COLLEGE 0F FOOD TECHNOLOGY ASHTI BEED

FOOD CHEMIESTRY & NUTRITION

THEORY NOTES

COURSE NO: FCN 368

COURSE TITLE: ENZYMES IN FOOD INDUSTRY

CREDITS: 2(1+1)

DEPARTMENT OF FOOD CHEMISTRY AND NUTRITION

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SYLLABUS INDEX

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(1–3) Introduction: classification and nomenclature, mechanism of enzyme action,
enzyme kinetics, factors affecting the rate of enzymic reactions, sources of enzymes

The human body is composed of different types of cells, tissues, and other complex organs. In order to
function efficiently, there are certain chemicals released by our body to speed up certain biological
processes like digestion, respiration, excretion, and other metabolic activities in order to maintain a
healthy life. Thus, enzymes play an important role in all higher multicellular organisms including plants
by regulating all the biological processes.

Let us have a detailed look at what are enzymes, their structure, types, mechanism and different factors
affecting its activity.

What Are Enzymes?

“Enzymes can be defined as biological polymers that catalyze biochemical reactions.”

The vast majority of enzymes are proteins with catalytic capabilities that are essential for maintaining
various life processes. Metabolic processes and other chemical reactions in the cell are carried out by a
set of enzymes that are necessary to sustain life.

The initial stage of metabolic process depends upon the enzymes, which react with a molecule and is
called substrate. Enzymes convert the substrates into other distinct molecules and are called the
products.

The regulation of enzymes has been a key element in clinical diagnosis because of their role in
maintaining life processes. The macromolecular component of all enzymes consists of protein, except in
the class of RNA catalysts called ribozymes. The word ribozyme is derived from the ribonucleic acid
enzyme. Many ribozymes are molecules of ribonucleic acid which catalyze reactions in one of their own
bonds or among other RNAs.

Enzymes exist in all fluids and tissues of the body. Intracellular enzymes catalyze all the reactions that
occur in metabolic pathways. The enzymes in plasma membrane regulate catalysis in the cells in
response to cellular signals and enzymes in the circulatory system regulate clotting of blood. Almost all
the significant life processes are based on the enzyme functions.

Enzyme Structure :-

Enzymes are a linear chain of amino acids that generate the three-dimensional structure. The sequence
of amino acids enumerates the structure which in turn identifies the catalytic activity of the enzyme. The

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structure of the enzyme denatures when heated, leading to loss of enzyme activity, which is typically
connected to the temperature.

Enzymes are larger than their substrates and their size vary, which range from sixty-two amino acid
residues to an average of two thousand five hundred residues present within fatty acid synthase. Only a
small section of the structure is involved in catalysis and are situated next to binding sites. The catalytic
site and binding site together constitute the enzyme’s active site. A small number of ribozymes exists
which serves as an RNA-based biological catalyst. It reacts in complex with proteins.

Enzymes Classification :-

Types of Enzymes

Earlier, enzymes were assigned names based on the one who discovered it. With further researches,
classification became more comprehensive.

According to the International Union of Biochemists (I U B), enzymes are divided into six functional
classes and are classified based on the type of reaction in which they are used to catalyze. The 6 types
of enzymes are oxidoreductases, hydrolases, transferases, lyases, isomerases, ligases.

Oxidoreductases :-

These catalyze oxidation and reduction reactions,e.g. pyruvate dehydrogenase, which catalyzes the
oxidation of pyruvate to acetyl coenzyme A.

Transferases :-

These catalyze the transfer of a chemical group from one compound to another. An example is a
transaminase, which transfers an amino group from one molecule to another.

Hydrolases :-

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They catalyze the hydrolysis of a bond. For example, the enzyme pepsin hydrolyzes peptide bonds in
proteins.

Lyases :-

These catalyze the breakage of bonds without catalysis, e.g. aldolase (an enzyme in glycolysis)
catalyzes the splitting of fructose-1, 6-bisphosphate to glyceraldehyde-3-phosphate and
dihydroxyacetone phosphate.

Isomerases :-

They catalyze the formation of an isomer of a

compound, example, phosphoglucomutase
catalyzes the conversion of glucose-1-phosphate
to glucose-6-phosphate (transfer of a phosphate
group from one position to another in the same
compound) in glycogenolysis (conversion of
glycogen to glucose for quick release of energy.

Ligases :-

Ligases catalyze the joining of two

molecules. For example, DNA ligase
catalyzes the joining of two fragments of
DNA by forming a phosphodiester bond.

Cofactors :-

Co-factors are non-proteinous substances

that associate with enzymes. A cofactor is
essential for the functioning of an enzyme.
An enzyme without a cofactor is called an
apoenzyme. An apoenzyme and its cofactor
together constitute the holoenzyme.

There are three kinds of cofactors present in

enzymes:

• Prosthetic groups: These are cofactors tightly bound to an enzyme at all times. A fad is a
prosthetic group present in many enzymes.

• Coenzyme: A coenzyme is bound to an enzyme only during catalysis. At all other times, it is
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 detached from the enzyme. NAD+ is a common coenzyme.

• Metal ions: For the catalysis of certain enzymes, a metal ion is required at the active site to form
coordinate bonds. Zn2+ is a metal ion cofactor used by a number of enzymes.

Nomenclature of Enzyme :-

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 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; and (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.

Enzyme Kinetics :-

Enzyme kinetics means the way in which enzymes react with substrate and form products. Enzymes are
biological catalysts. They are specific to one type of reaction and to one or a small group of reactants
called substrates. A catalyst speeds up the rate of a reaction without being changed itself. They are
necessary as most biological reactions are very slow.

Kinetics is the study of reaction rates. This will be considered in the context of enzymes where the rate
of the reaction means the rate of product formation. In this article we will look at the structure, function
and clinical significance of enzyme kinetics.

Enzyme Structure :-

Most enzymes are globular proteins with the exception of a few RNA enzymes (ribozymes). They have
an active site made up of a few amino acids. This is where the reaction occurs. The rest of the enzyme
acts as a scaffold, bringing these key amino acids together.

The active site forms a cleft or crevice that the substrate can sit in during the reaction. The cleft creates
a better environment for the reaction to take place. They may do this, for example, by excluding water.

The active site is almost complementary to the substrate’s shape. Therefore, when the substrate binds,

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the enzyme must change shape slightly to fit it. This forms the enzyme-substrate complex, also called
“ES”. This is the induced fit model, which is an addition to the lock and key hypothesis. Only weak bonds
between the enzyme and substrate hold them in place. This is necessary to allow dissociation later on.

Enzymes have an optimum temperature and pH where they work best. Changes in pH can alter critical
ionization states, while changes in temperature can disrupt important bonds. Deviations from these
optimum conditions will disrupt the enzyme’s structure and impact on its kinetics, eventually completely
denaturing and disabling.

Enzyme Function :- Enzymes lower the activation energy, Ea, of a particular reaction. They can do this
because they have a high affinity for a transition state. The activation energy is the minimum energy
needed for a reaction to occur. Enzymes assist in the reaction so that less energy is needed. This means
the reaction can occur more easily. This speeds up the rate of the reaction as it allows the product to be
formed faster.

An enzyme has a high affinity for the transition state (even higher than for its substrate). Therefore when
the substrate binds, it is quickly forced into the transition state. This is a state that exists between the
substrate and the product. The enzyme is said to facilitate the formation of the transition state.

The transition state has a high energy, making it very unstable. It can only exist transiently. The
transition state spontaneously turns into the more stable product with lower energy. The enzyme will
have a low affinity for the product and so the product is released.

Rate Limiting Steps :- The rate limiting step in any reaction is its slowest step. It sets the pace for the
entire reaction. After all, a production line can only be as productive as its slowest worker. In enzymatic
reactions, the conversion of the enzyme-substrate complex to the product is normally rate limiting. The
rate of this step (and therefore the entire enzymatic reaction) is directly proportional to the concentration
of ES.

The concentration of ES changes as the reaction progresses. Therefore, the rate of product formation
also changes over time. When the reaction reaches equilibrium (steady state) the concentration of ES
(and therefore the rate) remains relatively constant.

Reaction Kinetics :-

When an enzyme is added to a lot of substrate, the reaction that follows occurs in three stages with
distinct kinetics:

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The pre-steady state phase is very short as equilibrium is reached within microseconds. If you measure
the rate in the first few seconds of a reaction (V0) you are actually measuring the steady state. This is
the rate used in Michaelis-Menten Kinetics.

Michaelis-Menten Kinetics :-

Two terms that are important within Michaelis-Menten Kinetics are:

Vmax – the maximum rate of reaction when all enzyme active sites are saturated with substrate

Km – the substrate concentration that gives half maximal velocity. Km is a measure of the affinity an
enzyme has for its substrate, as a lower Km means that less of the substrate is required to reach half of
Vmax.

This equation concerns the steady state of an enzymatic reaction with one substrate, and is given by:

It describes how the initial rate of reaction, V0, is affected by the initial substrate concentration, [S]0. It
only looks at the start of the reaction. This allows it to ignore the reverse reaction where substrate is
formed from product. This is because at the start of the reaction there is no product present to become
substrate.

The plot of rate against concentration has the shape of a rectangular hyperbola. However, a more useful
representation of Michaelis–Menten kinetics is a graph called a Lineweaver–Burk plot. The equation used
to generate this plot is given by:
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This allows an easier interpretation of various quantities from the graph, such as the presence of an
inhibitor. This is shown in figure 3, where an inhibitor decreases Km and therefore shifts the original line
upwards.

Mechanism of Enzyme Reaction :-

Enzyme kinetics is a part of enzyme mechanism. Any two molecules have to collide for the reaction to
occur along with the right orientation and a sufficient amount of energy. The energy between these
molecules needs to overcome the barrier in the reaction. This energy is called activation energy.

Enzymes are said to possess an active site. The active site is a part of the molecule that has the definite
shape and the functional group for the binding of reactant molecules. The molecule binding to the enzyme
is called the substrate group. The substrate and the enzyme form an intermediate reaction with low
activation energy without any catalysts.

reactant(1)+reactant(2)→product

reactant(1)+enzyme→intermediate

intermediate+reactant(2)→product+enzyme

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The basic mechanism of enzyme action is to catalyze chemical reactions, which begin at the binding of
the substrate with the active site of the enzyme. This active site is a specific area that combines with
the substrate.

Enzyme-Substrate Interactions

Enzymes are the biocatalysts with high molecular weight proteinous compound. It enhances the reactions
which occur in the body during various life processes. It helps the substrate by providing the surface for
the reaction to occur. The enzyme comprises of hollow spaces occupying groups such as - SH, -COOH,
etc., on the outer surface. The substrate which has the opposite charge of the enzyme fits into these
spaces just like a key fits into a lock. This substrate binding site is called the active site of anenzyme
(E).

The favourable model of enzyme-substrate interaction is called the induced-fit model. This model states
that the interaction between substrate and enzyme is weak and these weak interactions induce
conformational changes rapidly and strengthen binding and bring catalytic sites close enough to substrate
bonds.

There are four major possible mechanisms of catalysis:

Catalysis by Bond Strain

The induced structural rearrangements in this type of catalysis produce strained substrate bonds that
attain transition state more easily. The new conformation forces substrate atoms and catalytic groups
like aspartate into conformations that strain substrate bonds.

Covalent Catalysis

The substrate is oriented to active place on the enzymes in such a manner that a covalent intermediate
develops between the enzyme and the substrate, in catalysis that occurs by covalent mechanisms. The
best example of this is involving proteolysis by serine proteases that have both digestive enzymes and
various enzymes of the blood clotting cascade. These proteases have an active site serine whose R
group hydroxyl produces a covalent bond with a carbonyl carbon of a peptide bond and causes
hydrolysis of the peptide bond.

Catalysis Involving Acids and Bases

Other mechanisms also contribute to the completion of catalytic events that are initiated by strain
mechanism like the use of glutamate as a general acid catalyst.

Catalysis by Orientation and Proximity

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Enzyme-substrate interactions bring reactive groups into proximity with one another. Also, groups like
aspartate are chemically reactive and their proximity and towards the substrate favours their
involvement in catalysis.

Action and Nature of Enzymes :-

Once substrate (S) binds to this active site, they form a complex (intermediate-ES) which then produces
the product (P) and the enzyme (E) The substrate which gets attached to the enzyme has a specific
structure and that can only fit in a particular enzyme. Hence by providing a surface for the substrate, an
enzyme slows down the activation energy of the reaction. The intermediate state where the substrate
binds to the enzyme is called the transition state. By breaking and making the bonds, the substrate binds
to the enzyme (remains unchanged), which converts into the product and later splits into product and
enzyme. The free enzymes then bind to other substrates and the catalytic cycle continues until the
reaction completes.

The enzyme action basically happens in two steps: Step1:

Combining of enzyme and the reactant/substrate.E+S →

[ES]

Step 2: Disintegration of the complex molecule to give the product.

[ES]→E+P

Thus, the whole catalyst action of enzymes is summarized as:E

+ S → [ES] → [EP] → E + P

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Factors affecting Enzyme Activity :-

There are several factors that affect the speed of an enzyme’s action, such as the concentration of the
enzyme, the concentration of the substrate, temperature, hydrogen ion concentration (pH), and the
presence of inhibitors.

Factor 1: Concentration of Enzyme

As the concentration of the enzyme is increased, the velocity

of the reaction proportionately increases. This property is used
for determining the activities of serum enzymes during the
diagnosis of diseases.

Factor 2: Concentration of Substrate

In the presence of a given amount of enzyme, the rate of enzymatic

reaction increases as the substrate concentration increases until a
limiting rate is reached, after which further increase in the substrate
concentration produces no significant change in the reaction rate.
At this point, so much substrate is present that essentially all of the
enzyme active sites have substrate bound to them.
In other words, the enzyme molecules are saturated with substrate. The excess substrate molecules
cannot react until the substrate already bound to the enzymes has reacted and been released (or been
released without reacting).

Factor 3: Effect of Temperature

The protein nature of the enzymes makes them extremely

sensitive to thermal changes. Enzyme activity occurs within a
narrow range of temperatures compared to ordinary chemical
reactions. As you have seen, each enzyme has a certain
temperature at which it is more active. This point is called the
optimal temperature, which ranges between 37 to 40C°.

The enzyme activity gradually lowers as the temperature rises more than the optimal temperature until it
reaches a certain temperature at which the enzyme activity stops completely due to the change of its
natural composition.

On the other hand, if the temperature lowers below the optimal temperature, the enzyme activity lowers
until the enzyme reaches a minimum temperature at which the enzyme activity is the least. The enzyme

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activity stops completely at 0C°, but if the temperature rises again, then the enzyme gets reactivated
once more.

Factor 4: Effect of pH

The potential of hydrogen (pH) is the best measurement for determining the concentration of hydrogen
ion (H+)in a solution. It also determines whether the liquid is acidic, basic or neutral. Generally, all liquids
with a pH below 7 are called acids, whereas liquids with a pH above 7 are called bases or alkalines.
Liquids with pH 7 are neutral and equal the acidity of pure water at 25 C°. You can determinepH of any
solution using the pH indicators.

Enzymes are protein substances that contain acidic carboxylic groups (COOH–) and basic amino groups
(NH2). So, the enzymes are affected by changing the pH value.

Each enzyme has a pH value that it works at with maximum efficiency called the optimal pH. If the pH is
lower or higher than the optimal pH, the enzyme activity decreases until it stops working. For example,
pepsin works at a low pH, i.e, it is highly acidic, while trypsin works at a high pH, i.e, it is basic. Most
enzymes work at neutral pH 7.4.

Factor 5: 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.

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Sources of Enzymes :-

Without enzymes, there would be no life. Enzymes are the worker bees that make things happen.
Enzymes act as biological catalysts. They accelerate biochemical reactions within our cells and affect
every function of the body, from digestion to breathing. Functionally, enzymes facilitate cellular reactions
that may not otherwise occur, by lowering the threshold of energy required for those reactionsto take
place. Some enzymes are even capable of reversing a standard reactionary response by sufficiently
lowering the required activation energy enough that the reaction favors the opposite direction.

Tens of thousands of different kinds of enzymes are believed to exist in the human body, each with a
specific purpose. There are three general categories of enzymes: digestive enzymes, metabolic
enzymes, and food or plant enzymes. The digestive enzymes category consists of the enzymes
produced within your own body to help break down food into its basic components for digestion.
Metabolic enzymes are found throughout our entire body – in our organs, bones, blood, and even within
the cells that produce them. They function in support of our heart, lungs, kidneys and brain. Food and
plant enzymes are naturally present in raw food. They generally serve the same function as digestive
enzymes, but these are the enzymes that we may take in through our diets, as opposed to the ones our
bodies produce. We can obtain these enzymes through eating fresh, raw and uncooked foods like fruits,
vegetables, eggs, unpasteurized dairy, meat and fish.

The modern diet generally revolves around processed and cooked food, but these processes destroy the
naturally occurring enzymes contained in the food. This places a heavy burden on our bodies to subsidize
the enzyme requirement for breaking down that food.

Raw food contains the necessary proportion and types of enzymes required to digest itself. This remains
one of the biggest benefits of a diet centered around raw food. The major components of the food (sugar,
protein, starch, fat) and their respective caloric amounts determine what type and quantityof enzymes
are also present. For example, the enzyme amylase is found in high carbohydrate fruits likeapples and
peaches. Fruits that are high in fat, such as avocados, contain the enzyme lipase.

Below, we will focus on enzymes we obtain from food sources (animal, plant and fungal) and their
respective usefulness.

Animal-Based Enzymes :-

There is a homeopathic theory called the “Law of Similars” which some believe may apply to animal
sourced enzymes. Although the source of animal-based enzymes we consume don’t originate from a
human body, it is thought they may be similar enough that the human body might do a better job
recognizing and utilizing them. However, it is important to note that this is merely a theory.

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Reliance on obtaining digestive enzymes from animal sources is challenging, because a majority of the
meat and other animal byproducts we consume are processed, pasteurized and/or cooked, which
destroys the natural enzymes. For vegetarians or vegans, animal enzymes are hardly an option, and
consuming raw meat or eggs is a dangerous endeavor, due to the risk of bacterial contamination.

From a digestive perspective, there are several important disadvantages associated with animal-based
enzyme sources. Temperature sensitivity is one of these. The human body does not generally have the
same temperature as the animal host of these enzymes, which can be destructive to the enzyme upon
entering the gastrointestinal tract.

Animal-based enzymes also function exclusively within a limited pH level range, which renders them fairly
ineffective in the gut. They become unstable in a low pH level (acidic) environment, resulting in theenzyme
being destroyed before it can perform its function. This pretty much eliminates the stomach as an
operational environment. As a result, to take in animal enzymes, they are better delivered into the body
within a protective enteric (polymer) coating capable of withstanding the stomach’s acidity. This means
that the enzymes don’t become available to the body until they reach the small intestines. The most
common type of animal enzymes used for dietary supplementation are pancreatic enzymes.
However, for the reasons outlined above, the general consensus is the best sources of enzymes are
plant and fungal.

Plant-Based Enzymes :-

Fruits and vegetables are commonly consumed in their raw, natural form. This alleviates the overarching
issue with animal-based enzymes by preserving the integrity of the enzymes themselves. Additionally,
plant-based digestive enzymes are effective over a broad scope of pH levels. This range is generally
believed to be between 3.0 and 9.0, which is highly compatible with the human gastrointestinal
environment. As a result, plant-based enzymes are well-suited for supporting comprehensive digestive
health.

Four important enzymes often found in plants are protease, amylase, lipase and cellulose. Protease
breaks down protein that can be present in meat, fish, poultry, eggs, cheese and nuts. Amylase assists
your body with the breakdown and subsequent absorption of carbohydrates and starches. Lipase aids the
digestion of fat. When your diet includes lipase-rich foods, it eases the production burden on the gall
bladder, liver and pancreas. Cellulase is present in many fruits and vegetables, and it breaks down food
fibers, which increases their nutritional value to our bodies. The presence of cellulase in plant-based
sources is important, because it is not naturally present in the human body.

Fruits and vegetables are an ideal source for enzymes. They are enzyme-rich and easily consumed
without needing to be cooked or processed, ultimately preserving the full functionality of the enzymes.

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Fungal Enzymes :-

Fungal Enzymes have numerous uses. They are critical in the production and preparation of many food
products, like beer, soy sauce, miso, baked goods, dairy and processed fruit. One of the oldest known
applications is the role of yeast in alcohol fermentation. Fungal enzymes are commonly produced from a
fungal source called Aspergillus. For example, Aspergillus oryzae is used in the preparation of sake and
soy sauce, while Aspergillus sojae is also used in soy sauce preparation, as well as in miso soup.

One of the most popular and well known culinary fungi is the mushroom. Some mushroom species
produce enzymes, including hydrolases, esterases, and phenol oxidases. Fungi and their enzymes can
also be found in yeast spreads and certain types of cheeses, such as Camembert and blue cheeses.

Fungi can contain a variety of enzymes, such as protease, amylase, lipase, cellulase and tilactase
(supports lactose absorption). Like plant enzymes, fungal enzymes are acid stable and can survive
within the pH range of the stomach. They are also suitable for a vegetarian diet, unlike animal-sourced
enzymes.

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(4-5) Enzyme Kinetics: enzyme concentration, substrate
concentration, environmentalconditions, inhibitors, activators and cofactors.

Enzyme Concentration :-

In order to study the effect of increasing the enzyme

concentration upon the reaction rate, the substrate must
be present in an excess amount; i.e., the reaction must be
independent of the substrate concentration. Any change in
the amount of product formed over a specified period of
time will be dependent upon the level of enzyme present.
Graphically this can be represented as: -->

These reactions are said to be "zero order" because the rates are independent of substrate
concentration, and are equal to some constant k. The formation of product proceeds at a rate which is
linear with time. The addition of more substrate does not serve to increase the rate. In zero order
kinetics, allowing the assay to run for double time results in double the amount of product.

The amount of enzyme present in a reaction is measured by the activity it catalyzes. The relationship
between activity and concentration is affected by many factors such as temperature, pH, etc. An enzyme
assay must be designed so that the observed activity is proportional to the amount of enzyme present in
order that the enzyme concentration is the only limiting factor. It is satisfied only when the reaction is
zero order.

In Figure 5, activity is directly proportional to concentration in the area AB, but not in BC. Enzyme
activity is generally greatest when substrate concentration is unlimiting.

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When the concentration of the product of an enzymatic reaction is plotted against time, a similar curve
results, Figure 6.

Between A and B, the curve represents a zero order reaction; that is, one in which the rate is constant
with time. As substrate is used up, the enzyme's active sites are no longer saturated, substrate
concentration becomes rate limiting, and the reaction becomes first order between B and C.

To measure enzyme activity ideally, the measurements must be made in that portion of the curve where
the reaction is zero order. A reaction is most likely to be zero order initially since substrate concentration
is then highest. To be certain that a reaction is zero order, multiple measurements of product (or
substrate) concentration must be made.

Figure 7 illustrates three types of reactions

which might be encountered in enzyme assays
and shows the problems which might be
enountered if only single measurements are
made

B is a straight line representing a zero order

reaction which permits accurate determination
of enzyme activity for part or all of the reaction
time. A represents the type of reaction that was
shown in Figure 6. This reaction is zero order
initially and then slows, presumably due to
substrate exhaustion or product inhibition. This
type of reaction is sometimes referred to as a
"leading" reaction. True "potential" activity is
represented by the dotted line.
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Curve C represents a reaction with an initial "lag" phase. Again the dotted line represents the potentially
measurable activity. Multiple determinations of product concentration enable each curve to be plotted
and true activity determined. A single end point determination at E would lead to the false conclusion that
all three samples had identical enzyme concentration.

Substrate Concentration :-

It has been shown experimentally that if the

amount of the enzyme is kept constant and the
substrate concentration is then gradually
increased, the reaction velocity will increase untilit
reaches a maximum. After this point, increasesin
substrate concentration will not increase the
velocity (delta A/delta T). This is represented
graphically in Figure 8.

It is theorized that when this maximum velocity had been reached, all of the available enzyme has been
converted to ES, the enzyme substrate complex. This point on the graph is designated Vmax. Using this
maximum velocity and equation (7), Michaelis developed a set of mathematical expressions to calculate
enzyme activity in terms of reaction speed from measurable laboratory data.

The Michaelis constant Km is defined as the substrate concentration at 1/2 the maximum velocity. This is
shown in Figure 8. Using this constant and the fact that Km can also be defined as: Km=K-1 + K2 / K+1.

K+1, K-1 and K+2 being the rate constants from equation (7). Michaelis developed the following
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Michaelis constants have been determined for many of the commonly used enzymes. The size of Km
tells us several things about a particular enzyme.
A small Km indicates that the enzyme requires only a small amount of substrate to become saturated.
Hence, the maximum velocity is reached at relatively low substrate concentrations.
A large Km indicates the need for high substrate concentrations to achieve maximum reaction velocity.
The substrate with the lowest Km upon which the enzyme acts as a catalyst is frequently assumed to be
enzyme's natural substrate, though this is not true for all enzymes.

Environmental conditions :-

Effects of Temperature :- Like most chemical

reactions, the rate of an enzyme-catalyzed reaction
increases as the temperature is raised. A ten degree
Centigrade rise in temperature will increase the
activity of most enzymes by 50 to 100%. Variations
in reaction temperature as small as 1 or 2 degrees
may introduce changes of 10 to 20% in the results. In
the case of enzymatic reactions, this is complicated
by the fact that many enzymes are adversely affected
by high temperatures. As shown in Figure 13, the
reaction rate increases with temperature to a
maximum level, then abruptly declines with further
increase of temperature.

Because most animal enzymes rapidly become denatured at temperatures above 40°C, most enzyme
determinations are carried out somewhat below that temperature.

Over a period of time, enzymes will be deactivated at even moderate temperatures. Storage of enzymes
at 5°C or below is generally the most suitable. Some enzymes lose their activity when frozen.

Effects of pH :- Enzymes are affected by changes

in pH. The most favorable pH value - the point
where the enzyme is most active - is known as the
optimum pH. This is graphically illustrated in
Figure 14

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In addition to temperature and pH there are other
factors, such as ionic strength, which can affect the
enzymatic reaction. Each of these physical and
chemical parameters must be considered and
optimized in order for an enzymatic reaction to be
accurate and reproducible.

Effects of Inhibitors on Enzyme Activity :-

Enzyme inhibitors are substances which alter the catalytic

action of the enzyme and consequently slow down, or in some
cases, stop catalysis. There are three common types of
enzyme inhibition - competitive, non-competitive and
substrate inhibition.

Most theories concerning inhibition mechanisms are basedon

the existence of the enzyme-substrate complex ES. As
mentioned earlier, the existence of temporary ES structures
has been verified in the laboratory.

Competitive inhibition occurs when the substrate and a

substance resembling the substrate are both added to the
enzyme. A theory called the "lock-key theory" of enzyme
catalysts can be used to explain why inhibit ion occurs.

The lock and key theory utilizes the concept of an "active site." The concept holds that one particular portion of
the enzyme surface has a strong affinity for the substrate. The substrate is held in such a way that its conversion
to the reaction products is more favorable. If we consider the enzyme as the lock and the substrate the key (Figure
9) - the key is inserted in the lock, is turned, and the door is opened and the reaction proceeds. However, when an
inhibitor which resembles the substrate is present, it will compete with the substrate for the position in the enzyme
lock. When the inhibitor wins, it gains the lock position but is unable to open the lock. Hence, the observed reaction
is slowed down because some of the available enzyme sites are occupied by the inhibitor. If a dissimilar substance
which does not fit the site is present, the enzyme rejects it, accepts the substrate, and the reaction proceeds
normally.

Non-competitive inhibitors are considered to be substances which when added to the enzyme alter the enzyme in
a way that it cannot accept the substrate. Figure 10.
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Substrate inhibition will sometimes occur when excessive amounts of substrate are present. Figure 11 shows the
reaction velocity decreasing after the maximum velocity has been reached.

Additional amounts of substrate added to the reaction mixture after this point actually decrease the reaction rate.
This is thought to be due to the fact that there are so many substrate molecules competing for the active sites on
the enzyme surfaces that they block the sites (Figure 12) and prevent any other substrate molecules from
occupying them.

This causes the reaction rate to drop since all of the enzyme present is not being used.
 22
Effects of Cofactors :-

A cofactor is a non-protein chemical compound or metallic ion that is required for an enzyme's activity as a catalyst,
a substance that increases the rate of a chemical reaction. Cofactors can be considered "helper molecules" that
assist in biochemical transformations. The rates at which these happen are characterized by in anarea of study
called enzyme kinetics.

Cofactors can be divided into two types, either inorganic ions, or complex organic molecules called coenzymes.[1]
Coenzymes are mostly derived from vitamins and other organic essential nutrients in small amounts. (Note that
some scientists limit the use of the term "cofactor" to inorganic substances; both types are included here

Enzymes are proteins that catalyze or speed up specific chemical reactions so they go faster than they would
without the catalyst. Some enzymes require the presence of an additional molecule or metal ion called a cofactor
before they can work their magic. Without this cofactor, the enzyme is no longer able to catalyze the reaction.

Function of cofactors

By definition, a cofactor is a nonprotein ion or molecule required by the enzyme for its function. If the cofactor is
removed, the enzyme will not be able to do its job and will no longer work as a catalyst. 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.

Types of cofactors

Cofactors may be positively charged metal ions, such as iron, magnesium and zinc, or they may be small carbon-
based molecules like vitamin B12. Small molecule cofactors are sometimes called coenzymes. Many of the
vitamins you need in your diet act as enzyme cofactors or precursors to enzyme cofactors. Some enzymes bind
their cofactors very tightly so that the cofactor is basically part of the enzyme; in these cases the cofactor is
sometimes called a prosthetic group. For other enzymes, the cofactor is only loosely bound or connected.

Mechanism of cofactors

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. With carbonic anhydrase, for example, the zinc ion is stuck in a
cleft in the protein called the active site. Since it's positively charged and electron-poor, it can form a bond with a
passing water molecule, enabling the water molecule to lose a hydrogen ion so that it becomes a hydroxide ion,
OH-. This hydroxide ion can now attack the carbon atom in a molecule of carbon dioxide to form carbonic acid. By
binding the water molecule and enabling it to lose a hydrogen ion, the zinc ion has helped the enzyme facilitate the
reaction.
 23
Enzyme Activators :-

Enzyme activators are chemical compounds that increase a velocity of enzymatic reaction. Their actions are
opposite to the effect of enzyme inhibitors. Among activators we can find ions, small organic molecules, as well
as peptides, proteins, and lipids.

There are many enzymes that are specifically and directly activated by small inorganic molecules, mainly by cations
such as Ca2+ which is a the second messenger (among enzymes activated by Ca2+, we can find different regulatory
enzymes, in particular phospholipases II, protein kinases C, adenylyl cyclases, etc.). These enzymes usually have
special site for Ca2+ binding; the binding of Ca2+ with it results in the change of enzyme conformation that
increase enzyme activity [33].

Cations can bind not only with enzyme but also with the substrate increasing its affinity to the enzyme that activate
enzyme. For example, magnesium ions interact with ATP or with other nucleotides that are negatively charged
molecules, decreasing their charge that provides effective binding of nucleotides in substrate binding site of
various enzymes and increasing their activity

https://t.me/Food_tech_notes
 24
(6 – 7) Undesirable and desirable enzymic reactions in foods

Their are Two types of Browning reaction which may be desirable or undesirable

Browning is the process of food turning brown due to the chemical reactions that take place within. The
process of browning is one of the chemical reactions that take place in food chemistry and represents
an interesting research topic regarding health, nutrition, and food technology. Though there are many
different ways food chemically changes over time, browning in particular falls into 2 main categories:
enzymatic versus non-enzymatic browning processes.

Browning has many important implications on the food industry relating to nutrition, technology, and
economic cost.[1] Researchers are especially interested in studying the control (inhibition) of browning
and the different methods that can be employed to maximize this inhibition and ultimately prolong the
shelf life of food.[2]

Enzymatic browning :-

Enzymatic browning is one of the most important reactions that takes place in most fruits and vegetables
as well as in seafood.[3] These processes affect the taste, color, and value of such foods.[3] Generally, it
is a chemical reaction involving polyphenol oxidase, catechol oxidase, and other enzymes that create
melanins and benzoquinone from natural phenols. Enzymatic browning (also called oxidation of foods)
requires exposure to oxygen. It begins with the oxidation of phenols by polyphenol oxidase into
quinones,[4] whose strong electrophilic state causes high susceptibility to a nucleophilic attack from other
proteins.[4] These quinones are then polymerized in a series of reactions, eventually resulting in the
formation of brown pigments (melanosis) on the surface of the food.[5] The rate of enzymatic browning is
reflected by the amount of active polyphenol oxidases present in the food.[1] Hence most research
investigating methods to inhibit enzymatic browning has focused on hindering polyphenol oxidase
activity.[1] However, not all browning of food produces negative effects.[1]

Enzymatic browning is a chemical process which occurs in fruits and vegetables by the enzyme
polyphenoloxidase, which results in brown pigments. Enzymatic browning can be observed in fruits
(apricots, pears, bananas, grapes), vegetables (potatoes, mushrooms, lettuce) and also in seafood
(shrimps, spiny lobsters and crabs).

Enzymatic browning is detrimental to quality, particularly in post-harvest storage of fresh fruits, juices and
some shellfish. Enzymatic browning may be responsible for up to 50% of all losses during fruit and
vegetables production. On the other hand enzymatic browning is essential for the colour and taste of tea,
coffee and chocolate.
 25
Examples of beneficial enzymatic browning:

Developing color and flavor in Coffee, Cocoa beans, and tea.[6]

Developing color and flavor in dried fruit such as figs and raisins.

A non-desirable enzymatic browning reaction is involved in the

formation of brown spots on the peel of bananas.

Examples of non-beneficial enzymatic browning:

Fresh fruit and vegetables, including apples, potatoes, bananas and avocados.

Polyphenols oxidases is the major reaction in the formation of Melanosis in crustaceans such as
shrimp.[7]

Control of Enzymatic Browning :-

A variety of methods are used to prevent or slow down enzymatic browning of foods, each method aimed
at targeting specific steps of the chemical reaction. The control of enzymatic browning has always been
a challenge for the food industry. In addition, the use of chemicals to inhibit browning, suchas sulfite (a
powerful antibrowning chemical) have been reconsidered due to the potential hazards[clarification
needed] it causes along with its activity.[5] Much research has been conducted regarding the exact types
of control mechanisms that take place when confronted with these enzymatic process. The different types
of enzymatic browning control can be classified into different groups.

Lemon juice and other acids lower the pH and remove the copper cofactor necessary for the responsible
enzymes to function

Blanching or roasting of foods, to denature the enzymes, and to destroy the responsible reactants, as
used in the "kill green" phase of tea processing.

Low temperatures can also prevent enzymatic browning by reducing rate of reaction.

Use of ascorbic acid in certain pH's to control browning of apples under certain conditions by changing
their phenolase activity. Different pH values affect phenolase activity of apples differently.[8]

Proteins can exert an inhibitory effect on PPO activity by Chelating the essential copper at the active
site of PPO through competitive inhibition, inhibiting its activity [6]

During wine synthesis, the use of Ion-exchange filtration is used to remove the brown color sediments in
the solution.
 26
Arctic Apples have been genetically modified to silence the expression of polyphenol oxidase, thereby
delaying a browning effect, and improving apple eating quality.

Non-Enzymatic Browning :-

The second type of browning, nonenzymatic browning, is a process that also produces the brown
pigmentation in foods, but without the activity of enzymes. The two main forms of non-enzymatic browning
are caramelization and the Maillard reaction. Both vary in the reaction rate as a function of water activity
(in food chemistry, the standard state of water activity is most often defined as the partialvapor pressure
of pure water at the same temperature).

Caramelization is a process involving the pyrolysis of sugar. It is used extensively in cooking for the
desired nutty flavor and brown color. As the process occurs, volatile chemicals are released, producing
the characteristic caramel flavor.

The other non-enzymatic reaction is the Maillard reaction. This reaction is responsible for the production
of the flavor when foods are cooked. Examples of foods that undergo Maillard reaction include breads,
steaks, and potatoes. It is a chemical reaction that takes place between the amine group of a free amino
acid and the carbonyl group of a reducing sugar,[1] usually with the addition of heat. The sugar interacts
with the amino acid, producing a variety of odors and flavors. The Maillard reaction is the basis for
producing artificial flavors for processed foods in the flavoring industry,[11] since the type of amino acid
involved determines the resulting flavor.

Melanoidins are brown, high molecular weight heterogeneous polymers that are formed when sugars and
amino acids combine through the Maillard reaction at high temperatures and low water activity.
Melanoidins are commonly present in foods that have undergone some form of non-enzymatic browning,
such as barley malts (Vienna and Munich), bread crust, bakery products and coffee. They are also present
in the wastewater of sugar refineries, necessitating treatment in order to avoid contamination around the
outflow of these refineries

Browning of grapes during winemaking :-

Like most fruit, grapes vary in the number of phenolic compounds they have. This characteristic is used
as a parameter in judging the quality of the wine.[4] The general process of winemaking is initiated by the
enzymatic oxidation of phenolic compounds by polyphenol oxidases.[4] Contact between the phenolic
compounds in the Vacuole of the grape cell and the Polyphenol oxidase Enzyme (located in thecytoplasm)
triggers the oxidation of the grape. Thus, the initial browning of grapes occurs as a result of
"compartmentalization modification" in the cells of the grape
 27
Implications of Browning reaction :-

Enzymatic browning affects the color, flavor, and nutritional value of foods, causing huge economic loss
when not sold to consumers on time.[1] It is estimated that more than 50% of produce is lost as a result
of enzymatic browning.[2] The increase in human population and consequential depletion in our natural
resources has prompted many biochemists and Food engineers alike to find new and improved
techniques to preserve food longer, by using methods to inhibit the browning reaction, and effectively
increase the shelf life of foods. A better understanding of the enzymatic browning mechanisms,
specifically, understanding the properties of the enzymes and substrates that are involved in the reaction,
may help food technologists to control certain stages in the mechanism and inhibit browning.

Apples are fruits commonly studied by researchers due to their high phenolic content, which make them
highly susceptible to enzymatic browning.[3] In accordance with other findings regarding apples and
browning activity, a correlation has been found between high phenolic amount and enzymatic activity of
apples.[3] This provides a hope for food industries in an effort to genetically modify foods to decrease
polyphenol oxidase activity and thus decrease browning. An example of such accomplishments in food
engineering is in the production of Arctic Apples. These apples, engineered by Okanagan Specialty Fruits
Inc, are a result of gene splicing, a technique that has allowed for the reduction in polyphenol oxidase.

Another type of issue that is closely studied is the browning of seafood.[7] Seafood, in particular shrimp,
is a delicacy consumed by people all over the world. The browning of shrimp which is actually referred to
as Melanosis, creates a great concern for food handlers and consumers. Melanosis mainly occurs during
postmortem handling and refrigerated storage.[7] Recent studies have found a plant extract that acts as
an anti melatonin polyphenol oxidase inhibitor and serves the same function as sulfites but without the
health risks.
 28
(8-9)Sources of enzymes: different sources, extraction of enzymes and purification,
enzyme technology and application

Sources of Enzymes :- We have already the see sources of enzymes.

Extraction of enzyme and Purification :-. In enzyme production there is a very unfavorable ratio
between input of raw material and output of product. This requires the installation of con-centration
procedures. For economic reasons of enzyme application a con-centration up to 10-fold is usually
satisfactory for industrial enzyme prepa-rations.

For example- enzyme products employed in detergents contain about 5-10% protease while amylase
preparations for use in flour treat-ment contain only about 0.1% pure a-amylase. However, in applications
where high purity enzymes are required, e.g., in enzymic analysis, 1000- fold purification is quite
common.

In some applications, such as baking and dextrose manufacture, the presence of contaminating enzymes
must be very low or rigidly controlled. Moreover, the raw enzyme solutions obtained from microbial
cultures con-tain—independent of their source—different types of by-products. Separa-tion ofall these
substances may be necessary because of the possibility of undesired effects.

Considering enzyme stability there is another reason for treatment of crude enzyme preparations. Since
the trend in enzyme applications is to-ward use of liquid preparations, stabilization is an important
procedure.

Techniques for the large-scale isolation and (partial) purification of enzymes from micro-bial sources
make use mainly of traditional procedures. Most of the equip-ment can be found in food-processing
plants. Large-scale equipment specific for enzyme isolation is not marketed.

Nearly all process operations are carried out at low temperatures (prefer-ably 0°-10°C), with the
exception of drying

Separation processes are usually conducted in batches rather than con-tinuously. However, the scale-up
of batch operations inherently causes extended processing times which for many enzymes result in
increased losses of activity due to denaturation of the enzyme protein.

For this reason the application of continuous operations seems to be useful, but the neces-sity for highly
reliable machines and ingenious process control delays introduction of continuous methods. In
 29
addition the value of continuous processing is lost when a single process step is conducted batch wise,
per-haps during precipitation.

Extraction Methods:

The first step in the isolation of enzymes is their extraction. Techniques that fall into this group are
employed either to separate enzymes from solid substrate culture or to release enzymes from the
interior of microbial cells.

Extraction of Solid Substrate Cultures:

Enzymes produced by solid sub-strate cultivation used to be of the extracellular type. It is therefore
easily conceived that extraction of mold brans is rather a washing out process. Countercurrent
techniques of percolation are the most frequently used unit operation.

In many cases the mold bran is dried prior to extraction. This is conve-nient when the utilization of the
particular enzyme preparation is seasonal. The cultures can be produced in relatively small equipment
all the year round, while the extraction is conducted in times of enzyme demand.

On the other hand, it is easily seen that extraction from dried bran will yield solutions with higher
enzyme concentrations. And last, drying avoids inter-ference caused by the activity of living cells of
fresh cultures. This argu-ment, however may not apply in continuously operated culture plants.

In all cases the extractant is water which, however, may contain acids (inorganic or organic), salts,
buffer, or other substances to facilitate solu-bilization of the enzyme or to improve its stability in
solution, or to exclude or minimize undesired effects caused by contaminating by-products or
microorganisms.

Extraction of Cells:

The decision on whether to employ whole cells for a biochemical process or to use isolated enzymes
depends on many factors. Technical difficulties and the related cost of large-scale isolation play an
important role.

There are a number of methods for cell disruption, as reviewed by Hughes et al. (1971). Chemical and
biochemical methods, such as autolysis, treat-ment with solvents, detergents, or lytic enzymes, have the
disadvantage of being in principle batch operations. Their conduct is difficult to standardize and optimize.
More recommendable are mechanical techniques.

At present, the APV-Manton-Gaulin homogenizer seems to be the most versatile type for cell
disintegration. In this machine the cell suspension passes a homogenizing valve at the selected
operating pressure and im-pinges on an impact ring. The strong shearing forces combined with the
 30
sudden decompression lead to a disruption of the cell wall. Dunnill and Lilly (1972), who examined the
disruption of yeast, found that release of protein can be described by a first order rate equation-

Where R is the amount of soluble protein released in g per kg cell mass, Rm the maximum amount of
soluble protein released, K a temperature-dependent rate constant, N the number of times the cell
suspension has passed the homogenizer, and P the operating pressure. With industrial models,
be-tween 50 and 9000 liters of bacterial suspension per hr can be treated, depending on the size of the
machine. Ball mills available on the market have a volume capacity of 0.6-250 liters.

Separation Methods:

It is possible here to give only the barest outline of methods that find wider application in the large-
scale production of enzymes.

Solids Separation Techniques:

Such methods are involved in the clari-fication of culture liquors and extracts, in the separation of
precipitates, and in the sterilization of liquid enzyme preparation by mechanical methods.

The solids to be separated may have a number of properties which make separation processes difficult.
For instance, they may be greasy, sometimes colloidal, and often density differences between solid
particles and liquid phase are very small. Therefore, pretreatment of the liquor is usually inevitable, as
conducted by acidification, addition of water miscible sol-vents or liquid polyions, mild heating, etc.

The problems of large-scale solid-liquid separation are complex and di-verse. There are two approaches
- centrifugation and filtration. Industrial centrifuges are not ideal for removal of finely divided biological
solids. Disc type centrifuges without solid discharge have proved most efficient for separation of easily
settling suspensions of greasy particles. Decanters are used in cases where solids content is high but
easily settling, e.g., in the production of dried acetone precipitates.

In cases of poorly settling protein precipitates, hollow bowl centrifuges are employed for separation
 31
from low solids suspensions as obtained during fractionated enzyme precipitation. In all cases flow rates
must be determined empirically. Sometimes (e.g., with precipitates) the throughput is reduced toless
that 10% of the nominal capacity. This requires the integration of cooling devices.

Most frequently filters are more suitable for separation of biological particles. Generally large
proportions of filter aids are required. In continu-ous processes vacuum drum filters are used, with
diatomaceous earth, wood-meal, or starch as pre-coat materials. Batch operations are conducted with
filter presses.

Membrane Separation Techniques:

Membrane processes allow separa-tion of solutes from one another or from a solvent, with no phase
change or interphase mass transfer. There are many different kinds of membrane processes, the
classification of which is based on the driving forces that cause the transfer of solutes through the
membrane. Such a force may be trans-membrane differences in concentrations, as in dialysis; electric
poten-tial, as in electro-dialysis; or hydrostatic pressure, as in microfiltration, ultrafiltration, and reverse
osmosis.

At present, ultrafiltration is the only membrane process of importance in large-scale enzyme production.
From normal filtration processes it differs just by the size range of the particles to be separated (molecular
weight cutoffs between 500 and 300,000). Two types of ultrafiltration membranes are used, which differ
in their transport mechanisms and their separation properties.

Isotropic porous membranes are the type most similar to conventional filters. They possess a spongy
structure with extremely small random pores the average size of which is in the range of 0.05 to 0.5
microns. Molecules with a diameter smaller than that of the smallest pore will pass the mem-brane
quantitatively, whereas particles larger than the largest pore will be retained at the filter surface.
Molecules of intermediate size, however, will only pass to some extent.

Another proportion of these particles will be retained within the structure of the membrane. This leads,
first, to a decrease in retention (or vice versa, in permeation) with a resulting fouling of the membrane, and
secondly, to a reduced discrimination among solutes of different size. In order to minimize fouling it is
useful to use membranes with a mean pore size well below that of the solute to be retained. Therefore,
porous membranes are advisable for the concentration of high molecular weight solutes (molec. Weight
< 1 x 106).

Diffusive membranes are capable of more selective molecular discrimina-tion. They are essentially
homogeneous hydrogel layers, through which the solvent as well as the solute is transported by
molecular diffusion under the driving force of a concentration or a chemical potential gradient. The
transportation of a molecule through the membrane requires considerable kinetic energy.
 32
This depends, of course, on the dimensions of the diffusing molecule and on the mobility of the single
polymer chains within the membrane matrix. As a rule, the rate of diffusion is high when the polymer
segments of the matrix are only loosely interlaced, i.e., when the gel matrix is highly hydrated. For this
reason all membranes made from hydrophilic polymers and capable of swelling in water to a certain
degree are princi-pally suited as pressure filtration membranes for aqueous solutions.

In addition to the retention potential of the membrane the flux is impor-tant for economic reasons. Since
in both the porous and the diffusive filter types the flux depends largely on the thickness of the membrane,
it is necessary to keep the membrane as thin as possible.

This requirement has been fulfilled by the construction of anisotropic membranes. They consist of a
highly consolidated but very thin (0.1-5 μm) active layer on a compara-tively thick (1 to 20 microns)
highly porous support. The advantage of these anisotropic membranes is that there is no reduction in
solvent permeability at constant hydrostatic pressure because there is no blockage within the
membrane.

In any ultrafiltration system, accumulation of solutes at the membrane surface occurs, which leads to
formation of a “slime” that increasingly impedes solvent flow through the membrane, until convective
transport of solute toward the membrane is equal to the rate of back diffusive transport away from the
membrane. This phenomenon is called “concentration polar­ization”.

Proteins and colloidal particles build up solid or thixotropic gels when concentrated beyond a certain
point. The solute concentration on the membrane surface reaches an upper limit which is typically
between 20 and 70% solute by volume. In order to reduce the polarization effect, in industrial
ultrafiltration equipment the feed solution passes the membrane surface at high flow rate.

When a macromolecular solution is ultra-filtered, flux of solvent is de-scribed by the relationship-

here A is the membrane constant (dependent on temperature, indepen-dent of pressure over the normal
operating range), ΔP is the hydrostatic pressure driving force, and Δπ is the osmotic pressure difference
across the membrane. For macromolecular solutions with concentrations over 1% w/v, osmotic pressures
exceeding 10 to 50 psi are not uncommon.

There are several basic types of ultra-filters – thin channel, tubular, helical tubes, spiral wrapped, and
hollow fiber systems. Suitability of a single type depends on the properties of the system to be treated.
 33
Gel Filtration:

The technique of ultrafiltration has the advantage of combining both separation of impurities and
concentration of the desired enzyme. However, due to its principle, it is a rather nonselective process.
Better results, regarding separation of molecules from each other, can be obtained by gel filtration, but
in many cases its application is not economically feasible.

In principle, gel filtration is the diffusional partitioning of solute mole-cules between the readily mobile
solvent phase and that confined in spaces within the porous gel particles that make up the stationary
phase. Diffusional exchange of solutes takes place between the stationary and mobile phases.

Gel filtration works rapidly and preservingly, without mechanical stress as in ultrafiltration. However,
precipitation of proteins within the gel column may occur as a consequence of desalting. In some cases
it has been observed that the metal bridges of enzyme quaternary structures were uncoupled.

Adsorption Techniques:

Because of the possibility of highly selective separations, adsorption processes are increasingly used.
Properties of en-zyme molecules as different as, e.g., lipophily, electric charge, specificity, etc., are the
basis of separation. This results in a great number of adsor-bents, such as active carbon, hydroxyapatite,
ion exchangers, carrier fixed substrate analogs, and so forth.

A common feature of all adsorption tech-niques is the principle of adsorption followed by desorption or
elution. Separation is achieved by adsorption and elution of either enzyme or impu-rities. Two methods
are available, batch wise adsorption or column chroma-tography. The latter process has greater
separation efficiency and, in addi-tion, offers the possibility of semi-continuous operation.

Among the different adsorption techniques, affinity chromatography is of very great interest, but far from
being applicable on a large scale. Ion exchangers available for large-scale processes are of the resin,
large-pore gels, or cellulose types. In particular, ion exchange resins exhibit useful properties for
industrial production of enzymes.

It must, however, be taken into consideration that the proximity of a resin matrix with a high charge
density can affect the structural integrity of enzymes. Large-pore ion exchangers and cellulose
exchangers have a number of properties which make them very suitable for enzyme separation
processes, but the former are very costly. Obviously, they are only suitable for batch processes
be-cause they are compressed in columns.
 34
Precipitation Techniques:

Separation from solution by salting out is one of the oldest and yet most important procedures of
concentration and purification of enzymes.

This means that the electrolyte concentration required for protein precipi-tation varies with protein
concentration.

The influence of the most important precipitation parameters can be outlined shortly as follows – Higher
valency salts produce higher ionic strength than lower valency salts. At a constant ion strength, protein
solubility increases with increasing distance (in both directions) from its isoelectric point. As a result,
lower ionic strength is required for precipita-tion when carried out at the isoelectric point of the protein.

The commonly used salt for precipitation is ammonium sulfate. The reasons can be found in the high
solubility of this salt and in its low price. In addition, ammonium sulfate is nontoxic for most enzymes
and in many cases it acts as a stabilizing agent. In ammonium sulfate solutions precipitated enzymes
are often storable for years without significant loss when kept at low temperatures.

In contrast to neutral salts, solvents are less customary for large-scale precipitation of enzymes. The
reason is higher costs of raw materials and equipment. Explosion proof equipment and recycling of the
solvents are inevitable requirements.

Solvent precipitation is based on the fact that the solubility of enzymes decreases with the decreasing
dielectric constant (ϵ) of the solvent. The concentration required is lower the less hydrophilic the solvent
is. Thus, an increasing precipitating effect can be achieved in the series methanol (ϵ25 = 33), ethanol
(ϵ25 = 24), isopropanol (ϵ25 = 18). Besides aliphatic alcohols, acetone (ϵ25 = 20) is often used as a
precipitant.

Solvent precipitates are distinguished from salt precipitates by the ease with which they settle. However,
at temperatures above 4°C denaturation of the enzyme protein can occur. Therefore, it is quite normal to
work at temperatures below zero. This, however, requires large cooling capacity in industrial
manufacture.

All these difficulties can be avoided by using polyethyleneglycol with a molecular weight of 6000. This
precipitant does not effect enzyme denatur-ation and is relatively independent of temperature and
electrolyte concen-tration. However, there is a strong dependence on hydrogen ion concentra-tion. The
best results are obtained at the isoelectric point of the enzyme to be precipitated.

As the solubility of a protein molecule is lowest at its isoelectric point, successive precipitation of different
enzymes from a solution can be achieved by changing the pH. These precipitates settle easily and can
easily be separated from the solution by centrifugation.
 35
Conversion enzyme to Storage (storable) Form:

Storability of an enzyme requires the preparation of a suitable storage form. Commercial enzyme
products are available either in solution or in solid state. Generally, users prefer solutions because of
their easier han-dling, but enzymes are usually very unstable in aqueous solution.

For this reason stabilization of dissolved enzymes is a very important step in the manufacture of liquid
enzyme preparations. The storage stability is af-fected by the following two factors- microbial deterioration
of the enzyme solution and denaturation of the enzyme protein. These two problems seemto be closely
related to each other.

Many treatments have been tried in order to prevent growth of microor-ganisms. The methods include,
for instance, incorporation of chemical pre-servatives, pasteurization, addition of salts and polyhydric
alcohols, and irradiation. But some of those treatments are undesirable due to legal aspects. Therefore,
the most suitable method to repress microbial growth is to dissolve the enzyme in a highly concentrated
solution of salts and sugars.

With liquid preparations, storage at low temperatures and at suitable pH is essentially inevitable. It is
well known that substrates almost invariably protect the corresponding enzyme against physical,
chemical, or physicochemical agents. This can be attributed to either conformational stabiliza-tion or
steric or competitive protection.

From a number of publications it can be seen that almost any effect on enzyme stability may a priori be
an allosteric one due to attack at sites other than the active site of the enzyme. For example - in
thermolysin, a bacterial protease, Ca ions stabilize the enzyme molecule, while Zn ions are required for
activity. The enormous value of Ca ions in stabilizing bacterial α-amylase has long been known.

A number of techniques are available for stabilization.

Some of them are presented in the following list, immobilization methods excluded:

(1) Conformational or charge stabilization and/or protection from dilution-dissociation by using buffers,
glycerol, substrates, or inhibitors.

(2) Protection of active site thiol via disulfide exchange by thiols, redox dyes, oxygen-binding agents, or
chelating agents.

(3) Miscellaneous methods include, e.g., inhibition or removal of proteo-lytic enzymes; protection from
light by photosensitive dyes; lowering activity of water by viscosity effectors, salts, or sugars; lowering
surface energy by antifoams; cooling and crystallization protection by antifreeze; removal of harmful
agents; and sterilization for protection against microbial attack.
 36
Commercially available solid enzyme preparations are dried mold brans, dried precipitates, or dried
solutions. Spray drying is the preferred method for removal of water from enzyme solutions due to
economic reasons. How-ever, it is only applicable to enzymes sufficiently resistant to the tempera-ture
conditions of this process. On the other hand, freeze drying is most preserving, but its use is limited by
cost considerations as well as by the fact that unless the salt concentrations of the enzyme solution are
sufficiently reduced, eutectic mixtures may be formed.

This may lead to incomplete drying or to severe foaming and protein denaturation. A specific method of
drying sometimes used is granulation in a fluidized bed with milk sugar or maltodextrin as carrier. In this
case, of course, sufficiently high specific activity of the enzyme is required in order to ensure.

Enzymes are the biocatalysts synthesized by living cells. They are complex protein molecules that bring
about chemical reactions concerned with life. It is fortunate that enzymes continue to function (bring out
catalysis) when they are separated from the cells i.e. in vitro. Basically, enzymes are non-toxic and
biodegradable. They can be produced in large amounts by microorganisms for industrial applications.

Enzyme technology broadly involves production, isolation, purification and use of enzymes (in soluble or
immobilized form) for the ultimate benefit of humankind. In addition, recombinant DNA technology and
protein engineering involved in the production of more efficient and useful enzymes are also a part of
enzyme technology.

The commercial production and use of enzymes is a major part of biotechnology industry. The
specialties like microbiology; chemistry and process engineering, besides biochemistry have largely
contributed for the growth of enzyme technology.

Applications of Enzymes:

Enzymes have wide range of applications. These include their use in food production, food processing
and preservation, washing powders, textile manufacture, leather industry, paper industry, medical
applications, and improvement of environment and in scientific research.

As per recent estimates, a great majority of industrially produced enzymes are useful in processes
related to foods (45%), detergents (35%), textiles (10%) and leather (3%).

Commercial Production of Enzymes:

Microbial enzymes have been utilized for many centuries without knowing them fully. The first enzyme
produced industrially was taka-diastase (a fungal amylase) in 1896, in United States. It was used as a
pharmaceutical agent to cure digestive disorders.

In Europe, there existed a centuries old practice of softening the hides by using feces of dogs and
 37
pigeons before tanning. A German scientist (Otto Rohm) demonstrated in 1905 that extracts from
animal organs (pancreases from pig and cow) could be used as the source of enzymes-proteases, for
leather softening.

The utilization of enzymes (chiefly proteases) for laundry purposes started in 1915. However, it was not
continued due to allergic reactions of impurities in enzymes. Now special techniques are available for
manufacture, and use of enzymes in washing powders (without allergic reactions). Commercial enzymes
can be produced from a wide range of biological sources. At present, a great majority (80%) of them are
from microbial sources.

The different organisms and their relative contribution for the production of commercial enzymes are
given below:

Fungi – 60%

Bacteria – 24%

Yeast – 4%

Streptomyces – 2%

Higher animals – 6%

Higher plants – 4%

Enzymes from animal and plant sources:

In the early days, animal and plant sources largely contributed to enzymes. Even now, for certain
enzymes they are the major sources.

Animal organs and tissues are very good sources for enzymes such as lipases, esterases and proteases.
The enzyme lysozyme is mostly obtained from hen eggs. Some plants are excellent sources for certain
enzymes-papain (papaya), bromelain (pineapple).

Limitations:

There are several drawbacks associated with the manufacture of enzymes from animal and plant sources.
The quantities are limited and there is a wide variation in their distribution. The most importantlimitations
are the difficulties in isolating, purifying the enzymes, and the cost factor. As regards extraction of
industrial enzymes from bovine sources, there is a heavy risk of contamination with bovine spongiform
encephalo-pathy (BSE is prion disease caused by ingestion of abnormal proteins). For these
 38
reasons, microbial production of enzymes is preferred.

Enzymes from mammalian cell cultures:

There exists a possibility of producing commercial enzymes directly by mammalian cell cultures. But the
main constraint will the cost factor which will be extremely high. However, certain therapeutic enzymes
such as tissue plasminogen activator are produced by cell cultures.

Enzymes from microbial sources:

Microorganisms are the most significant and convenient sources of commercial enzymes. They can be
made to produce abundant quantities of enzymes under suitable growth conditions. Microorganisms can
be cultivated by using inexpensive media and production can take place in a short period.

In addition, it is easy to manipulate microorganisms in genetic engineering techniques to increase the

production of desired enzymes. Recovery, isolation and purification processes are easy with microbial
enzymes than that with animal or plant sources.

In fact, most enzymes of industrial applications have been successfully produced by micro-organisms.
Various fungi, bacteria and yeasts are employed for this purpose.

List of Industrially Produced Enzymes

Aspergillus niger— A unique organism for production of bulk enzymes:

Among the microorganisms, A. niger (a fungus) occupies a special position for the manufacture of a
large number of enzymes in good quantities. There are well over 40 commercial enzymes that are
conveniently produced by A. niger. These include a-amylase, cellulase, protease, lipase, pectinase,
phytase, catalase and insulinase.

The Technology of Enzyme Production—General Considerations:

In general, the techniques employed for microbial production of enzymes are comparable to the
methods used for manufacture of other industrial products .The salient features are briefly described.

1. Selection of organisms

2. Formulation of medium

3. Production process

4. Recovery and purification of enzymes.

 39
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 appropriate methods (mutagens, UV rays). From the organism chosen, inoculum can be
prepared in a liquid medium.

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.

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.

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.

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
 40
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. 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.

Removal of cell debris:

Filtration or centrifugation can be used to remove cell debris.Removal

of nucleic acids:

Nucleic acids interfere with the recovery and purification of enzymes. They can be precipitated and
removed by adding poly-cations such as polyamines, streptomycin and polyethyleneimine.

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.

Liquid-liquid partition:

Further concentration of desired enzymes can be achieved by liquid-liquid extraction using polyethylene
glycol or polyamines.

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.
 41
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.

Regulation of Microbial Enzyme Production —General Considerations:

A maximal production of microbial enzymes can be achieved by optimising the fermentation conditions
(nutrients, pH, O2, temperature etc.). For this purpose, a clear understanding of the genetic regulation of
enzyme synthesis is required. Some of the general aspects of microbial enzyme regulation are briefly
described.

Inducible Enzymes :-

The inducer compounds are expensive and their handling (sterilization, addition at specific time) also is
quite difficult. In recent years, attempts are being made to develop mutants of microorganisms in which
inducer dependence is eliminated.

Feedback repression:

Feedback regulation by the end product (usually a small molecule) significantly influences the enzyme
synthesis. This occurs when the end product accumulates in large-quantities. Large scale production of
feedback regulated enzymes is rather difficult. However, mutants that lack feedback repression have
been developed to overcome this problem.

Nutrient repression:

The native metabolism of microorganism is so devised that there occurs no production of unnecessary
enzymes. In other words, the microorganisms do not synthesize enzymes that are not required by them,
since this is a wasteful exercise. The inhibition of unwanted enzyme production is done by nutrient
repression. The nutrients may be carbon, nitrogen, phosphate or sulfate suppliers in the growth medium.
For large scale production of enzymes, nutrient repression must be overcome.

Glucose repression is a classical example of nutrient (more appropriately catabolite) repression. That is
in the presence of glucose, the enzymes needed for the metabolism of rest of the compounds are not
synthesized. Glucose repression can be overcome by feeding of carbohydrate to the fermentation medium
in such a way that the concentration of glucose is almost zero at any given time. In recent years,
 42
attempts are being made to select mutants that are resistant to catabolite repression by glucose. For
certain microorganisms, other carbon sources such as pyruvate, lactate, citrate and succinate also act
as catabolite repressors.

Nitrogen source repression is also observed in microorganisms. This may be due to ammonium ions or
amino acids. Most commonly inexpensive ammonium salts are used as nitrogen sources. The repression
by ammonium salts can be overcome by developing mutants resistant to this nitrogen source.

Genetic Engineering for Microbial Enzyme Production:

Enzymes are the functional products of genes. Therefore, theoretically, enzymes are good candidates for
improved production through genetic engineering. During the past 15 years, the advances in the
recombinant DNA technology have certainly helped for increasing the microbial production of commercial
enzymes. It is now possible to transfer the desired enzyme genes from one organism to the other. Once
an enzyme with a potential use in industry is identified, the relevant gene can be cloned andinserted into
a suitable production host.

Cloning strategies:

A diagrammatic representation of a cloning strategy for industrial production of enzymes is given in Fig.
21.2. This involves the development of cDNA library for the mRNA, and creation of oligonucleotide
probes for the desired enzyme. On hybridization with oligonucleotide probes, the specific cDNA clones
can be identified.

Cloning Strategy for Industrial Production. :- The next step is the transformation of industrially important
host organism (e.g. Aspergillus oryzae) for the production of the desired enzyme. By this approach, it is
possible to manufacture high quality industrial enzymes. A couple of enzymes produced by employing
cloning strategies are described below.

1. The enzyme lipolase, found in the fungus Humicola languinosa is very effective to remove fat stains
in fabrics. However, industrial production of lipolase by this organism is not possible due to a very low
level of synthesis. The gene responsible for lipolase was isolated, cloned and inserted into Aspergillus
oryzae.

Thus, large scale production of this enzyme was successfully achieved. Lipolase is very stable and
resistant to degradation by proteases that are commonly used in detergents. All these properties make
lipolase a strong candidate for its use fabric washing.

2. Rennet (chymosin) is an enzyme widely used in making cheese. It is mainly obtained from the
stomachs of young calves. Consequently, there is a shortage in its supply. The gene for the synthesis of
chymosin has been cloned for its large scale production.
 43
(10) Enzymes in milk and cheese industries: enzymes in milk processing
and cheeseproduction

In the dairy industry, some enzymes are required for the production of cheeses, yogurt, and other dairy
products, while others are used in a more specialized fashion to improve texture or flavor. Five of the
more common types of enzymes and their role in the dairy industry are described below.

Rennet :-

Milk contains proteins, specifically caseins, that maintain its liquid form. Proteases are enzymes that
are added to milk during cheese production, to hydrolyze caseins, specifically kappa casein, which
stabilizes micelle formation preventing coagulation. Rennet and rennin are general terms for any
enzyme used to coagulate milk. Technically rennet is also the term for the lining of a calf's fourth
stomach.

The most common enzyme isolated from rennet is chymosin. Chymosin can also be obtained from several
other animals, microbial or vegetable sources, but indigenous microbial chymosin (from fungi orbacteria)
is ineffective for making cheddar and other hard cheeses.

Limited supplies of calf rennet have prompted genetic engineering of microbial chymosin by cloning calf
prochymosin genes into bacteria. Bioengineered chymosin may be involved in the production of up to 70%
of cheese products. While the use of bioengineered enzymes spares the lives of calves, it presents ethics
issues for those opposed to eating foods prepared with GEMs.

Lactalbumin and Lactoglobulin :-

Milk contains a number of different types of proteins, in addition to the caseins. Cow milk also contains
whey proteins such as lactalbumin and lactoglobulin. The denaturing of these whey proteins, using
proteases, results in a creamier yogurt product. Destruction of whey proteins is also essential for cheese
production.

During the production of soft cheeses, whey is separated from the milk after curdling and may be sold
as a nutrient supplement for bodybuilding, weight loss, and lowing blood pressure, among other things.
There have even been reports of dietary whey for cancer therapies, and having a role in the induction of
insulin production for those with Type 2 diabetes.

Proteases are used to produce hydrolyzed whey protein, which is whey protein broken down into shorter
polypeptide sequences. Hydrolyzed whey is less likely to cause allergic reactions and is used to prepare
supplements for infant formulas and medical uses.
 44
Lactase :-

Lactase is a glycoside hydrolase enzyme that cuts lactose into its constituent sugars, galactose, and
glucose. Without sufficient production of lactase enzyme in the small intestine, humans become lactose
intolerant, resulting in discomfort (cramps, gas, and diarrhea) in the digestive tract upon ingestion of milk
products.

Lactase is used commercially to prepare lactose-free products, particularly milk, for such individuals. It is
also used in the preparation of ice cream, to make a creamier and sweeter tasting product. Lactase is
usually prepared from Kluyveromyces sp. of yeast and Aspergillus sp. of fungi.

Catalase :-

The enzyme Catalase has found limited use in one particular area of cheese production. Hydrogen
peroxide is a potent oxidizer and toxic to cells. It is used instead of pasteurization, when making certain
cheeses such as Swiss, in order to preserve natural milk enzymes that are beneficial to the end product
and flavor development of the cheese.

These enzymes would be destroyed by the high heat of pasteurization. However, residues of hydrogen
peroxide in the milk will inhibit the bacterial cultures that are required for the actual cheese production,
so all traces of it must be removed. Catalase enzymes are typically obtained from bovine livers or
microbial sources and are added to convert the hydrogen peroxide to water and molecular oxygen.

Lipases :-

Lipases are used to break down milk fats and give characteristic flavors to cheeses. Stronger flavored
cheeses, for example, the Italian cheese, Romano, are prepared using lipases. The flavor comes from
the free fatty acids produced when milk fats are hydrolyzed. Animal lipases are obtained from kid, calf,
and lamb, while microbial lipase is derived by fermentation with the fungal species Mucor meihei.

Although microbial lipases are available for cheese-making, they are less specific in what fats they
hydrolyze, while the animal enzymes are more partial to short and medium-length fats. Hydrolysis of the
shorter fats is preferred because it results in the desirable taste of many kinds of cheese. Hydrolysis of
the longer chain fatty acids can result in either soapiness or no flavor at all
 45
(11) Enzymes in Meat industry: enzymes in tenderization of meat

Tenderness is a quality of meat gauging how easily it is chewed or cut. Tenderness is a desirable quality,
as tender meat is softer, easier to chew, and generally more palatable than harder meat. Consequently,
tender cuts of meat typically command higher prices. The tenderness depends on a number of factors
including the meat grain, the amount of connective tissue, and the amount of fat.[1] Tenderness can be
increased by a number of processing techniques, generally referred to as tenderizing or tenderization.

As the name implies, these enzymes are used to treat tough cuts of meat and make them more
acceptable to consumers. In addition to beef, pork, and chicken, these enzymes can also be used on
seafood such as squid or clams. Enzymes for Meat Tenderizing.

The two most often used meat tenderizing enzymes are Papain and Bromelain. Both are derived from
plant sources. These are the papaya fruit and the pineapple plant. To a much lesser extent, Ficin,
derived from fig tree latex is also used. Other sources of enzymes have been cited for meat
tenderization such as Bacillus subtilis, Aspergillus oryzae and even pancreatin derived from the
pancreas gland (typically hog).

PAPAIN :-

Papain is usually produced as a crude, dried material by collecting the latex from the fruit of the papaya
tree. The latex is collected after scoring the neck of the fruit whereupon it may either dry on the fruit or
drip into a container. This latex is then further dried. It is now classified as a dried, crude material. A
purification step is necessary to remove contaminating substances. This purification consists of the
solubilization and extraction of the active papain enzyme system through a government registered
process. This purified papain may be supplied as dried powder or as a liquid.

BROMELAIN :-

Bromelain is prepared from the stump or root portion of the pineapple plant after harvest of the fruit.
This stump or root portion is collected from the fields, peeled and crushed to extract the juice containing
the soluble Bromelain enzyme. Further processing includes precipitation of the enzyme to further purify
it. This process is carried out in factories under strictly controlled conditions to assure microbiological
quality and enzyme purity. The Bromelain products are all supplied as powders. The other enzymes
mentioned are produced using selected micro-organisms, such as Bacillus subtilis and Aspergillus
oryzae in commercial enzyme production facilities.

Roughly 95 plus percent of the meat tenderizing enzymes consumed in the United States are from the
plant proteases – Papain and Bromelain. The microbial tenderizers constitute a minimal portion and
have never been successfully applied on a large scale
 46
(12) Enzymes in baking industry

There are two scenarios regarding the use of enzymes, either the enzymes are used to convert the raw
material into the main product, or the enzymes are used as additives to alter a functional characteristic
of the product. In the first case, the enzymatic process is undertaken in optimized and controlled
conditions to enhance the catalytic potential of the enzyme, whereas in the second situation it is more
difficult to assure optimal conditions and to control the enzymatic reaction. In this article let’s talk about
Enzymes Use In Baking Industry!

Bakery Enzymes Market :-

The development of the bread process was an important event in mankind. An important aspect that
contributed to the evolution of the baking market was the introduction of industrial enzymes in the
baking process, where bakery enzymes represent a relevant segment of the industry

Main Constituents Of Baked Products :-

Baking is a common name for the production of baked goods, such as bread, cake, pastries, biscuits,
crackers, cookies, pies and tortillas, where wheat flour is both the most essential ingredient and a key
source of enzyme substrates for the product. Even though based on cereals other than wheat, baked
goods such as gluten-free products or rye bread are also considered to be baked products.

Bread is usually made from wheat flour as raw material, which is a mixture of starch, gluten, lipids, non-
starch polysaccharides, and enzymes. After flour, yeast, and water are mixed, complex biochemical and
biophysical processes begin, catalyzed by the wheat enzymes and by the yeast, characterizing the dough
phase. These processes go on in the baking phase, giving rise to bread

Extra enzymes added to the dough improve control of the baking process, allowing the use of different
baking processes, reducing process time, slowing-down staling, compensating for flour variability and
substituting chemical additives

Starch is the main component of products such as bread and other bakery goods and is added to different
foods, acting as a thickener, water binder, emulsion stabilizer, gelling agent and fat substitute. It is the
most abundant constituent and most important reserve polysaccharide of many plants, including cereals,
occurring as intracellular, semi-crystalline granules. On a molecular level, its major components are the
glucose polymers amylose and amylopectin.
 47
Importance of Enzymes in Bakery1.Dough

conditioning :-

Flour contains 2.5-3.5% non-starch polysaccharides, which are large polymers (mainly pentosans) that
play an important role in bread quality due to their water absorption capability and interactions with
gluten. Although the true mechanism of hemicellulase, pentosanase or xylanase in bread-making has
not been clearly demonstrated, it is well known that the addition of certain types of pentosanases or
xylanases at the correct dosage can improve dough machinability yielding a more flexible, easier-to-
handle dough. Consequently, the dough is more stable and gives better ovenspring during baking,
resulting in a larger volume and improved crumb texture.

Normal wheat flour contains 1-1.5% lipids, both polar and non-polar. Some of these lipids, especially the
polar lipids such as phosphorlipids and galactolipids are able to stabilise the air bubbles in the gluten
matrix. The addition of functional lipases modifies the natural flour lipids so they become better at
stabilizing the dough. This ensures a more stable dough in case of over-fermentation, a larger loaf volume,
and significantly improved crumb structure. Because of the more uniform and smaller crumb cells, the
crumb texture is silkier and the crumb colour appears to be whiter. It also reduces the need foraddition of
emulsifiers like DATEM and SSL that otherwise are commonly added to dough in order to stabilise it. This
in turn means that emulsifiers can be removed from the label.

Chemical oxidants such as bromates, azodicarbonamide and ascorbic acid have been widely used to
strengthen the gluten when making bread. As an alternative, oxidases such as glucose oxidase can
partially replace the use of these chemical oxidants and achieve better bread quality. glucose oxidase
and fungal alpha-amylase can be used not only to replace bromate but also to give a greater bread
volume.

2.The synergistic effects of enzymes :-

Each of the enzymes mentioned above has its own specific substrate in wheat flour dough. For example,
lipases work on the lipids, xylanase works on the pentosans, and amylases work on the starch. Because
the interaction of these substrates in dough and bread is rather complex, the use of enzyme combinations
can have synergistic effects that are not seen if only one enzyme is used, not even at high dosages. Quite
often an overdosage of enzymes will have a detrimental effect on either the dough or the bread. For
instance, an overdose of fungal alpha-amylase or hemicellulase / xylanase may result in a dough that is
too sticky to be handled by the baker or baking equipment. It is therefore beneficial for some types of
bread formulation to use a combination of lower dosages of alpha-amylase and xylanase with low dosages
of lipase or glucose oxidase to achieve optimum dough consistency stability and bread quality. Another
example is to use maltogenic alpha-amylase in combination with fungal alpha- amylases and xylanase or
lipase to secure optimum crumb softness as well as optimum bread quality in
 48
terms of crumb structure, bread volume, etc.

3.Reduction of acrylamide content in food products :-

During recent years it has been shown that the amount of the potentially carcinogenic substance
acrylamide is relatively high in a number of cereal and potato based products like biscuits, crackers,
crisp bread, French fries and potato crisps. This is a substance that is formed at high temperatures
when the amino acid asparagine reacts with a reducing sugar like glucose. To meet this issue the
enzyme asparaginase have been developed in order to reduce the formation of acrylamide.
Asparaginase converts asparagine to aspartic acid which does not take part in the formation of
acrylamide. The use of an asparginase is able to reduce the formation of acrylamide with up to 90%.

4.Flour supplementation

Malt flour and malt extract can be used as enzyme supplements because malt is rich in alpha-amylases.
Commercial malt preparations can differ widely in their enzyme activity, whereas an industrial enzyme is
supplied with a standardised activity.

The alpha-amylases degrade the damaged starch in wheat flour into small dextrins, which allows yeast
to work continuously during dough fermentation, proofing and the early stage of baking. The result is
improved bread volume and crumb texture. In addition, the small oligosaccharides and sugars such as
glucose and maltose produced by these enzymes enhance the Maillard reactions responsible for the
browning of the crust and the development of an attractive baked flavour.

Enzymes used in Backery :-

 49
1. Amylases

The most commonly used enzyme in baking is amylase. Amylase converts starch to dextrins,
oligosaccharides, and the sugar maltose. Maltose provides a fermentable sugar for the yeast—a critical
function before adding sugar to bread became common. Other benefits are improved loaf volume and
symmetry, darker crust color, better flavor, and shelf life extension.5

2. Proteases

Protease breaks down proteins such as gluten. This reduces mix times, increases dough extensibility
and flow, improves gas retention, and produces a darker crust as well as a better crumb grain and
texture.

3. Lipases

Lipase breaks down lipids into monoglycerides, diglycerides, and free fatty acids. This increases dough
tolerance, improves loaf volume, and allows for a reduction in the use of shortening.7 Some new lipase
ingredients can replace the chemical dough improvers DATEM and SSL.8

4. Pentosanases and cellulases

These enzymes work on pentosans and cellulose, fibers that are naturally present in wheat and rye. The
result is a softening of the dough and a reduced water absorption capacity, which can allow for a shorter
bake time in certain baked goods like crackers.

5. Hemicellulases

(or cellulases and pentosanases) are used to improve the baking properties of stiff rye flours.
Hemicellulases can also be used to improve dough properties and bread quality in the production of
wheat breads.

Other enzymes are available for baking such as phospholipases, oxidases, and xylanases and, in fact,
it’s very common to use blends. This helps achieve the best finished product for the current processing
conditions. Opportunities exist for enzyme use in other baked goods as well such as cakes, muffins,
pizza crusts, and tortillas.
 50
(13) Enzymes in production of beverages and fruit juices: enzymes in tea,
cocoa, wine,beer, whiskey, cider, etc

The most important field of application for enzymes in the beverage industry is the extraction of fruit
juice and vegetable juice. Pectinases, in particular, are employed for apple and pear juice and for juices
made from berries and tropical fruits. They break down pectins found in the plant cell walls as supporting
substances. This increases the quality of juice extracted and reduces fruit waste.

Enzymes can be used in winemaking to increase the preliminary juice extraction and to obtain more high
-quality wine. Pecto-lytic (pectin degrading) enzymes ensure that the necessary clarification and filtration
can be carried out. In the manufacturing of red wine, enzymes support the release of color andthus
improve color extraction. They can also influence the taste of the wine.

A further field in the manufacturing of beverages where enzymes are employed is in the cleaning of ultra
-filtration membranes. In the case of UF membranes which are increasingly being used in fruit juice
industry, the combination of cellulase and protease produces excellent cleaning effects.

Enzymes in Brewing :-

Even for an old industry like beer brewing new industrial processes benefit from using enzymes developed
from microbial sources. Future aspects focus on a wider application of enzymes to brew with high
amounts of inexpensive raw materials like barley.

Barley contains starch that has to be broken down to fermentable sugars before the yeast can make
alcohol. Therefore, traditional brewing contains an extra step compared with wine-making, namely
malting in which enzymes needed for the degradation of starch into fermentable sugars are produced.

Some enzymes are already present in the barley, e.g. β-amylases, but the majority of enzymes are
produced during the germination, e.g. α-amylases and proteases, and in the final malt all the enzymes
needed for the conversion of “grains” into a fermentable liquid (wort) is present.

The malt enzymes do have some limitations. They can only work at certain temperatures, pH values etc.,
and the activities might be too low to do a proper job in proper time. In contrast, commercial exogenous
enzymes can be designed to work at preferred temperatures and pH values and to have more enzymatic
power. Addition of exogenous enzymes at various steps during the process can therefore make brewing
easier, faster and more consistent.

The traditional source of enzymes used for the conversion of cereals into beer is barley malt. If too little
 51
enzyme activity is present in the mash, there will be several undesirable consequences: the extract yield
will be too low; wort separation will take too long; the fermentation process will be too slow; too little
alcohol will be produced; the beer filtration rate will be reduced; and the flavor and stability of the beer
will be inferior.

Exogenous enzymes are used to supplement the malt’s own enzymes in order to prevent these problems.
Furthermore, industrial enzymes are used to ensure better adjunct liquefaction, to produce low-
carbohydrate beer (‘light beer’), to shorten the beer maturation time, and to produce beer from cheaper
raw materials. The various steps of the brewing operations, where microbial enzymes are occasionally
added, are shown in the following table.
 52
Enzymes in Juices and Wine :-

Enzymes increase processing capacity and improve economy in the fruit juice and wine industries. The
most commonly used enzymes in these industries are pectinase. Pectinase increase juice yields and
accelerate juice clarification. They produce clear and stable single-strength juices, juice concentrates
and wines, from not only core-fruits such as apples and pears, but also stone fruits, berries, grapes,
citrus-fruits, tropical fruits and vegetables like carrots, beets and green peppers.

Fruit and Vegetable Juice :-

Pectinase not only increase juice yields, but also increase the color and health-promoting antioxidants in
fruit and vegetable juices. They also increase color extraction and juice volume by reducing fruit and
vegetable mash viscosity and improving solid/liquid separation, Pectinase and Amylase enzyme solutions
speed up filtration and prevent storage or post-packaging haze formation by depectinizing andreducing
starch in raw juices.

Wine :-

Winemakers supplement naturally occurring grape enzymes with commercial enzymes to increase
production capacity of clear and stable wines with enhanced body, flavor and bouquet. When added to
grapes or musts, Pectinase that contain hemicellulase enzyme products increase free-run juice volume
and extraction of color, fermentable sugars and flavor components, as well as reduce pressing and
fermentation timeβ-glucanase containing Pectinase depectinize grape-musts during fermentation or
young wines prior to fining and filtration. Grape musts and wines treated with B-glucanase containing
Pectinase are less viscous. They ferment, settle and mature more quickly.

A beta-glucanase containing pectinase is also used to degrade Botrytis-glucan. Wines made from
overripe grapes infected with Botrytis cinerea mold are often difficult to clarify and filter due to high
concentrations of Botrytis-produced glucan polysaccharides. The use of B-glucanase containing
Pectinase can speed up clarification and filtration.
 53

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(14) Enzymes in sugar industries: Types of enzymes in sugar industry;
isolation,purification and assay of enzymes,

Sugar Processing Enzymes:- Less number of enzymes are used in sugar industry,but they are
important.

Biotechnology has contributed in increasing the Sugar processing efficiency and energy saving by virtue
of hydrolytic Enzymes such as Dextranase and Amylase.

Hydrolytic enzymes are used as aids to sugar production and refining by removing materials which inhibit
crystallization or cause high viscosity. In some parts of the world, sugar cane contains significantamounts
of starch, which becomes viscous, thus slowing filtration processes and making the solution hazy when
the sucrose is dissolved. This problem can be overcome by using the most thermostable Alpha Amylase
Enzyme which is entirely compatible with the high temperatures and pH values that prevail during the
initial vacuum evaporation stage of sugar production.

Other persistent problem involves dextran. A dextran is produced by the action of dextransucrase from
Leuconostocmesenteroides on sucrose and found as a slime on damaged cane and beet tissue,
especially when processing has been delayed in hot and humid climates. Dextran has the sucrose
molecule as part of its structure and thus inhibit sucrose crystal growth.

This produces needle-like crystals which are not readily harvested by equipment designed for the
approximately cubic crystals otherwise obtained. Dextran can produce extreme viscosity in process
streams and even bring plant to a stop. Dextran problems are solved by the use of Dextranase Enzyme.

1.Alpha Amylase Enzyme for Hydrolyzing Starch

Alpha Amylase Enzyme to hydrolyze Starch to increase Sugar Recovery and improve overall performance.

2.Dextranase Enzyme for Hydrolysing Dextran

Dextranase Enzyme to hydrolyze Dextran to increase Sugar Recovery and improve overall performance.

3.Enzymes Based Biocide

Enzymes based Biocide for Mill Sanitation.

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Enzyme Assays :-

Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of
enzyme kinetics and enzyme inhibition.

Enzyme units :-

The quantity or concentration of an enzyme can be expressed in molar amounts, as with any other
chemical, or in terms of activity in enzyme units.

Enzyme activity :-

Enzyme activity = moles of substrate converted per unit time = rate × reaction volume. Enzyme activity is
a measure of the quantity of active enzyme present and is thus dependent on conditions, which should be
specified. The SI unit is the katal, 1 katal = 1 mol s−1, but this is an excessively large unit. A more practical
and commonly used value is enzyme unit (U) = 1 μmol min−1. 1 U corresponds to 16.67 nanokatals.

Enzyme activity as given in katal generally refers to that of the assumed natural target substrate of the
enzyme. Enzyme activity can also be given as that of certain standardized substrates, such as gelatin,
then measured in gelatin digesting units (GDU), or milk proteins, then measured in milk clotting units
(MCU). The units GDU and MCU are based on how fast one gram of the enzyme will digest gelatin or milk
proteins, respectively. 1 GDU equals approximately 1.5 MCU.

An increased amount of substrate will increase the rate of reaction with enzymes, however once past a
certain point, the rate of reaction will level out because the amount of active sites available has stayed
constant.

Specific activity :-

The specific activity of an enzyme is another common unit. This is the activity of an enzyme per milligram
of total protein (expressed in μmol min−1 mg−1). Specific activity gives a measurement of enzyme purity
in the mixture. It is the micro moles of product formed by an enzyme in a given amount oftime (minutes)
under given conditions per milligram of total proteins. Specific activity is equal to the rate of reaction
multiplied by the volume of reaction divided by the mass of total protein. The SI unit is katal/kg, but a
more practical unit is μmol/mgmin.

Specific activity is a measure of enzyme processivity (the capability of enzyme to be processed), at a

specific (usually saturating) substrate concentration, and is usually constant for a pure enzyme.

An active site titration process can be done for the elimination of errors arising from differences in
cultivation batches and/or misfolded enzyme and similar issues. This is a measure of the amount of

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active enzyme, calculated by e.g. titrating the amount of active sites present by employing an irreversible
inhibitor. The specific activity should then be expressed as μmol min−1 mg−1 active enzyme. If the
molecular weight of the enzyme is known, the turnover number, or μmol product per second per μmol of
active enzyme, can be calculated from the specific activity. The turnover number can be visualized as the
number of times each enzyme molecule carries out its catalytic cycle per second.

Related terminology :-

The rate of a reaction is the concentration of substrate disappearing (or product produced) per unit time
(mol L−1 s−1).

The % purity is 100% × (specific activity of enzyme sample / specific activity of pure enzyme). The
impure sample has lower specific activity because some of the mass is not actually enzyme. If the
specific activity of 100% pure enzyme is known, then an impure sample will have a lower specific
activity, allowing purity to be calculated and then getting a clear result.

Types of assay :-

All enzyme assays measure either the consumption of substrate or production of product over time. A
large number of different methods of measuring the concentrations of substrates and products exist
and many enzymes can be assayed in several different ways. Biochemists usually study enzyme-
catalysed reactions using four types of experiments:

Initial rate experiments. When an enzyme is mixed with a large excess of the substrate, the enzyme-
substrate intermediate builds up in a fast initial transient. Then the reaction achieves a steady-state
kinetics in which enzyme substrate intermediates remains approximately constant over time and the
reaction rate changes relatively slowly. Rates are measured for a short period after the attainment of the
quasi-steady state, typically by monitoring the accumulation of product with time. Because the
measurements are carried out for a very short period and because of the large excess of substrate, the
approximation that the amount of free substrate is approximately equal to the amount of the initial
substrate can be made. The initial rate experiment is the simplest to perform and analyze, being relatively
free from complications such as back-reaction and enzyme degradation. It is therefore by far the most
commonly used type of experiment in enzyme kinetics.

Progress curve experiments. In these experiments, the kinetic parameters are determined from
expressions for the species concentrations as a function of time. The concentration of the substrate or
product is recorded in time after the initial fast transient and for a sufficiently long period to allow the
reaction to approach equilibrium. Progress curve experiments were widely used in the early period of
enzyme kinetics, but are less common now.

Transient kinetics experiments. In these experiments, reaction behaviour is tracked during the initial
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fast transient as the intermediate reaches the steady-state kinetics period. These experiments are more
difficult to perform than either of the above two classes because they require specialist techniques (such
as flash photolysis of caged compounds) or rapid mixing (such as stopped-flow, quenched flow or
continuous flow).

Relaxation experiments. In these experiments, an equilibrium mixture of enzyme, substrate and product
is perturbed, for instance by a temperature, pressure or pH jump, and the return to equilibrium is
monitored. The analysis of these experiments requires consideration of the fully reversible reaction.
Moreover, relaxation experiments are relatively insensitive to mechanistic details and are thus not
typically used for mechanism identification, although they can be under appropriate conditions.

Enzyme assays can be split into two groups according to their sampling method: continuous assays,
where the assay gives a continuous reading of activity, and discontinuous assays, where samples are
taken, the reaction stopped and then the concentration of substrates/products determined

Factors to control in assay :-

Salt Concentration: Most enzymes cannot tolerate extremely high salt concentrations. The ions interfere
with the weak ionic bonds of proteins. Typical enzymes are active in salt concentrations of 1-500 mM. As
usual there are exceptions such as the halophilic algae and bacteria.

Effects of Temperature: All enzymes work within a range of temperature specific to the organism.
Increases in temperature generally lead to increases in reaction rates. There is a limit to the increase
because higher temperatures lead to a sharp decrease in reaction rates. This is due to the denaturating
(alteration) of protein structure resulting from the breakdown of the weak ionic and hydrogen bonding
that stabilize the three-dimensional structure of the enzyme active site.[12] The "optimum" temperature
for human enzymes is usually between 35 and 40 °C. The average temperature for humans is 37 °C.
Human enzymes start to denature quickly at temperatures above 40 °C. Enzymes from thermophilic
archaea found in the hot springs are stable up to 100 °C.[13] However, the idea of an "optimum" rate of
an enzyme reaction is misleading, as the rate observed at any temperature is the product of two rates,
the reaction rate and the denaturation rate. If you were to use an assay measuring activity for one second,
it would give high activity at high temperatures, however if you were to use an assay measuring product
formation over an hour, it would give you low activity at these temperatures.

Effects of pH: Most enzymes are sensitive to pH and have specific ranges of activity. All have an optimum
pH. The pH can stop enzyme activity by denaturating (altering) the three-dimensional shape ofthe enzyme
by breaking ionic, and hydrogen bonds. Most enzymes function between a pH of 6 and 8; however pepsin
in the stomach works best at a pH of 2 and trypsin at a pH of 8.
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Substrate Saturation: Increasing the substrate concentration increases the rate of reaction (enzyme activity).
However, enzyme saturation limits reaction rates. An enzyme is saturated when the active sites of all the
molecules are occupied most of the time. At the saturation point, the reaction will not speed up, no matter
how much additional substrate is added. The graph of the reaction rate will plateau.

Level of crowding, large amounts of macromolecules in a solution will alter the rates and equilibrium
constants of enzyme reactions, through an effect called macromolecular crowding.

https://t.me/Food_tech_notes

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(15) Enzymes in fats, oil, flavour and fragrances

Lipase, any of a group of fat-splitting enzymes found in the blood, gastric juices, pancreatic secretions,
intestinal juices, and adipose tissues. Lipases hydrolyze triglycerides (fats) into their component fatty
acid and glycerol molecules.

Initial lipase digestion occurs in the lumen (interior) of the small intestine. Bile salts reduce the surface
tension of the fat droplets so that the lipases can attack the triglyceride molecules. The fatty acid and
glycerol molecules are then taken up into the epithelial cells that line the intestinal wall, where they are
resynthesized into triglycerides for transport to muscles and adipose tissues. At these sites lipases in
the bloodstream hydrolyze the triglycerides, and the resulting fatty acids and glycerol are taken up by the
cells of these tissues. In the adipose tissues triglycerides are re-formed for storage until the energy
needs of the animal increase under conditions of stress or exercise. Lipases in the cells of adipose tissues
break down the triglycerides so that fatty acids can reenter the bloodstream for transport to energy-
requiring tissues.

Enzymes for oil extraction: A better way

Using enzymes in oil processing is easier – and increasingly more established – than you might think.
Our enzymes give you higher oil extraction yields and higher protein levels in your meal, enabling a
significant increase in your profit . And it’s a more sustainable process.

Better use of seeds with oil degumming enzymes

Why enzymatic oil extraction?

Enzymatic degumming of vegetable oils is a more sustainable and economic alternative to both caustic
refining or water degumming. It transforms the gums into oil and makes it easier to separate them in a
more environment-friendly manner.

How does it work?

Our enzymes uncouple the oil and water-soluble parts of phospholipids, increasing oil yield by capture of
diglycerides in the oil phase. With that, our enzymes dramatically reduce the gum volume and reduce their
ability to form an emulsion. Less emulsion formation means less oil yield loss due to entrained oil, while
a lower gum content means cleaner separation of oil from the heavy phases, further reducing yield loss

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Flavors and fragrances are constant companions in our daily life. We find them in food and in cosmetics,
and of course, the demand for natural ingredients instead of chemicals is increasing. A special group of
enzymes allows us to produce these valuable compounds from raw materials.

Flavors and fragrances play a big role in our daily lives. We wake up, loving the smell of hot coffee or tea.
We enjoy a fruit juice, our cereals or some of us prefer the joy of smoky bacon and eggs. We then switch
to brushing our teeth with toothpaste filling our mouths with freshness. We get a shower with musk shower
cream and almond shampoo, we dry ourselves with towels with a scent of lavender. Finally, when properly
dressed, we decorate ourselves with a puff of perfume. Many of us wish that the products, we consume on
the daily basis are flavored with natural ingredients rather than chemicals and xenobiotics. And this is,
where biocatalysis can kick in: instead of chemical steps, we let natural enzymes catalyze reactions in the
transformation of raw materials to valuable compounds.

Most smells are based on an aldehyde :-

Many compounds we know from their pleasant smell contain an aldehyde group. Some examples are
vanillin, the typical smell of vanilla, or cinnamaldehyde, a constituent of cinnamon smell. A totally different
aldehyde compound has the smell of grass and there are many, many more. Scientists from acib recently
searched for enzymes which are able to produce aldehyde compounds in a very selective manner. The
team of Margit Winkler built on the work of researchers from the late 1960s: they had described a promising
enzyme, which was isolated from the fungus Neurospora crassa. To be able to use this enzyme in modern
processes, the first essential step was to filter the amino acid sequence from the myriad of sequences in
today’s database jungle.

CAR – the tool for aldehyde production :-

The next step was to find a good method to produce this enzyme, which is called by its nick-name CAR
for simplicity to avoid the lengthy full name carboxylate reductase. The trick for the rather complicated
120 kDa three-domain protein was its heterologous expression in E. coli using autoinduction conditions
at 20°C. Once the CAR was accessible in satisfying amounts, the team challenged the CAR with many
different substrates and conditions to understand it in depth and to learn how to use it in the best possible
way. The new concept using an engineered E. coli strain allows the preparation of aldehyde in high
amounts and almost without the formation of undesired impurities, which is the achievement of a
collaboration with the Massachussetts Institute of Technology (MIT), Boston, USA. With this strategy,
the team filled their laboratory in Graz with the overly pleasant smell of piperonal: a mixture of vanilla,
almond and cherry, which is also known as heliotropin from the tropical flower heliotrope.

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(16) Immobilized enzymes in food processing

An immobilized enzyme is an enzyme attached to an inert, insoluble material—such as calcium alginate

(produced by reacting a mixture of sodium alginate solution and enzyme solution with calcium chloride).
This can provide increased resistance to changes in conditions such as pH or temperature. It also lets
enzymes be held in place throughout the reaction, following which they are easily separated from the
products and may be used again - a far more efficient process and so is widely used in industry for enzyme
catalysed reactions. An alternative to enzyme immobilization is whole cell immobilization

Commercial Uses:-

Immobilized enzymes are very important for commercial uses as they possess many benefits to the
expenses and processes of the reaction of which include:

1. Convenience: Minuscule amounts of protein dissolve in the reaction, so workup can be much easier.
Upon completion, reaction mixtures typically contain only solvent and reaction products.

2. Economy: The immobilized enzyme is easily removed from the reaction making it easy to recycle the
biocatalyst. This is particularly useful in processes such as the production of Lactose Free Milk, as the
milk can be drained from a container leaving the enzyme (Lactase) inside ready for the next batch.

3. Stability: Immobilized enzymes typically have greater thermal and operational stability than the
soluble form of the enzyme.

In the past, biological washing powders and detergents contained many proteases and lipases that
broke down dirt. However, when the cleaning products contacted human skin, they created allergic
reactions. This is why immobilization of enzymes are important, not just economically.

Methods of Immobilization :-

There are various ways by which one can immobilize an enzyme:

Affinity-tag binding: Enzymes may be immobilized to a surface, e.g. in a porous material, using non-
covalent or covalent Protein tags. This technology has been established for protein purification
purposes. This technique is the generally applicable, and can be performed without prior enzyme
purification with a pure preparation as the result. Porous glass and derivatives thereof are used, where
the porous surface can be adapted in terms of hydrophobicity to suit the enzyme in question.[4]

Adsorption on glass, alginate beads or matrix: Enzyme is attached to the outside of an inert material. In
general, this method is the slowest among those listed here. As adsorption is not a chemical reaction,

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the active site of the immobilized enzyme may be blocked by the matrix or bead, greatly reducing the
activity of the enzyme.

Entrapment: The enzyme is trapped in insoluble beads or microspheres, such as calcium alginate beads.
However, these insoluble substances hinder the arrival of the substrate, and the exit of products.

Cross-linkage: Enzyme molecules are covalently bonded to each other to create a matrix consisting of
almost only enzyme. The reaction ensures that the binding site does not cover the enzyme's active site,
the activity of the enzyme is only affected by immobility. However, the inflexibility of the covalent bonds
precludes the self-healing properties exhibited by chemoadsorbed self-assembled monolayers. Use of a
spacer molecule like poly(ethylene glycol) helps reduce the steric hindrance by the substrate in this case.

Covalent bond: The enzyme is bound covalentely to an insoluble support (such as silica gel or
macroporous polymer beads with epoxide groups). This approach provides the strongest
enzyme/support interaction, and so the lowest protein leakage during catalysis.
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What is enzyme immobilization?

Immobilization is defined as the imprisonment of cell or enzyme in a distinct support or matrix. The
support or matrix on which the enzymes are immobilized allows the exchange of medium containing
substrate or effector or inhibitor molecules. The practice of immobilization of cells is very old and the
first immobilized enzyme was amino acylase of Aspergillus oryzae for the production of L-amino acids
in Japan.

Advantages of immobilized enzymes:

(1). Increased functional efficiency of enzyme

(2). Enhanced reproducibility of the process they are undertaking

(3). Reuse of enzyme

(4). Continuous use of enzyme

(5). Less labour input in the processes

(6). Saving in capital cost and investment of the process

(7). Minimum reaction time

(8). Less chance of contamination in products

(9). More stability of products

(10). Stable supply of products in the market

(11). Improved process control

(12). High enzyme substrate ratio

Disadvantages of enzyme immobilization:

(1). Even though there are many advantages of immobilized enzymes, there are some disadvantages
also.

(2). High cost for the isolation, purification and recovery of active enzyme (most important
disadvantage)
 64
(3). Industrial applications are limited and only very few industries are using immobilized enzymes or
immobilized whole cells.

(4). Catalytic properties of some enzymes are reduced or completely lost after their immobilization on
support or carrier.

(5). Some enzymes become unstable after immobilization.

(6). Enzymes are inactivated by the heat generated in the system

Applications of enzyme immobilization:

(1). Industrial production: Industrial production of antibiotics, beverages, amino acids etc. uses
immobilized enzymes or whole cells.

(2). Biomedical applications: Immobilized enzymes are widely used in the diagnosis and treatment of
many diseases. Immobilized enzymes can be used to overcome inborn metabolic disorders by the supply
of immobilized enzymes. Immobilization techniques are effectively used in drug delivery systems
especially to oncogenic sites.

(3). Food industry: Enzymes like pectinases and cellulases immobilized on suitable carriers are
successfully used in the production of jams, jellies and syrups from fruits and vegetables.

(4). Research: A Research activity extensively uses many enzymes. The use of immobilized enzyme allow
researcher to increase the efficiency of different enzymes such as Horse Radish Peroxidase (HRP)in
blotting experiments and different Proteases for cell or organelle lysis.

(5). Production of bio-diesel from vegetable oils.

(6). Waste water management: treatment of sewage and industrial effluents.

(7). Textile industry: scouring, bio-polishing and desizing of fabrics.

(8). Detergent industry: immobilization of lipase enzyme for effective dirt removal from cloths.
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Supports or Matrix used in immobilization technology:

The matrix or support immobilizes the enzyme by holding it permanently or temporarily for a brief period
of time. There are a wide variety of matrixes or carriers or supports available for immobilization. The matrix
used should be cheap and easily available. Their reaction with the components of the medium or with the
enzyme should be minimum as possible. The matrixes or supports for immobilization of enzymes or whole
cells are grouped into three major categories

(1). Natural polymers

(2). Synthetic polymers

(3). Inorganic materials

(1). Natural polymers:

(a). Alginate: A natural polymer derived from the cell wall of some algae. Calcium or magnesium
alginate is the most commonly used matrix. They are inert and have good water holding capacity.

(b). Chitosan and chitin: They are structural polysaccharides occurring naturally in the cell wall of fungi
and the exoskeleton of Arthropods. The various functional groups in enzymes can bind to the – OH
group of chitin and can form covalent bonds.

(c). Collagen: It is the protenaceous support with good porosity and water holding capacity. The side
chains of the amino acids in the collagen and that of enzyme can form covalent bonds to permanently
hold the enzyme to the support.

(d). Carrageenan: It is a sulfated polysaccharide obtained from some red algae. Their good gelling
properties together with its high protein holding capacity makes it good support for immobilizing
enzymes.

(e). Gelatin: Gelatin is the partially hydrolyzed collagen with good water holding capacity.

(f). Cellulose: Most abundant polymer of nature and it is the cheapest support available as carrier of
enzymes. The hydroxyl group of the monomer units (glucose) can form covalent bonds with that of the
amino acids of enzyme.

(g). Starch: A natural polymer of amylose and amylo-pectin. It has good water holding capacity.

(h). Pectin: It is a structural polysaccharide of plants found in their primary cell wall and they also acts
as the inter-cellular cementing material in plant tissues. Pectin is a gelling agent with good water
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holding capacity.

(2). Synthetic polymers:

They are ion exchange resins or polymers and are insoluble supports with porous surface. Their porous
surface can trap and hold the enzymes or whole cells. Example: Diethylaminoethyl cellulose (DEAE
cellulose), Polyvinyl chloride (PVC), UV activated Polyethylene glycol (PEG)

(3). Inorganic materials:

(a). Zeolites: They are microporous, aluminosilicate minerals with good adsorbing properties and
extensively used for immobilizing enzymes and whole cells.

(b). Ceramics: They are nonmetallic solids consisting of metal and nonmetal atoms held in ionic and
covalent bonds. The composition and bonding pattern varies with different types.

(c). Diatomaceous earth: They are silicious sedimentary rocks formed by fossilized accumulations of
the cell wall of diatoms. Celite is the trade name of diatomaceous earth. It is a good adsorbent and are
resistant to high pH and temperature.

(d). Silica:

(e). Glass:

(f). Activated carbon

(g). Charcoal.

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