
similar in action to the amylases of malt and pancreas, or to the proteases of the
pancreas and papaya fruit. This led to the development of processes for producing
such microbial enzymes on a commercial scale. Dr. Jokichi Takamine (1894, 1914)
was the first person to realize the technical possibility of cultivated enzymes and to
introduce them to industry. He was mainly concerned with fungal enzymes, whereas
Boidin and Effront (1917) in France pioneered in the production of bacterial enzymes
about 20 years later. Technological progress in this field during the last decades has
been so great that, for many uses, micro' Presented at Symposium, Society for
Industrial Microbiology, Storrs, Connecticut, August, 1956.
bial cultivated enzymes have replaced the animal or plant enzymes. For example, in
textile desizing, bacterial amylase has largely replaced malt or pancreatin. At present,
only a relatively small number of microbial enzymes have found commercial
application, but the number is increasing, and the field will undoubtedly be much
expanded in the future.(17)
Enzyme Production and purification:
1.Selection of organism
The most important criteria for selecting the microorganism are that the
organism should produce the maximum quantities of desired enzyme in a short time
while the amounts of other metabolite produced are minimal. Once the organism is
selected, strain improvement for optimising the enzyme production can be done by
- 13 -
appropriate methods(figure2) (mutagens, UV rays). From the organism chosen,
inoculum can be prepared in a liquid medium.
2.Formulation of medium
The culture medium chosen should contain all the nutrients to support adequate
growth of microorganisms that will ultimately result in good quantities of enzyme
production. The ingredients of the medium should be readily available at low cost and
are nutritionally safe. Some of the commonly used substrates for the medium are
starch hydrolysate, molasses, corn steep liquor, yeast extract, whey, and soy bean
meal. Some cereals (wheat) and pulses (peanut) have also been used. The pH of the
medium should be kept optimal for good microbial growth and enzyme
production.(22)
3.Production process
Industrial production of enzymes is mostly carried out by submerged liquid
conditions and to a lesser extent by solid-substrate fermentation. In submerged culture
technique, the yields are more and the chances of infection are less. Hence, this is a
preferred method. However, solid substrate fermentation is historically important and
still in use for the production of fungal enzymes e.g. amylases, cellulases, proteases
and pectinases.
The medium can be sterilized by employing batch or continuous sterilization
techniques. The fermentation is started by inoculating the medium.
The growth conditions (pH, temperature, O2 supply, nutrient addition) are
maintained at optimal levels.
The froth formation can be minimised by adding antifoam agents.
- 14 -
The production of enzymes is mostly carried out by batch fermentation and to a
lesser extent by continuous process.
The bioreactor system must be maintained sterile throughout the fermentation
process. The duration of fermentation is variable around 2-7 days, in most production
processes.
Besides the desired enzyme(s), several other metabolites are also produced. The
enzyme(s) have to be recovered and purified.
4.Recovery and purification of enzymes:
The desired enzyme produced may be excreted into the culture medium
(extracellular enzymes) or may be present within the cells (intracellular enzymes).
Depending on the requirement, the commercial enzyme may be crude or highly
purified. Further, it may be in the solid or liquid form.
The steps involved in downstream processing i.e. recovery and purification
steps employed will depend on the nature of the enzyme and the degree of purity
desired.
In general, recovery of an extracellular enzyme which is present in the broth is
relatively simpler compared to an intracellular enzyme. For the release of intracellular
enzymes, special techniques are needed for cell disruption.
The reader must invariably refer them now and learn all the details, as they
form part of enzyme technology(17).
Microbial cells can be broken down by physical means (sonication, high
pressure, glass beads).
The cell walls of bacteria can be lysed by the enzyme lysozyme. For yeasts,
the enzyme β-glucanase is used. However, enzymatic methods are expensive.
The recovery and purification (briefly described below) steps will be the same for
both intracellular and extracellular enzymes, once the cells are disrupted and
intracellular enzymes are released.
The most important consideration is to minimise the loss of desired enzyme
activity.(19)
5.Removal of cell debris
Filtration or centrifugation can be used to remove cell debris.
6.Removal of nucleic acids
Nucleic acids interfere with the recovery and purification of enzymes.
- 15 -
They can be precipitated and removed by adding poly-cations such as polyamines,
streptomycin and polyethyleneimine.
7.Enzyme precipitation
Enzymes can be precipitated by using salts (ammonium sulfate) organic solvents
(isopropanol, ethanol, and acetone). Precipitation is advantageous since the
precipitated enzyme can be dissolved in a minimal volume to concentrate the enzyme.
8.Liquid-liquid partition
Further concentration of desired enzymes can be achieved by liquid-liquid
extraction using polyethylene glycol or polyamines.
9.Separation by chromatography
There are several chromatographic techniques for separation and purification of
enzymes. These include ion-exchange, size exclusion, affinity, hydrophobic
interaction and dye ligand chromatography .Among these, ion- exchange
chromatography is the most commonly used for enzyme purification.
10.Drying and packing
The concentrated form of the enzyme can be obtained by drying. This can be
done by film evaporators or freeze dryers (lyophilizers). The dried enzyme can be
packed and marketed. For certain enzymes, stability can be achieved by keeping them
in ammonium sulfate suspensions.
All the enzymes used in foods or medical treatments must be of high grade
purity, and must meet the required specifications by the regulatory bodies. These
enzymes should be totally free from toxic materials, harmful microorganisms and
should not cause allergic reactions.
Types of Culture Media:
A culture media is a special medium used in microbiological laboratories to
grow different kinds of microorganisms. A growth or a culture medium is composed
of different nutrients that are essential for microbial growth.
Since there are many types of microorganisms, each having unique properties
and requiring specific nutrients for growth, there are many types based on what
nutrients they contain and what function they play in the growth of
microorganisms.(18)
- 16 -
A culture may be solid or liquid. The solid culture media is composed of a
brown jelly like substance known as agar. Different nutrients and chemicals are added
to it to allow the growth of different microorganisms.(19-21)
A.Broth cultures
One method of bacterial culture is liquid culture, in which the desired bacteria
are suspended in a liquid nutrient medium, such as Luria Broth, in an upright flask.
This allows a scientist to grow up large amounts of bacteria for a variety of
downstream applications.
Liquid cultures are ideal for preparation of an antimicrobial assay in which the
experimenter inoculates liquid broth with bacteria and lets it grow overnight (they
may use a shaker for uniform growth). Then they would take aliquots of the sample to
test for the antimicrobial activity of a specific drug or protein (antimicrobial peptides).
B.Agar plates
Microbiological cultures can be grown in petri dishes of differing sizes that
have a thin layer of agar-based growth medium. Once the growth medium in the petri
dish is inoculated with the desired bacteria, the plates are incubated at the optimal
temperature for the growing of the selected bacteria (for example, usually at 37
degrees Celsius, or the human body temperature, for cultures from humans or
animals(22), or lower for environmental cultures).
After the desired level of growth is achieved, agar plates can be stored upside
down in a refrigerator for an extended period of time to keep bacteria for future
experiments.
C.Stab cultures
Stab cultures are similar to agar plates, but are formed by solid agar in a test
tube. Bacteria is introduced via an inoculation needle or a pipette tip being stabbed
into the center of the agar.
Bacteria grow in the punctured area.
Stab cultures are most commonly used for short-term storage or shipment of
cultures.
1.The Preservation Culture Media
This is composed of all the basic nutrients required for a microbial growth
and is used to preserve a specific type of microorganism, preferably bacteria or a set
of different microbial entities for a long period of time.
- 17 -
The basic purpose of this culture is to let these microorganisms grow safely
in an ensured environment that has all the important nutrients and to protect them
against any environmental damage so these organisms can be used when needed.
2.The Enrichment Culture Media
This is a liquid medium which allows the microorganisms to multiply and has
the essential nutrients that are required for it.
It is usually composed of bacteria taken from a liquid source such as pond
water. The basic nutrient broth is the most commonly used.
3.Selective Culture Media
This is a special type of media which allows the growth of certain
microorganisms while inhibits the growth of the others.
For example if we want to isolate a specific bacteria let’s say that can with stand an
acidic environment from a sample of pond water and get rid of others(23), a selective
media with a low pH will be taken which will allow the growth of only those
organisms that can withstand acidity and will kill the others that cannot.
Examples of commonly used selective media includes: PALCAM agar
medium or Mac conkey agar medium.
4.Differential Culture Media
This is a media that is used for differentiating between bacteria by using an
identification marker for a specific type of microorganism.
The selective and differential culture media are opposites to each other in a
way that one inhibits the growth of other organisms(24)while allowing the growth of
some while the other does not kill the others but only highlights one type.
Blood agar is a common differential culture medium used to identify
bacteria that causes haemolysis in blood.
5.Resuscitation Culture Media
This is a special type of media which is used for growing microorganisms
that are damaged and have lost the ability to produce due to certain harmful
environmental factors.
This culture allows the organisms to regain their metabolism by providing
the nutrients that the organisms had been deprived of. For example, a type of bacteria
that requires histamine for its growth is subjected to a medium lacking this essential
component its growth will be inhibited.
- 18 -
If the same bacteria is then placed in a medium consisting of histamine it will start to
grow again. In this case the media containing histamine will act as resuscitation
media. An example of a commonly used resuscitation culture media is the tryptic soya
agar.
6.General Purpose MediaThe general purpose media is a media that has a multiple
effect, i.e. it can be used as a selective,
or a resuscitation media.
Figure3:deferential
batch culture
7.Isolation Culture Media
An isolation culture medium is a simple agar containing solid medium that allows the
growth of microorganisms in the direction of the streaks.
For example the bacteria will only grow on the pattern made on the solidified agar
during the streak plate method. This is the most commonly used medium in
microbiological labs.
8.Fermentation Media
The fermentation culture media is a liquid selective media which is used to obtain a
culture of a specific organism more likely yeast or a particular toxin.
The fermentation media can also be differential but mostly it is selective in nature that
is allowing the growth of one type while inhibiting the growth of others.
Microbial culture techniques
A.Indoors:allows
control
over
illumination,
temperature,
nutrient
level,
contamination, but it expensive.
B.outdoors:make it difficult to grow specific species for extended period, and it is
cheap.
C.Open culture:such as in uncovered ponds and tanks.
D.closed culture:such as tubes, flasks, carboys, bags....etc
Examples:1.Batch culture
Consist of single inoculation of cells into a container of media followed by a growing
for several days under suitable condition and finally harvested when growth reaching
maximum denisty(figure3).(26)(27)
- 19 -
Advantages of batch culture
1.simple
2.allow to change species
3.remedy defects in the system rapidly
Disadvantages of batch culture
1.difficulty to prevent contamination
2.require a lot of labour to harvest, clean,sterilized, refill and inoculate containers
3.efficient and quality may be inconsistent
2.Continuous culture
Nutrients are supplied to the cell culture at a constant rate in order to maintain
constant volume(28). they may be:A.Turbidostate:fresh media is delivered only when cell denisity reach predetermined
point, fresh media is added and an equal volume of culture is removed.
B.Chemostate:nutrient medium is delivered to the culture at a constant rate by a
pump.
Advantages of continuous culture
1.more predictable quality
2.amenable to technological control and reduce need for labour
Disadvantages of continuous culture
1.high cost and complexity
2.require high control so it is feasible for relatively small proudction scales
3.Semi-continuous culture
Prolongs the use of large tank cultures by partial periodic harvesting and used for
large scale production.
A. Fed-batch culture
It is, in the broadest sense, defined as an operational technique in biotechnological
processes where one or more nutrients (substrates) are fed (supplied) to the bioreactor
during cultivation and in which the product(s) remain in the bioreactor until the end of
- 20 -
the run (figure4). An alternative description of the method is that of a culture in which
"a base medium supports initial cell culture and a feed medium is added to prevent
nutrient depletion". It is also a type of semi-batch culture. In some cases, all the
nutrients are fed into the bioreactor. The advantage of the fed-batch culture is that one
can control concentration of fed-substrate in the culture liquid at arbitrarily desired
levels (in many cases, at low levels).Generally speaking, fed-batch culture is superior
to conventional batch culture when controlling concentrations of a nutrient (or
nutrients) affect the yield or productivity of the desired metabolite.
AMYLASE
Among different types of enzymes obtained from microbial sources, amylases
are the most widely used in industries. In the present study, bacteria were isolated
from air exposure and screened for the production of amylase. Among four bacterial
isolates, one isolate produced maximum zone of starch hydrolysis. The bacterial
isolate was identified as Bacillus sp. and was later used for further characterization.
Maximum yield of amylase was obtained after 48hrs of incubation. The optimum pH
for enzyme activity was found to be at pH 6.8 and the optimum temperature for the
activity was found to be at 37 ºC.
All amylases are glycoside hydrolases and act on α-1,4-glycosidic bonds.(30-39)
α-Amylase:
The α-amylases : (alternative names: 1,4-α-D-glucan glucanohydrolase;
glycogenase) are calciummetalloenzymes. By acting at random locations along the
starch
chain,
α-amylase
breaks
down
long-chain carbohydrates,
yielding maltotriose and maltose from amylose,
or
ultimately
maltose, glucose and "limit
dextrin" from amylopectin. Because it can act anywhere on the substrate, α-amylase
tends to be faster-acting than β-amylase. In animals, it is a major digestive enzyme,
and its optimum pH is 6.7–7.0.
In human physiology, both the salivary and pancreatic amylases are α-amylases.
The α-amylases form is also found in plants, fungi (ascomycetes and basidiomycetes)
and bacteria (Bacillus)
- 21 -
β-Amylase:
Another form of amylase, β-amylase (alternative names: 1,4-α-D-glucan
maltohydrolase;
glycogenase;
saccharogen
amylase)
is
also
synthesized
by bacteria, fungi, and plants. Working from the non-reducing end, β-amylase
catalyzes the hydrolysis of the second α-1,4 glycosidic bond, cleaving off two glucose
units (maltose) at a time. During the ripening of fruit, β-amylase breaks starch into
maltose, resulting in the sweet flavor of ripe fruit.
Both α-amylase and β-amylase are present in seeds; β-amylase is present in an
inactive form prior to germination, whereas α-amylase and proteases appear once
germination has begun. Many microbes also produce amylase to degrade extracellular
starches. Animal tissues do not contain β-amylase, although it may be present in
microorganisms contained within the digestive tract. The optimum pH for β-amylase
is 4.0–5.0[8]
γ-Amylase :
(alternative names: Glucan 1,4-α-glucosidase; amyloglucosidase; Exo-1,4-αglucosidase; glucoamylase; lysosomal α-glucosidase; 1,4-α-D-glucan glucohydrolase)
- 22 -
will cleave α(1–6) glycosidic linkages, as well as the last α(1–4)glycosidic linkages at
the nonreducing end of amylose and amylopectin, yielding glucose. The γ-amylase
has most acidic optimum pH of all amylases because it is most active around pH 3.
Isolation of Amylase Producing Microorganisms:
Soil samples were collected from different environment sources. Serial
dilution was made by One gram of soil sample was serially diluted in sterilized
distilled water to get a concentration range from 10-1 to 10-6 and volume of 0.1 ml of
each dilution was transferred aseptically to starch agar plates. The sample was spread
uniformly. The plates were incubated at 37°C for 24 hrs. The bacterial isolates were
further sub cultured to obtain pure culture. Pure isolates on starch agar slants were
maintained at 4ºC.
Screening of potent amylase producing bacteria by starch hydrolysis test:
Bacterial isolates were screened for amylolytic activity by starch hydrolysis
test on starch agar plate. The microbial isolates were streaked on the starch agar plate
and incubated at 37°C for 48 hrs. After incubation iodine solution was flooded with
dropper for 30 seconds on the starch agar plate. Presence of blue colour around the
growth indicates negative result and a clear zone of hydrolysis around the growth
indicates positive result. The isolates produced clear zones of hydrolysis were
considered as amylase producers and were further investigated.
Morphological and Biochemical Characteristics:
Gram staining, motility, indole production, methyl red, Vogues Proskauer's,
citrate utilization, triple sugar iron, nitrate reduction, catalase, oxidase, gelatin
liquefaction, urease, hydrolysis of casein, hydrolysis of starch were carried out.
- 23 -
Enzyme production media:
Production medium contained (g/l) Trypticase 10gm, peptone 5gm,
(NH4)2SO4 3gm, K2HPO4 2gm, L-Cysteine HCl 0.5gm, MgSO4 0.2gm.10 ml of
medium was taken in a 100 ml conical flask. The flasks were sterilized in autoclave at
1210 C for 15 min and after cooling the flask was inoculated with overnight grown
bacterial culture. The inoculated medium was incubated at 37oC in shaker incubator
for 24 hr. At the end of the fermentation period, the culture medium was centrifuged
at 5000 rpm for 15 min to obtain the crude extract, which served as enzyme
source.(37-40)
PURIFICATION OF ENZYME:
The crude enxyme was purified using organic solvent and ammonium
sulphate precipitation method.
Acetone method:
The crude extract is treated with different concentration of acetone. In this
30% and 50% acetone was used for purification. Acetone is slowly added to the
extract to precipitate out the enzyme. This is done on an ice bath and kept for 1hr
under continuous stirring. The mixture is the centrifuged at 3000 rpm for 10 min. The
supernatant and pellet are separated and checked for enzyme activity.
Ammonium sulphate precipitation:
The crude extract is treated with different concentration of Ammonium
sulphate. In this 30% and 50% Ammonium sulphate was used for purification.
- 24 -
Ammonium sulphate is slowly added to the extract to precipitate out the enzyme.
This is done on an ice bath and kept for 1hr under continuous stirring. The mixture is
the centrifuged at 3000 rpm for 10mins. The supernatant and pellet are separated and
checked for enzyme activity.
Mechanism of amylase action on glucoside starch bonds.
Functional groups of glucoamylase and alpha-amylase from Asp. awamori,
alpha-amylase from Asp. oryzae and alpha- and beta-amylases from barley malt are
identified. Kinetic curves of the activity dependency on pH, values of ionization heats
and photooxidative inactivation draw to the conclusion that carboxyl-imidazole
system enters into the active site of the enzymes. A hypothetic mechanism of
hydrolysis of alpha-1,4-glucoside bond in starch molecule by alpha- and betaamylases and of alpha-1,4- and alpha-1,6-glucoside bonds by glucoamylase is given.
A theory of induced correspondence of enzyme and substrate satisfactorily explains
the specificity of the enzyme action and the cause of complete starch convertion into
glucose under glucoamylase action and of terminal starch hydrolysis by alpha- and
beta-amylases.(40-47)
Enzyme activity:
Enzyme activity is determined by DNS method. 0.5ml of isolated enzyme is
incubating for 10 min at 370 C with 0.5 ml of substrate (ET) & 0.5 ml of substrate for
(EC). After 10 min arrest the reaction by adding 1 ml of DNS to both the tube
followed by 0.5 ml of enzyme to control tube only. Then kept the tubes in boiling
water bath for 15 min. The solution is then dilute with 8 ml of distilled water & read
the absorbance at 540 nm.
- 25 -
Optimization of Amylase Production Using Solid State Fermentation:
Alpha-amylase [EC 3.2.1.1] cleaves the 1,4-α-D-glycosidic linkages between
adjacent glucose unit inside the linear starches, glycogen, and oligosaccharides in a
random manner [48]. Multifarious uses of alpha-amylases as a major starch degrading
agent in food, paper, textile, and brewing industry necessitates its prolific production
that can be effectively met up by solid state fermentation (SSF) [49]. Agrowastes like
wheat bran, rice bran, and coconut oil bran have replaced the high cost media
generally used in submerged fermentation for alpha-amylase preparation because of
their simplicity, low cost, easy availability, better productivity, and lesser water
output. Additionally it solves the pollution problem occurring due to their disposal in
the surrounding [50]. High starch content of almost all agrowastes (60–70% by
weight) can be effectively utilized as a major nutrient source by microorganisms like
bacteria, fungi, and so forth, for the synthesis of inducible alpha-amylase which is
under the control of catabolic repression.
Plethora of evidences exists in favor of wheat bran as the best sources among all the
agrosources for extracellular amylase production [51.52]. Based on the prior
knowledge of primary solid state fermentation culture condition, the present study
was initiated using wheat bran as a prime source of nutrient and B.
amyloliquefaciens (MTCC 1270) as the producer organism at pH 7 to increase the
alpha-amylase yield through media optimization.Earlier reports are also in agreement
with
the
fact
that
most
of
the Bacillus species,
namely,Bacillus
licheniformis and Bacillus stearothermophilus, are the most effective producers of
alpha-amylase [52-62].
Most of the amylases are metalloenzyme requiring Ca+2 for their activity, structural
integrity, and stabilization [50-55]. At least three calcium binding sites have been
located on barley alpha-amylase isoform that is also visible for plants, mammals,
fungi, and bacteria [60.-68]. For B. amyloliquefaciens, the calcium binding site is
contributed by three conserved regions of the polypeptide chain comprising residues
Gly97-Ala109, Ile217-His235, and Ser314-Ser334 [65]. Depletion of calcium ion from the
binding site abolishes amylase activity. Similar stabilization effect has been provided
- 26 -
by chloride and nitrate ions as reported by Aghajari et al. [66]. In this work major
emphasishas been given in search of conditions as well as for parameters like ions and
sugar alcohols whose presence in the fermentation media stimulates alpha-amylase
production from SSF.
Materials and Methods
Microorganism
Bacillus amyloliquefaciens (MTCC 1207, IMTECH, Chandigarh) was used as
working strain for solid state fermentation (SSF) extraction of alpha-amylases. All the
reagents are of analytical grade (SRL).
2.2. Preparation of Inoculum and Solid State Fermentation (SSF)
Wheat bran was collected from local market and solid state fermentation has been
carried out with 4 gm dry wheat bran in a 100 mL Erlenmeyer flask. The moisture
level of the wheat bran was adjusted to 50% (w/w) with autoclaved distilled water.
The contents of the flask were autoclaved prior to the solid state fermentation.
25 mL of nutrient broth was taken in a 100 mL flask and was inoculated with a loop
full of Bacillus amyloliquefaciens cells from a 24-hour-old slant and kept at 37°C in a
shaker. After 16 hours of growth, 1 mL inoculum (1.5–2 × 108 cfu/mL) from this
broth culture was added in the WB. It was fermented for various fermentation periods
(24 and 48 hours) at different temperatures (30°, 33°, 37°, and 42°C).
Enzyme Extraction
After 24 and 48 hours of fermentation, the fermented media containing wheat
bran were mixed with 25 mL 20 mM phosphate buffer (pH = 7.0) for 30 minutes at
4°C in a rotary shaker at 150 rpm. The suspension was then centrifuged at 8000 rpm
for 15 min at 4°C. The supernatant has been collected and used for amylase assay.
Amylase Assay
Alpha-amylase activity of the extract was measuredby DNS method [19]. In
briefthe reaction mixture containing 1% soluble starch, 20 mM phosphate buffer (pH
= 7), and fermented extract was taken and incubated at 37°C for 20 minutes followed
by the addition of 3,5-dinitrosalicylic acid (DNS). The amount of the reducing sugar
- 27 -
liberated during assay was estimated by measuring color development at 540 nm by
UV-VIS spectrophotometer. 1U of amylase activity is defined as the amount of
enzyme that liberated micromole of maltose per minute under standard assay
condition.
Protein Estimation
The protein content of the extract was determined following Lowry’s method
[67].
2.6. Starch Hydrolysis
A 2% starch agar plate (beef extract—0.3%, soluble starch—1%, and agar—2%) has
been prepared and streaked from a 24-hour-old culture of Bacillus amyloliquefaciens.
The plate was grown for 48 hours in 37°C. To check the starch hydrolysis property of
alpha-amylase the plate was flooded with iodine solution.
Optimization of Media
4 gram of WB was supplemented with various concentrations of ions like
Ca+2, , and (0.1, 0.2, and 0.4 M) from 0.5 M respective stocks of CaCl2, NaCl, and
NaNO3 salt solutions for a comparative analysis regarding the yield of alpha-amylase
with that of the control WB. The relative humidity was kept constant at a level of 50%
(w/w) with autoclaved distilled water. The content of the flask was autoclaved and
tested for solid state fermentation for 48 hours at 37°C with the addition of 1 mL
inoculum (1.5–2 × 108 cfu/mL) from the broth culture. The extraction of the enzyme
was performed following the same procedure as described earlier. Similar protocol of
SSF has been followed for 0.5 and 1% D-inositol and D-mannitol supplementation
into the WB, with proper moisture level adjusted. Control WB was autoclaved and
kept for solid state fermentation under similar experimental condition without any salt
and sugar supplementation with equal inoculums size as earlier. The alpha-amylase
activity has been calculated according to DNS method [19].
Statistical Analysis
Effect of each parameter was studied in triplicate and graphically represented
as the mean ± SD () using Origin 5.
- 28 -
. Results
Amylase Is Able to Hydrolyze Starch
The starch agar plate was inoculated with B. amyloliquefaciens (MTCC 1270)
and kept for 48 hours at 37°C. The plate was flooded with iodine and clear zone of
starch hydrolysis has been observed (Figure 1). This ensures that this microorganism
secretes amylase that is capable of starch hydrolysis
.
Figure 8: Starch hydrolysis performed on a 2% starch agar plate using B.
amyloliquefaciens(MTCC 1270).
Production of Alpha-Amylase from B. amyloliquefaciens (MTCC 1270) Using
Solid State Fermentation
To optimize the appropriate fermentation period for high yield alpha-amylase
production,
the
study
had
been
initiated
with
wheat
bran
and B.
amyloliquefaciens (MTCC 1270) for 24, 48, and 72 hours. The values of specific
activity of alpha-amylase were 7.25 ± 0.25 U/mg, 14.25 ± 0.24 U/mg, and 13.5 ±
0.75 U/mg, respectively, after 24, 48, and 72 hours using SSF under identical
fermentation conditions (time and temperature) (Figure 2). Fermentation conducted
for longer period of time was accompanied with decline in the alpha-amylase activity
caused by denaturation and degradation of enzyme products.
- 29 -
Figure
9: α-Amylase
production
from
solid
state
fermentation
using B.
amyloliquefaciens(MTCC 1270) and wheat bran; black column: specific activity of αamylase from SSF extract during different fermentation time periods.
Influence of Temperature on Amylase Production from SSF
Temperature had profound effect on the growth of the microorganism as well
as on the enzyme activity.
Effect of temperature on alpha-amylase production through solid state fermentation
had been tested for two fermentation hours (24, 48) and at four different temperatures
(30°, 33°, 37°, and 42°C). A 24-hour SSF at 37°C yielded maximum alpha-amylase
production with an activity (7.25 ± 0.25 U/mg) that had been further enhanced with
longer fermentation period after 48 hours at the same temperature. Although alphaamylase production was evident at all the four temperatures studied for fermentation,
37°C was the best among all to produce maximum amylase from SSF with a specific
activity of 14.25 ± 0.24 U/mg This result corroborated well with optimum
temperature of alpha-amylase (data not shown) that came around 40°C using standard
DNS assay method [56]. After 42°C alpha-amylase activity declined due to the
metabolic heat generated as an outcome of microbial growth in the solid state
fermentation medium.
- 30 -
Figure 10: Effect of temperature on α-amylase yield after various periods of
fermentation using B. amyloliquefaciens (MTCC 1270); white column: specific
activity of α-amylase from 24 hours SSF extract; black column: specific activity of αamylase from 48 hours SSF extract.
Effect of Ions Present in the SSF Media on Alpha-Amylase Production
Effect of calcium (Ca+2) on amylase production through solid state
fermentation had been checked for 48 hours fermentation at four different
temperatures (30, 33, 37, and 42°C). Effect of Ca+2 at a concentration of 100 mM had
been tested with a control (without any ion). Compared to control the yield of alphaamylase increased in presence of Ca+2 Among all the temperatures, 37°C solid state
fermentation carried out with calcium ion gave maximum alpha-amylase activity (27
± 1.05 U/mg) where as in absence of calcium it was about 50% less (15 ± 1.75 U/mg).
This indicated the supportive role of calcium (Ca+2) in the preservation of amylase
structural integrity and stability [50]. There was a gradual increase in the specific
activity of amylase from 30°C to 37°C in presence of calcium (Ca+2) with a downfall
of amylase activity at 42°C (9.5 ± 1.1 U/mg).
- 31 -
Figure 11: Effect of 0.1 mM calcium (Ca+2) on amylase production after 48 hours of
SSF at 37°C temperature; dotted column: specific activity of α-amylase in absence of
calcium ion from SSF extract; black column: specific activity of α-amylase in
presence of calcium ion from SSF extract.
Effect of chloride and nitrate ion at various concentration ranges (100, 200, and
400 mM) had been tested in order to check the effect of negative ions on the alphaamylase yield from SSF with a control (without any ion). The result was noteworthy
with respect to improved amylase activity in presence of both and salts in the SSF
media. Presence of 400 mM chloride () and () in the fermentation mixture improved
amylase yield from 14.5 ± 0.25 U/mg to 58 ± 3 U/mg and 68 ± 0.25 U/mg,
respectively, compared to control without any salt This observation can be correlated
well with an insight to the alpha-amylase crystal structure derived from porcine
pancreatic source at 5 Å resolutions. Chloride ion stabilized amylase structure by
making electrostatic interaction with the neighboring positively charged residues like
Arg 195, Lys 257, and Arg 337, which were on the other hand very close to the active
site cleft of amylase. This was in congruence with the observation by Lifshitz and
Levitzky, identifying one lysine residue close to the active site region that bonded
with the chloride ion if present in the vicinity of the enzyme [68].
Figure 12: Effect of anions (nitrate and chloride ions) on amylase production after 48
hours of SSF, (○): specific activity of α-amylase in presence of nitrate ion from SSF
extract; (●): specific activity of α-amylase in presence of chloride ion from SSF
extract.
3.5. Influence of Supplementation of Sugar Alcohol on Amylase Production from SSF
- 32 -
Being an inducible enzyme, alpha-amylase was sensitive to catabolite repression [65].
Addition
of
soluble
starch
encouraged
amylase
production
by B.
amyloliquefaciens [69]. SSF was conducted in presence and absence of D-inositol and
mannitol at 37°C for 48 hours and the alpha-amylase activity had been presented in
Figure 13. The increase in inositol and mannitol concentration in the fermentation
media was accompanied with the rise in amylase activity (Figure 14). 1% inositol and
mannitol had maximum amylase activity of 48.5 ± 1 U/mg and 51.24 ± 1.75 U/mg,
respectively, compared to control 14.5 ± 0.25 U/mg.
Figure 13: Effect of sugar alcohol (D-mannitol and D-inositol) on amylase production
after 48 hours of SSF; lined column: specific activity of α-amylase in presence of
inositol from SSF extract; black column: specific activity of α-amylase in presence of
mannitol from SSF extract.
In order to elucidate the role of all the supplements in enhancing alpha-amylase
activity in the fermented extract, the extract containing alpha-amylase was subjected
to thermal decay at 37°C temperature for various incubation periods ranging from 0 to
60 minutes in absence and presence of ions and sugar alcohols. D-Inositol and Dmannitol have offered considerable protection against heat induced denaturation at
37°C after one hour as manifested from the retention of residual enzyme activity
around 73 and 77% compared to 52% observed for amylase in extract alone in
absence of any stabilizer. Similar trend of stabilization of alpha-amylase activity in
presence of various salt ions (100 mM) like calcium, chloride, and nitrate has also
been noticed to be subjected under thermal denaturation under similar conditions as
before. All the salt ions have protected around 80% of amylase activity compared to
control without salts having activity around 52% (Figure 13).
- 33 -
Figure 14: Percentage residual activity of amylase in absence and presence of ions
and sugar alcohols.
ASSAY OF AMYLASE:
ENZYME Enzyme activity of crude enzyme was performed by using DNS
reagent. And the enzyme activity observed for this strain was found to be 9 U/ml.
Among physical parameters, pH of the growth medium plays an important role by
inducing morphological changes in microbes and inenzyme secretion. The pH change
observed during the growth of microbes also affected product stability in the medium.
As shown in table 2 the isolate was able to grow in the pH range of 5–8, but pH 7.0
was the optimum for the growth of the cultures. Temperature also plays the
significant role in the stability in enzyme activity. 35°C was found to be optimum
temperature at which enzyme activity was found to be higher.
Application:
Fermentation
Alpha and beta amylases are important in brewing beer and liquor made from
sugars derived from starch. In fermentation, yeast ingest sugars and excrete alcohol.
In beer and some liquors, the sugars present at the beginning of fermentation have
been produced by "mashing" grains or other starch sources (such as potatoes). In
traditional beer brewing, malted barley is mixed with hot water to create a "mash,"
which is held at a given temperature to allow the amylases in the malted grain to
convert the barley's starch into sugars. Different temperatures optimize the activity of
alpha or beta amylase, resulting in different mixtures of fermentable and
- 34 -
unfermentable sugars. In selecting mash temperature and grain-to-water ratio, a
brewer can change the alcohol content, mouthfeel, aroma, and flavor of the finished
beer.
In some historic methods of producing alcoholic beverages, the conversion of starch
to sugar starts with the brewer chewing grain to mix it with saliva.[9] This practice is
no longer widely in use.
Flour additive:
Amylases are used in breadmaking and to break down complex sugars, such as
starch (found in flour), into simple sugars. Yeastthen feeds on these simple sugars and
converts it into the waste products of alcohol and CO2. This imparts flavour and
causes the bread to rise. While amylases are found naturally in yeast cells, it takes
time for the yeast to produce enough of these enzymes to break down significant
quantities of starch in the bread. This is the reason for long fermented doughs such as
sour dough. Modern breadmaking techniques have included amylases (often in the
form of malted barley) into bread improver, thereby making the process faster and
more practical for commercial use.
Alpha amylase is often listed as an ingredient on commercially package milled flour.
Bakers with long exposure to amylase-enriched flour are at risk of developing
dermatitis or asthma.
Molecular biology:
In molecular biology, the presence of amylase can serve as an additional
method of selecting for successful integration of a reporter construct in addition to
antibiotic resistance. As reporter genes are flanked by homologous regions of the
structural gene for amylase, successful integration will disrupt the amylase gene and
prevent starch degradation, which is easily detectable through iodine staining.
Medical uses:
Amylase also has medical applications in the use of Pancreatic Enzyme
Replacement Therapy (PERT). It is one of the components in Sollpura (Liprotamase)
to help in the breakdown of carbohydrates into simple sugars.
- 35 -
Other uses:
An inhibitor of alpha-amylase, called phaseolamin, has been tested as a
potential diet aid.
When used as a food additive, amylase has E number E1100, and may be derived
from swine pancreas or mould mushroom.
Bacilliary amylase is also used in clothing and dishwasher detergents to dissolve
starches from fabrics and dishes.
Factory workers who work with amylase for any of the above uses are at increased
risk of occupational asthma. Five to nine percent of bakers have a positive skin test,
and a fourth to a third of bakers with breathing problems are hypersensitive to
amylase.
Hyperamylasemia:
Blood serum amylase may be measured for purposes of medical diagnosis. A
higher than normal concentration may reflect one of several medical conditions,
including acute inflammation of the pancreas (it may be measured concurrently with
the more specific lipase), but also perforated peptic ulcer, torsion of an ovarian
cyst, strangulation, ileus,
mesenteric
ischemia,
macroamylasemia
and mumps.
Amylase may be measured in other body fluids, including urine and peritoneal fluid.
A January 2007 study from Washington University in St. Louis suggests that saliva
tests of the enzyme could be used to indicate sleep deficits, as the enzyme increases
its activity in correlation with the length of time a subject has been deprived of sleep.
History:
In 1831, Erhard Friedrich Leuchs (1800–1837) described the hydrolysis of
starch by saliva, due to the presence of an enzyme in saliva, "ptyalin", an
amylase. The modern history of enzymes began in 1833, when French chemists
Anselme Payen and Jean-François Persoz isolated an amylase complex from
germinating barley and named it "diastase". In 1862, Alexander Jakulowitsch
Danilewsky (1838–1923) separated pancreatic amylase from trypsin.[22][23]
Human evolution
- 36 -
Carbohydrates are a food source rich in energy. Following the agricultural
revolution 12,000 years ago, human diet began to rely more on plant and animal
domestication in place of hunting and gathering. This shift also symbolizes the
beginning of a diet composed of 49% carbohydrates as opposed to the previous 35%
observed in Paleolithic humans.[citation needed] As such, starch became a staple of human
diet. Large polymers such as starch are partially hydrolyzed in the mouth by the
enzyme amylase before being cleaved further into sugars. Therefore, humans that
contained amylase in the saliva would benefit from increased ability to digest starch
more efficiently and in higher quantities. Despite the obvious benefits, early humans
did not possess salivary amylase, a trend that is also seen in evolutionary relatives of
the human, such as chimpanzees and bonobos, who possess either one or no copies of
the gene responsible for producing salivary amylase. This gene, AMY1, originated in
the pancreas. A duplication event of the AMY1 gene allowed it to evolve salivary
specificity, leading to the production of amylase in the saliva. In addition the same
event occurred independently in rodents, emphasizing the importance of salivary
amylase in organisms that consume relatively large amounts of starch.
However, not all humans possess the same number of copies of the AMY1
gene. Populations known to rely more on carbohydrates have a higher number of
AMY1 copies than human populations that, by comparison, consume little starch. The
number of AMY1 gene copies in humans can range from six copies in agricultural
groups such as European-American and Japanese (two high starch populations) to
only 2-3 copies in hunter-gatherer societies such as the Biaka, Datog, and Yakuts. The
correlation that exists between starch consumption and number of AMY1 copies
specific to population suggest that more AMY1 copies in high starch populations has
been selected for by natural selection and considered the favorable phenotype for
those individuals. Therefore, it is most likely that the benefit of an individual
possessing more copies of AMY1 in a high starch population increases fitness and
produces healthier, fitter offspring. This fact is especially apparent when comparing
geographically close populations with different eating habits that possess a different
number of copies of the AMY1 gene. Such is the case for some Asian populations
that have been shown to possess few AMY1 copies relative to some agricultural
population in Asia. This offers strong evidence that natural selection has acted on this
gene as opposed to the possibility that the gene has spread through genetic drift.[25]
- 37 -
Lipase
lipase is any enzyme that catalyzes the hydrolysis of fats (lipids). Lipases are
a subclass of the esterases. (80)
Lipases perform essential roles in the digestion, transport and processing of dietary
lipids
(e.g. triglycerides, fats, oils)
in
most,
if
not
all,
living organisms. Genes encoding lipases are even present in certain viruses.
Most lipases act at a specific position on the glycerol backbone of a
lipid substrate (A1, A2 or A3)(small intestine). For example, human pancreatic
lipase (HPL), which is the main enzyme that breaks down dietary fats in
the human digestive system, converts triglyceride substrates found in ingested oils
to monoglycerides and two fatty acids.
Several other types of lipase activities exist in nature, such as phospholipases
and sphingomyelinases, however these are usually treated separately from
"conventional" lipases.
Structure and catalytic mechanism
Although a diverse array of genetically distinct lipase enzymes are found in
nature; and, they represent several types of protein folds and catalytic mechanisms,
most of them are built on an alpha/beta hydrolase fold and employ a chymotrypsinlike hydrolysis mechanism using a catalytic triad consisting of a serine nucleophile,
a histidine base, and an acid residue (usually aspartic acid).(82-86)
Figure15: Structure of lipase
- 38 -
Production and purification of lipase enzyme
1. Isolation and characterization of bacterial strains
Petrol spilled soil sample was collected from petrol bunk situated in
Coimbatore, Tamilnadu, India. Serial dilution technique was used to isolate
bacterial strains. Isolated bacterial strains were subjected to Gram’s staining for
morphological identification and biochemical tests such as Indole production test,
Citrate utilization test, Carbohydrate fermentation test, Triple sugar iron test, Oxidase
test, Catalase test, Nitrate reduction
test, Hydrogen
sulphidetest, Methyl
red and
Voges–Proskauer test were performed according to Cappuccino et al. (1996).(89)
Screening of lipase producing bacterial strains
Isolated bacterial strains were screened for their lipolytic activity on the basis
of Tributyrin Agar plate assay method (TBA). The tributyrin agar was purchased from
Himedia. Tributyrin agar media along with 1.0% (v/v) olive oil were prepared and
sterilized at 121 °C for 15 min, and then sterilized media were poured into petriplate.
Isolated strains were streaked on the tributyrin agar plate and it was incubated at
37 °C for 24 h to observe zone.
Enzyme production media
Screened positive bacterial strains were cultivated in lipase producing media
for enzyme production. Lipase producing media consist of 3% yeast extract,
3% sucrose, 0.1 g (g/l) CaSO4, 0.5 g/l – KH2PO4, 0.1 g/l – MgSO4.7H20, 1% olive oil
and 100 ml distilled water in a 250 ml conical flask as submerged fermentation
method. Inoculated flaks were incubated at 37 °C for 24–48 h (Mobarak-Qamsari et
al., 2011).
Optimization of lipase producing media
Production media was supplemented with different carbon and nitrogen source
such as sucrose, glucose, lactose, peptone, yeast extract and ammonium sulphate at
different concentration (1–5%) to determine the highest yield of lipase enzyme.
Microbial growth was optimized by inoculating bacteria in an autoclaved medium that
had pH varying from 5 to 10 by dissolving components of the minimal medium in the
- 39 -
buffer of desired pH. Temperature optimization was carried out by growing bacterial
strains at temperature 32–40 °C in a shaking incubator. Effect of media components
on lipase activity was measured using photoelectric colorimeter at 610 nm. Similar
colorimetric measurement method was carried out by Schmidt and Blum (1978).
Partial purification of lipase enzyme
All purification steps were performed at room temperature. From the above
lipase produced media 20 ml of each bacterial strain medium was taken and the cells
were separated by centrifugation at 5000 rpm for 30 min. The supernatant was
collected and enzyme was concentrated using addition of 10–100% ammonium
sulphate. Fractionated enzyme samples were then subjected to dialysis process for
partial purification with the help of dialysis membrane.
Microorganism producing lipase
The bacterial strain Pseudomonas aeruginosa used in this study was isolated
from a wastewater at sidibel abbes, Algeria. The isolates were identified on the basis
of various morphological, physico-chemical, and biochemical characteristics.
Lipolytic bacteria are typically detected and secreened through the appearance of
clearing zones by using a selective medium. Which was containing Tween 80 or olive
oil as the only source of carbon? The diameter ratio of clear zone and colony was
measured.
- 40 -
Figure: Microorganisms producing lipase
Effect of physical parameters on lipase production
The effect of physical parameters (pH, tempera-ture and incubation period) on
lipase production was carried out on production medium containing the chemical
ingredients (g/l) (Peptone 10, olive oil 10, yeast extract- 5, NaCl -1, NaH2PO4 -6.08,
Na2HPO4- 8.63,at pH 7.4. One ml of sterile MgSO4.7H2O stock solution (500 g/l)
was added after autoclaving). 0.1% culture was inoculated into the autoclaved
medium. The flasks were incubated on rotary shaker at 100 rpm, at30°C for 2 days.
The culture were collected at different time intervals and centrifuged at 1000rpm for
10minat 4°C. Lipase activity was estimated by titrimetry method.(90)
Effect of incubation time and biomass on lipase activity
The effect of incubation time and biomass on lipase activity was determined
for 12-120 h. it was noted that a high biomass was obtained at 48 h of incubation and
high lipase activity was found in 72 h of incubation time around 1.2U/ml (Figure 17)
Effect of pH on lipase production
pH and temperature are the two important environmental factors which
influences the lipase production. The pH of the production medium plays a critical
- 41 -
role for the optimal physiological performances of the bacterial cell and the transport
of various nutrient components across the cell membrane aiming at maximizing the
enzyme yields. Bacillus sp. was inoculated in the lipase production medium and
incubated at different pH’s namely 5.5, 6, 6.5, 7, 7.5, 8 and 8.5. At pH 8, maximum
lipase activity of 1.4 U ml-1 was observed
Figure 18
Effect of Temperature lipase production
A comprehensive review of all bacterial lipase.states that maximum activity of
lipases at pH values higher than 7 has been observed in many cases. Bacterial lipases
have a neutral or alkaline optimum pH with the exception of lipase from P.
fluorescensSIK W1 that had an acidic optimum pH 4.8. For the temperature
optimization process, Bacillus sp. was inoculated in lipase production medium at
different temperatures namely 4, 15, 25, 35 and 45°C. The optimum temperature for
lipase production was found to be 30°C showing lipase units of0.62 U ml-1. The
temperature 30°C and pH 8 was found to be optimum for the lipase production by the
bacterial strain.
Figure 19
- 42 -
Effect of carbon sources
The culture environment has a dramatic influence on enzyme production
especially carbon and nitrogen sources playa crucial role in enzyme induction in
bacteria . The major factor for the expression of lipase activity has always been
carbon, since lipases are inducible enzymes and are thus generally produced in the
presence of a lipid source such as oil or any other inducer, such as triacylglycerols,
fatty acids, hydrolysable esters, tweens, bile salts and glycerol. However, their
production is significantly influenced by other carbon sources such as sugars,
polysaccharides, whey and other complex sources. Various carbon sources namely
glucose, sucrose, galactose, lactose and starch were administrated at 1% in the
production medium in this study. It was noted that lactose favoured high enzyme
production of 75 U ml-1. The present study is in contrast with the findings of
Lakshmi who reported that the production of lipase was high in medium containing
glucose supported by Banerjee who also reported that some bacteria showed higher
activities when grown in medium containing glucose.
(Figure 20)
Effect of Nitrogen source
Beside carbon source, the type of nitrogen source in the medium also
influenced the lipase titers in production medium. Generally, microorganisms provide
high yields of lipase when organic nitrogen sources are used, such as peptone and
yeast extract, which have been used for lipase production by various Bacillus sp. and
- 43 -
Pseudomonads sp. It was noted that among the different nitrogen sources used,
peptone was found to be the most suitable nitrogen source for Bacillus lipase
production.Sirisha et al has also reported peptone as the bestnitrogen source for lipase
production.reported that inorganic nitrogen sources such as ammonium chloride and
ammonium dihydrogen phosphate have been also reported to be effective in some
bacterial sp.
(Figure 21)
Dairy Industry
Lipases are extensively used in the dairy industry for the hydrolysis of
milk fat. Current applications include the flavour enhancement of cheeses, the
acceleration of cheese ripening , the manufacturing of cheese like products,
and the lipolysis of butterfat and cream. The free fatty acids generated by the
action of lipases on milk fat endow many diary products, particularly soft
cheeses, with their specific flavour characteristics. Thus the addition of lipases
that primarily release short chain (mainly C4 and C6) fatty acids lead to the
development of a sharp,tangyflavour, while the release of medium chain
(C12,C14) fatty acids tend to impart a soapy taste to the product. In addition,
the free fatty acids take part in simple chemical reactions, as well as being
converted by the microbial population of the cheese. This initiates the
synthesis of flavour ingredients such as acetoacetate, beta-keto acids, methyl
ketones, flavour esters and lactones.The intensive use of lipases in cheese
- 44 -
making started in the U.S.A after the Second World War. It was engendered
by the Food and Drug Administration's ban on the import of rennet paste from
Europe because of the impurity and unsatisfactory microbiology of the
product. This paste was used by the manufacturers of traditional Italian
cheeses ( Provolone, Romano, Mozzarela, Parmesan) and the first lipase
cocktails were introduced as a substitute to create the typical lipolyticflavour
of these varieties.The traditional sources of lipases for cheese flavour
enhancement are animal tissues, especially pancreatic glands(bovine and
porcine) and the pre-gastric tissues of young ruminants(kid,lamb,calf). The
latter are more commonly used in cheese making. The commercial pre-gastric
lipases are available in the form of liquid extracts, pastes and vaccum or freeze
dried powders. Each type of pre-gastric lipase gives rise to its own
charecteristicflavourprofile : a buttery and slightly peppery flavour(calf) ; a
sharp 'piccante' flavour (kid); a strong 'pecerino' also described as 'dirty sock'
flavour(lamb).A whole range of microbial lipase preparations has been
developed for the cheese manufacturing industry: Mucormeihei(Piccnate,
Gist-Brocades; Palatase M, Novo Nordisk), Aspergillusniger and A.oryzae
(Palatase A, Novo Nordisk; Lipase AP, Amano; Flavour AGE, Chr. Hansen)
and several others. These microbial lipases are used not only for flavour
enhancement and the acceleration of the ripening of specific cheeses such as
blue, but in some cases they have also successfully replaced pre-gastric lipases
. A range of cheeses of good quality was produced by using individual
microbial lipases or mixtures of several preparations.Apart from substitution
of rennet paste and flavour enhancement, lipases are widely used for imitation
of cheeses made from ewe's or goat's milk. Addition of lipases to cow's milk
generates a flavour rather similar to that of ewe/goat milk. This is used for
producing cheeses like Feta, Manchego and Romano from cow's milk. When
added to certain blue cheeses, lipase imitate the taste of Roquefort, which is
normally produced from sheep's milk. Similarly, the addition of lipases to
pasteurised milk leads to the development of the normal flavour of Ras or
Konpanisti, which traditionally are produced from raw milk.Lipases also play
a crucial role in the preparation of so-called enzyme modified cheeses (EMC).
EMC is a cheese that is incubated in the presence of enzymes at elevated
temparature in order to produce a concentrated flavour for use as an ingredient
- 45 -
in other products (dips,sauses,dressings,soups,snacks etc.). Typically the
concentration of free fatty acids is ten times higher in EMC than in that of the
corresponding young cheese. EMC technology is widely used in the U.S.A.
Detergents
Enzymes can be used in the laundry detergents and automatic dish
washing machines detergents. Enzymes can reduce the environmental load of
detergent products, since they save energy by enabling a lower wash
temperature to be used; allow the content of other, often less desirable,
chemicals in detergents to be reduced; are biodegradable, leaving no harmful
residues; have no negative impact on sewage treatment processes; and, do not
present a risk to aquatic life. Other enzymes are currently widely used in
household cleaning products. A great deal of research is currently going into
developing lipases which will work under alkaline conditions as fat stain
removers.
Oleochemical Industry
The scope for the application of lipases in the oleochemical industry is
enormous. Fats and oils are produced world wide at a level of approximately
60 million tonnes per annum and a substantial part of this(more than 2 million
tonnes per annum) is utilised in high energy consuming processes such as
hydrolysis, glycerolysis and alcoholysis. The conditions for steam fat splitting
and conventional glycerolysis of oils involve high temparatures of 240-260
degree C and high pressures (methanolysis is currently performed under
slightly milder conditions). The resulting products are often unstable as
obtained and require re-distillation to remove impurities and products of
degradation. In addition to this, highly unsaturated heat sensitive oils cannot
be used in this process without prior hydrogenation.The saving of energy and
minimisation of thermal degradation are probably the major attractions in
replacing the current chemical technologies with biological ones. However, in
spite of their apparent superiority, enzymic methods have not as yet attained a
level of commercial exploitation commensurate with their potential. There
have been several communications about relatively small scale enzymic fat
- 46 -
splitting processes for the production of some high value polyunsaturated fatty
acids and the manufacture of soap. For instance Miyoshi Oil & Fat Co., Japan,
reported the commercial use of Candida cylindracea lipase in the production
of soaps. The company claimed that the enzymic method yielded a superior
product and was cheaper overall than the conventional Colgate-Emery
process.There are probably several reasons for the generally disappointing
level of commercial applications of lipase in this sector at present. First of all,
the oleochemical industry is very conservative, owing to huge capital
investments involved. Therefore one cannot expect rapid changes. Secondly
until recently the high cost of lipases remained prohibitive for the
manufacturing of bulk products. The introduction of the new generation of
cheap and very thermostable enzymes should change the economic balance in
favour of lipase use. Thirdly, some concern has been expressed by chemical
engineers with regard to running and controlling enzymic processes on the
required scale. However the recent commercialisation of several lipase based
technologies has proved their feasibility unambiguosly.The future of lipases
look rather promising in the context of oleochemistry.Some fats are much
more valuable than others beacuse of their structure. Less valuable fats can be
converted into more useful species using blending of chemical methods but
these tend to give quite random products. Lipase catalysedtransesterification
of cheaper oils can be used, for example to produce cocoa butter from palm
mid-fraction.
Pharmaceutical Industry
The vast variety of synthetic pharmaceuticals and agrochemicals
containing one or more chiral centres, are stll being sold as racemates. This is
despite the fact that the desired biological activity resides in one particular
enantiomer. A single isomer is preferable to a racemate, but there are severe
technical and/or economic problems with the production of single isomers.The
usefulness of lipases in the preparation of chiral synthons is well recognised.
The resolution of 2-halopropionic acids, starting materials for the synthesis of
phenoxypropionate herbicides is being carried out on a 100-kg scale by
Chemie Linz Co.(Austria) under a license from the Massachusetts Institute of
- 47 -
Technology. The process is based on the selective esterification of (S)-isomers
with butanolcatalysed by porcine pancreatic lipase in anhydrous hexane.
Typically ,> 99% enantiomeric excess(e.e) is obtained at 75% of the
theoritical yield and the resolution is complete in several hours.Generally, the
lipase mediated resolution of 2-substituted propionic acids, and especially 2aryl derivatives, have been the subject of intensive investigations. A
substantial body of literature exists on the production of both (R) and (S)
isomers of alpha(*sub)-phenoxypropionic acids, which are useful synthons for
the preparation of enantiomerically pure herbicides and non-steroidal antiinflammatory drugs (naproxen,ibuprofen) respectively. The required optically
pure derivative can be obtained directly via the (trans)esterification or
hydrolysis of the corresponding ester. These resolutions have been performed
on a multi-kilogramme scale by several companies world-wide.Another
instance of commercial application of lipases to the resolution of racemic
mixtures is the hydrolysis of epoxy alcohol esters. The highly enantioselective
hydrolysis of (R,S)-glycidyl butyrate has been developed by DSM-Andeno
(the Netherlands). The reaction products, (R)-glycidyl esters and (R)-glycidol,
are readily converted to (R)-and (S)-glycidyltosylates, which are very
attractive intermediates for the preparation of optically active beta-blockers
and a wide range of other products.A similar technology has been
commercialized by Sepracor Inc.(USA). This company has successfully
operated a multi-kilogramme scale membrane bioreactor to produce the
2(R),2(S) methyl methoxyphenylglycidate, the key intermediate in the
manufacture of the optically pure cardiovascular drug diltiazem. 2(S),3(R)methoxyphenylglycidic acid, the product of enzymic hydrolysis, was found to
be unstable under the conditions of the reaction, and the resultant aldehyde
inhibited the lipase activity and reduced the lifetime of the enzyme. Both
problems were overcome by the introduction of a multi-phase membrane
reactor where the aldehyde by-product reacted in situ with bisulphite to form a
non-inhibitory, water soluble adduct, extracted into the aqueous phase.Lipases
have been found useful as industrial catalysts for the resolution of racemic
alcohols. Enantiomerically pure endo-2-norborn-2-ol is an important chiral
intermediate in the preparation of some prostaglandins, steroids and
carbocyclic nucleoside analogues. Bend Research Inc(USA) have developed a
- 48 -
two-step resolution process . The process involved acylation of the (R)-alcohol
with butyric anhydride, mediated by Candida cylindracea lipase, followed by
the hydrolysis of the (R)-ester catalysed by the same enzyme. The first
resolution resulted in the enantiomerically pure (S)-alcohol (e.e> 98%) and
(R)-ester(e.e-78%) which was further enriched by the back conversion to (R)alcohol(e.e> 98%). the resolution was performed on a multi-kilogramme scale
in a permselective membrane bioreactor specially designed to facilitate
product recovery and to minimise product inhibition.Lipases are currently
being used by many pharmaceutical companies world-wide for the preparation
of optically active intermediates on a kilo-gramme scale. A number of
relatively small biotechnological companies, such as Enzymatix in the U.K,
specialise in biotransformations and offer a whole variety of intermediates
prepared via lipase mediated resolution.Regioselective modifications of
polyfunctional organic compounds is yet another area of expanding lipase
application. In may cases, lipases have been shown to acylate or deacylate
selectively one or several hydroxyl groups of similar reactivity in
carbohydrates, polyhydroxylated alkaloids and steroids.Apart from the
synthesis of sugar based surfactants, lipases wer successfully applied in the
regioselective modification of castanospermine. a promising drug for the
treatment of AIDS.Thus lipases have become a conventional research tool in
many organic chemistry laboratories. As a result they are readily incorporated
into synthetic routes especially when optical purity of the final product is
essential.
Cosmetic Industry
Although the cost of lipase catalysed esterification remains too high
for the manufacturing of bulk products, the synthesis of several speciality
esters has found its way in the market place. Unichem International has
launched the production of isopropyl myristate, isopropyl palmitate and 2ethylhexylpalmitate for use as an emollient in personal care products such as
skin and sun-tan creams, bath oils etc. Immobilised Rhizomucormeihei lipase
was used as a biocatalyst in the solvent free esterification, which was driven to
completion by vaccum distillation of the water produced during the reaction.
- 49 -
The company claims that the use of the enzyme in place of the conventional
acid catalyst gives products of much higher quality, requiring minimum
downstream refining. Batches of several tonnes have been successfully
produced at Unichem's factory in Spain.Wax esters have similar applications
in personal care products and are also being manufactured enzymically The
company uses Candida cylindracea lipase in a batch bioreactor. According to
the manufaturer, the overall production cost is slightly higher the that of the
conventional method, but the cost is justified by the improved quality of the
final
product.
Medical applications
Possible medical applications of lipases are under consideration, for
example inhibition of the human enzyme as a method of reducing fatty acid
absorption is being investigated as a possible treatment for obesity.
- 50 -
summary
Enzymes are macromolecular biological catalysts. Enzymes accelerate chemical
reactions. The molecules upon which enzymes may act are called substrates and the
enzyme converts the substrates into different molecules known as products.they are
active in small amount, highly specific, sensitive, colloidal in nature. Almost all
metabolic processes in the cell need enzyme catalysis in order to occur at rates fast
enough to sustain life.
Most
enzymes
are
proteins,althoughafewarecatalyticRNAmolecules.Thelatterarecalledribozymes.Enzym
es' specificity comes from their unique three-dimensional structures.
Some enzymes do not need additional components to show full activity. Others
require non-protein molecules called cofactors to be bound for activity. Cofactors can
be either inorganic or organic compounds.
Enzymes activity affecting by different factors as enzyme concentration, substrate
concentration, temperature, pH, time, coenzymes concentration, presence of
inhibitors.
Enzymes can come from animals, plants and microbial organisms ,generally Microbes
are preferred to plants and animals as sources of enzymes as they are generally
cheaper to produce, their enzyme contents are more predictable and controllable,
reliable supplies of raw material of constant composition are more easily arranged,
and plant and animal tissues contain more potentially harmful materials than
microbes, including phenolic compounds (from plants), endogenous enzyme
inhibitors and proteases.
Production and purification of enzymes occure in multi-steps beginning with selection
of organism, formulation of medium, production process, recovery and purification of
enzymes, removal of cell debris, removal of nucleic acids, enzymes precipitation,
separation by chromatography and finally drying and packing.
Amylases are the major group of enzymes used for starch enzyme hydrolysis, which
is required prior to further fermentation for bioethanol production in biorefinery
industries. This chapter discusses the characteristics, sources, production, and
applications of amylases in various Industries. The main four industrially important
members of the amylase family of a wide range of applications are a‐amylase,
ß‐amylase, glucoamylase (GA), and pullulanase. In general, two main cultivation
- 51 -
strategies are applied for amylase production‐submerged fermentation (SMF) and
solid‐state fermentation. Amylases are the most important industrial enzymes based
on their wide range of applications in many industries. Production of stable amylases
at a high temperature, acidic pH, and calcium independence was successfully
achieved by using different approaches, such as production by extermophilic
microorganisms, production by recombinant microorganisms, protein engineering and
amino acids mutagenesis, a chemical stabilization method, a metal ion stabilization
method, and an immobilization method
Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) are part of the family of
hydrolases that act on carboxylic ester bonds. The physiologic role of lipases is to
hydrolyze triglycerides into diglycerides, monoglycerides, fatty acids, and glycerol.
These enzymes are widely found throughout the animal and plant kingdoms, as well
as in molds and bacteria. Of all known enzymes, lipases have attracted the most
scientific attention. In addition to their natural function of hydrolyzing carboxylic
ester bonds, lipases can catalyze esterification, interesterification, and
transesterification reactions in nonaqueous media. This versatility makes lipases the
enzymes of choice for potential applications in the food, detergent, pharmaceutical,
leather, textile, cosmetic, and paper industries. The most significant industrial
applications of lipases have been mainly found in the food, detergent, and
pharmaceutical sectors. Limitations of the industrial use of these enzymes have
mainly been owing to their high production costs, which may be overcome by
molecular technologies, enabling the production of these enzymes at high levels and
in a virtually purified form.
- 52 -
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