REGULATION OF ENZYME ACTIVITY AND SYNTHESIS

In cellular metabolism, groups of enzymes work together

in sequential pathways to carry out a given metabolic

process, such as the multireaction breakdown of glucose

to lactate or the multireaction synthesis of an amino acid

from simpler precursors. In such enzymic systems, the

reaction product of one enzyme becomes the substrate

of the next. Most of the enzymes in each metabolic pathway follow

the kinetic patterns we have already described.

Each pathway, however, includes one or more enzymes

that have a greater effect on the rate of the overall sequence.

The activities of regulatory enzymes are modulated

in a variety of ways. Allosteric enzymes function

through reversible, noncovalent binding of regulatory

compounds called allosteric modulators or allosteric

effectors, which are generally small metabolites or

cofactors. Other enzymes are regulated by reversible

covalent modification. Both classes of regulatory

enzymes tend to be multisubunit proteins, and in some

cases the regulatory site(s) and the active site are on

separate subunits. Metabolic systems have at least two

other mechanisms of enzyme regulation. Some enzymes

are stimulated or inhibited when they are bound by separate

regulatory proteins. Others are activated when

peptide segments are removed by proteolytic cleavage;

unlike effector-mediated regulation, regulation by proteolytic

cleavage is irreversible. Important examples of

both mechanisms are found in physiological processes

such as digestion, blood clotting, hormone action, and

vision.

Regulation of enzyme activity occur to coordinate the different metabolic Processes and homeostasis
i.e. to maintain the internal environment of the organism constant.

It can be achieved by two general mechanisms:

1- CONTROL OF ENZYME QUANTITY

Enzyme quantity is affected by:

 Altering the rate of enzyme synthesis and degradation,

 Induction
 Repression

Altering the rates of enzyme synthesis and degradation.

Enzymes are protein in nature, they are synthesized and degraded again from/ to amino acids

genetically controlled processes.

Enzyme quantity depends on the rate of enzyme synthesis and the rate of its

degradation.

• Increased or enzyme quantity may be due to an increase in the rate of

synthesis, a decrease in the rate of degradation or both.

• Decreased enzyme quantity may be due to a decrease in the rate of

synthesis, an increase in the rate of degradation or both.

• For example, the quantity of liver arginase enzyme increases after protein

rich meal due to an increase in the rate of its synthesis; also it increases in

starved animals due to a decrease in the rate of its degradation.

Induction

• Induction means an increase in the rate of enzyme synthesis by substances

called inducers, eg. induction of lactase enzyme in bacteria grown on glucose

media. Inducible enzymes, the concentration of these enzymes

depends on the presence of inducers

Constitutive enzymes, the concentration of these enzymes

does not depend on inducers.

Repression
• Repression means a decrease in the rate of enzyme synthesis by substances

called repressors.

• Repressors are low molecular weight substances that decrease the rate of

enzyme synthesis at the level of gene expression.

• they are usually end products of biosynthetic reaction, so repression

is sometimes called feedback regulation.

• For example, dietary cholesterol decreases the rate of synthesis of HMG

CoA reductase (β-hydroxy β-methyl glutaryl CoA reductase), which is a

key enzyme in cholesterol biosynthesis

Concentration of substrates, coenzymes and metal ion activator

The susceptibility of the enzyme to degradation depends on its conformation.

Presence of substrate, coenzyme or metal ion activator causes changes in the

enzyme conformation decreasing its rate of degradation.

2- CONTROL OF CATALYTIC EFFICIENCY OF THE ENZYME

 Allosteric regulation
 Feedback inhibition
 Proenzyme (zymogen)
 Covalent modification
 Protein – Protein interaction

Allosteric Regulation
Some enzymes are reversibly inhibited or activated by the
presence of metabolites that are not their substrate or product. These metabolites, if
inhibitory are normally distant products of the pathway, thereby providing negative
feedback for the activity of the pathway.
The enzymes controlled in this way usually have additional binding site other than the
active or substrate binding site. The binding of inhibitor or activators at distant site from
active site often brought about conformational changes in the enzyme molecule which
may decrease or increase its catalytic activity. Effector

or allosteric activator increases it activity, it is called positive e.g. ADP is allosteric activator for
phosphofructokinase enzyme.

Negative effector or allosteric inhibitor causes a decrease in its activity

e.g. ATP and citrate are allosteric inhibitors for phosphofructokinase enzyme. Glucose-6-phosphate
is allosteric inhibitor for hexokinase enzyme as shown in the following diagram.
 Kinetics of allosteric enzymes
• One of the common characteristics of an allosteric enzyme is that it shows a

sigmoid plot when velocity is plotted against substrate concentration

• Allosteric enzymes generally do not follow the Michaelis-Menten equation.

• The Lineweaver-Burk plot is concave upward.

Feedback Inhibition
This is a means by which biosynthetic pathways are
regulated and involves the process whereby and products or near end products control the
metabolic flux by inhibiting one or more of the enzyme at the early functionally irreversible step

specific to a particular biosynthetic pathway.

Often maximum feedback inhibition is attained only by the combined actions of multiple
end products.

Feedback inhibition may occur by simple feedback loop. as in the following diagram

Where A is the substrate, E is the end product, B, C, D are intermediate metabolites, E1,

E2, E3 and E4 are enzymes in biosynthetic pathway.

Feedback inhibition can occur by multiple feedback inhibition loops as occurs in

 branched biosynthetic pathways.Feedback inhibition could be (1) sequential (2) concerted
(3)cumulative (4) co-operative

N.B.: Feedback regulation is different from feedback inhibition.

Feedback regulation:

• It means that an end product in the reaction decreases the rate of enzyme

synthesis at the level of gene expression.

• It does not affect the enzyme activity.

• It decreases the enzyme quantity through the action on the gene that

encodes the enzyme.

• It is a complicated process that takes hours to days.

• For example, inhibition of HMG CoA reductase enzyme by dietary

cholesterol.

Feedback inhibition

• It means that an end product directly inhibits an enzyme early in

biosynthetic pathways.

• It does not affect enzyme quantity

• It decreases enzyme activity.

• It is a direct and rapid process that occurs in seconds to minutes.

eg, CTP inhibits aspartate- transcarbamylase enzyme in

pyrimidine synthesis

Proenzymes (Zymogens)

Enzyme regulation is an important matter to cells, and

evolution has provided a variety of additional options, including zymogens,
isozymes, and modulator proteins.
Some enzymes are secreted in inactive forms called proenzymes or zymogens because it contains an
additional polypeptide chain that

masks (blocks) the active site of the enzyme.

Examples for zymogens include pepsinogen, trypsinogen, chymotrypsinogen,

prothrombin and clotting factors.

Activation of zymogen occurs by removal of the polypeptide chain that masks

the active site as shown in the following figure.

Activation of zymogens can occur by one of the following methods:

1- Activation by HCl

HCl

Pepsinogen ▬▬▬▬▬▬▬► Pepsin

2- Activation by other enzymes

Enterokinase

Trypsinogen ▬▬▬▬▬▬▬► Trypsin

Thrombokinase + Ca++

Prothrombin ▬▬▬▬▬▬▬▬▬► Thrombin

3- Auto activation i.e. the enzyme activates itself.

Pepsin

Pepsinogen ▬▬▬▬▬▬▬► Pepsin

Biological importance of zymogens

1. Some enzymes are secreted in zymogen form to

protect the tissues of origin from auto digestion.

2. Another biological importance of zymogens is to

insure rapid mobilization of enzyme activity at the

time of needs in response to physiological

demands.

Protein-protein interaction /Modulator Proteins

Modulator proteins are yet another way that cells mediate metabolic activity.

Modulator proteins are proteins that bind to enzymes, and by binding, influence

the activity of the enzyme.

In enzymes that are formed from of many protein subunits, the enzyme may be
 present in an inactive form through interaction between its protein subunits.The whole enzyme,
formed of regulatory and catalytic subunits, is inactive.

Activation of the enzyme occurs by separation of the catalytic subunits from the

regulatory subunits.

Protein kinase A enzyme is an example for regulation of enzyme activity through

protein interaction.

This enzyme is formed of 4 subunits, 2 regulatory (2R) and 2 catalytic (2C)

subunits. The whole enzyme (2R2C) is inactive. cAMP (cyclic adenosine

monophosphate) activates the enzyme by binding to the 2 regulatory (2R)

subunits releasing the 2 catalytic (2C) subunits and hence activating the enzyme.

Covalent modification

It means modification of enzyme activity through formation of

covalent bonds such as:.

1. Methylation (addition of methyl group).

2. Hydroxylation (addition of hydroxyl group).

3. Adenylation (addition of adenylic acid).

4. Phosphorylation (addition of phosphate group)

Phosphorylation is the most covalent modification used to regulate enzyme activity.

Phosphorylation of the enzyme occurs by addition of phosphate group to the enzyme at

the hydroxyl group of serine, threonine or tyrosine. This occurs by protein kinase

enzyme.

Dephosphorylation of the enzyme occurs by removal of phosphate group from the

hydroxyl group of serine, threonine or tyrosine. This occurs by phosphatase enzyme.

The phosphorylated form is the active form in some enzymes, while the

dephosphorylated form is the active form in other enzymes.

Examples of enzymes activated by phosphorylation. These are usually enzymes of

degradative reactions e.g.

1. Glycogen phosphorylase that breaks down glycogen into glucose.

2. Citrate lyase, which breaks down citrate.

3. Lipase that hydrolyzes triglyceride into glycerol and 3 fatty acids.

Examples of enzymes inactivated by phosphorylation. These are usually enzymes of

biosynthetic reactions e.g.

1. Glycogen Synthetase, which catalyzes biosynthesis of glycogen.

2. Acetyl CoA carboxylase, an enzyme in fatty acid biosynthesis.

3. HMG CoA reductase, an enzyme in cholesterol biosynthesis.

ENZYME ASSAY TECHNIQUES

Assay simply means measurement of the enzymatic activity that is based on the
determination of either the rate of formation of the product or the rate of
utilization or disappearance of reactant or substrate under control conditions. Most
assays are carried out at 30°C-37°C. Adequate buffering capacity is always being
ensured. Apparatus used must be clean.
Analytical assays may be classified as (1) continous (2) discontinuous.
In order to standardize the report on enzyme activities, the commission of enzyme
of the international union of biochemistry defined a standard unit i.e International
unit (I.U) of enzyme as the amount of enzyme that catalysed the formation of 1
micro mole of product per minute under defined condition. The concentration of
enzyme in an impure preparation is expressed in terms of units/ml while the
specificity activity is expressed as units/mg protein.
In most cases, as the enzyme is being purified, its specific activity is expected to
increase to maximum.
Kcat: is defined as the amount of enzyme that catalyze I mole of substrate per
second.
Turnover number: is defined as the number of moles of substrate transformed per
minute per second.
There exist different enzyme assay techniques, however, irrespective of the
principle of the chosen method, the enzyme assay requires the use of excess
substrate (zero order kinetics at least equal to 10Km) and an appropriate control is
required. This control is in all respect the same as the test assay but lacks either the
enzyme or substrate. Both the test and the control must be subjected to the same
experimental condition.
TYPES OF ENZYME ASSAY TECHNIQUES
(1) VISIBLE AND UV SPECTROPHOTOMETRIC METHOD
(2) SPECTROFLUORIMETRY
(3) RADIOISOTOPE
(4) IMMUNOCHEMICAL METHOD
(5) MACRO CALORIMETRIC METHOD
(6) MANOMETRIC METHOD
(7) COUPLE ASSAY METHOD/TECHNIQUES

Enzyme purification:
Enzymes are purified by employing successive chemical or physical fractionation
procedures. However, it should be noted that there is no fast and hard rule in
 respect of the protocol to be controlled. It is usually by a trial and error study
based on a pilot study.
The utmost idea of each step is to get rid of the contaminants as possible and
retain much desired enzyme. The efficiency of each step is given by
(1) The yield or recovery (i.e percentage of the total enzyme activity originally
present that is retained.
(2) The purification factor (the factor by which the specific activity of the
preparation has increased).
The preparation of the cell free extract may be prepared by several means
depending on the nature of the starting material (tissue, or cell, or
organism) and the size of the preparation. For instance, cell breakage
method may be by autolysis freeze thaw, mechanical grinding and holistic
homogenization e.t.c. the resulting homogenate is usually centrifuged to
remove unbroken cells and large debris. At times, differential centrifugation
for mitochondria, chloroplast, microsome, ribosome, or nucleic acids could

be carried out.
The purity of the final preparation should be checked by several methods
before one can conclude if the final preparation is homogenous. Some of
the criteria are
(1) Homogenous enzyme preparation should elute from an ion-exchange or
gel filtration column as a single symmetrical activity and protein peak
with a constant specific activity throughout.
(2) Probably give a single bond (if not a dimer or polymer without artifacts
or background noise).
Enzyme purification has several purposes:
1. To increase catalytic activity of the enzyme, in order to produce an enzyme
preparation, that is of industrial significance.
2. To study the enzyme characteristics, namely the optimum pH, temperature, stability, Km, etc.,
3. To determine its structure using X-ray crystallography

Enzyme purification involves several steps that eliminate other proteins and chemicals
that may interfere with the enzyme’s catalytic activity.

METHODS/STAGES OF ENZYME ISOLATION AND PURIFICATION:

Steps involved in isolation of a protein or enzyme:
1. Cell disruption (which can done via a number of number of
different processes of choice e.g Detergents lysis, Osmolysis, freeze thaw
cycles, enzymatic lysis, ultrasonication, Homogenisation)
2. Centrifugation differential (at a specific speed depending on the organ,
tissue, organelle or fluid).
3) Removal of Supernatant (Decantation To Obtain Supernatant)

Steps involved in purification of a protein or enzyme

1. Salt precipitation (Salting out of proteins):
Salt precipitation is usually carried out using ammonium sulfate.
This step involves precipitation of
proteins, thus eliminating residual nutrients of the medium and other released by products.
Principle: Proteins are soluble in aqueous media because they have hydrophilic amino
acid side-chains which are provided by the basic amino acids, the acidic amino acids and
the neutral hydrophilic amino acids. A salt like ammonium sulfate interferes with these
interactions between amino acid side-chains and water, by reducing the available water
and will reduce the solubility of the protein.
This enables protein-protein interactions

instead of protein-water interactions and the protein will come out of solution. Depending
upon the hydrophilicity of the protein, different proteins would separate at different
ammonium sulfate saturation levels. Higher the hydrophilicity, higher ammonium sulfate
concentration would be required to break the protein-water interactions to enable its
precipitation.

2. Dialysis followed Ammonium sulfate precipitation, to remove the salt which

is bound to the protein. The protein/enzyme solution is placed in a bag of selectively

permeable membrane (e.g. gelatin/cellophane), immersed in a large volume of buffer,
under constant stirring at 4oC. The removal of ammonium salt is managed by the pore
size of the membrane that allows molecules of ammonium sulfate to cross, while
inhibiting the protein molecules to cross.

3. Chromatographic methods:
Depending upon the net charge of the
protein/enzyme of interest, the method to be employed can be either cation-exchange
chromatography or anion-exchange chromatography, which are differentiated on the
basis of the ion exchange resin.
Anion-exchange chromatography:
The resin used is diethylaminoethylcellulose
(DEAE- Cellulose), which is a weak base. The cationic nature of the resin
enables anionic proteins to bind while allowing cationic proteins to pass through. The
binding of the protein to the resin depends upon the pI value of the protein and the pH of
the buffer solution in which the DEAE-Cellulose is equilibrated. The unbound proteins
can be collected in the unadsorbed fraction. The proteins which are bound can be eluted
using a salt gradient (i.e. NaCl linearly increasing from 0 mM to 200 mM). While elution
using a salt gradient, the positive ions of the salt will compete with those of the proteins,
for binding to the column, thus enabling the separation of protein
Cation Exchange Chromatography:
The resin used is CM-cellulose
(Carboxymethyl cellulose) is a weak acid which binds cations and allows anions to pass

ENZYME APPLICATIONS
APPLICATION OF ENZYMES
Enzymes are attractive catalysts owing to mild reaction conditions, high product selectivity, and low
environmental impact, and thus have been employed for both simplified chemical synthesis routes
and improved chemical processes consequently, are often utilized for a broad range of applications
such as:
washing powders (e.g. proteases, lipases, amylases); textile manufacture (amylases and
catalase to remove the starch); the leather industry (proteases to hydrolyze proteins);
the paper industry; improvement of the environment; food production (enzyme modified
cheese/butter), processing (glucose oxidase for dough strengthening) and
preservation; and medical applications.

INDUSTRIAL APPLICATION
There is a shift toward increased technical
enzyme production including those used in textile, paper, leather, and biodiesel industries where
 excess waste generation incurs fines from environmental agencies . Such were largely driven by
process development in enzymatic production, which present
good opportunities for the scale-up of immobilized biocatalysts.

The Food-Water-Fuel Nexus

The large-scale production of biofuels i.e, fuels derived from biomass, animal fats, waste oils,
and other renewable resources that encompass chemical products such as bioalcohols, biodiesel,
biosynthetic oils, and biogas, has been recognized for its potential to supplement or replace fossil
fuels, particularly as oil reserves are depleted to meet global energy demands. The benefits
of economically feasible biofuel production are two-fold: biofuel offers improved sustainability
over traditional fuel sources as well as significantly reduces the environmental impact owing to
its lower emission of carbon monoxide, nitrogen oxides, sulfur oxides, and particulate matter.
According to British Petroleum (BP), global production of biofuels rose by an average of 14.1% from
2006 to 2016, illustrating the growing impact that biofuels have on the world energy landscape.
While government incentives have helped to drive industry-scale biofuel production, the economic
viability of biofuel production will be determined by the development of processes that efficiently use
waste from agriculture and industry as feedstock, thus side-stepping the ethical dilemma of using
freshwater and land resources for fuel production. Numerous technologies exist for the conversion of
raw biological materials to usable, high-energy bio products with most production routes requiring
either the transesterification of oils or the esterification of fatty acids. The traditional chemical process
uses sodium methoxide for conversion of plant oil triglycerides to fatty acid methyl esters(FAMEs),
which subsequently results in a product contaminated with high alkali salt content requiring costly
purification.
The use of lipase as a biocatalyst for esterification was researched for its efficiency at mild reaction
conditions and high-purity product yields and was proposed to eliminate the need for purification.
However, for biofuel production, economical implementation of such an enzymatic process requires
efficient recovery and reuse of lipase due to the required scale of production, therefore necessitating
an
immobilized enzyme.
A prominent trend in the production of biofuels is the design of processes based on inexpensive,
largely abundant starting materials because cheap feedstock is one of the biggest driving forces in the
profitability of biofuels processes. The biggest such feedstock is lignocellulosic biomass—made
up of lignin, cellulose, and hemicellulose—due to its massive abundance and wide range of sources
including crop residues, softwood and hardwood, herbaceous biomass, and municipal solid waste.
The most difficult technical barrier required to unlock lignocellulosic biomass is however the
extensive mechanical or chemical pretreatment required to further enable the processing of cellulose
in lignocellulosic biomass via hydrolysis to glucose and subsequent conversion to bioethanol via
whole-cell fermentation. Current research suggests that ionic liquids, i.e., salts that exist in
molten states at temperatures below 100 _C with strong chemical and thermal stabilities and
extremely low vapor pressures, will play a key role in the development of processes that viably
release cellulose from lignocellulosic material.
Chemical conversion of cellulose to glucose requires the use of diluted acids and high temperatures,
which implies high energy inputs to result in the generation of a significant amount of unwanted
by products. A more ideal approach is the use of cellulase, i.e., a mixture of hydrolytic enzymes
that act synergistically in the conversion of cellulosic material, for the selective enzymatic hydrolysis
of
cellulose to glucose, which requires longer reaction time but leads to improved yield from the
subsequent
fermentation due to a low generation of unwanted by product. The economically viable
scale-up of cellulase-catalyzed cellulose conversion to glucose for bioethanol production is limited by
poor
biocatalyst recovery, slow enzyme-catalyzed reaction rates, and low biocatalyst stability under
industrial
operation conditions. Therefore, immobilized cellulase is a requirement for industrial catalysis,
particularly considering the acid pretreatment required for cellulose.
.
Natural Gas Conversion
The composition of natural gas is 80–95% methane with varying degrees of heavier hydrocarbons,
but methane has a relatively low market value due to difficulties in storage and transportation as well
as limited use as fuel.
The traditional chemical conversion of methane to methanol, such as steam reformation
and the Fischer-Tropsch process, are limited by several significant drawbacks. The chemical
conversion
route requires the use of high-temperature, high-pressure unit operations as well as noble metal
catalysts,
resulting in a poor selectivity of methanol. The low yield of methanol necessitates a large process
through put in order to overcome large capital cost investments, thus the process is only profitable
at a massive scale, thus placing further constrictions on process employment due to the difficulty of
transporting methane from an extraction site to a production plant.
The use of the biocatalyst methane monooxygenase (MMO), for the conversion of methane to
methanol, has recently gained interest in the wave of expanding natural gas extraction. MMO has
been shown to convert methane to methanol at ambient conditions with selectivity approaching
100% and thus has been researched for the scale-up of methane conversion, considering its multiple
advantages over the chemical conversion route. The high selectivity of MMO-catalyzed
methanol production eases the intensity of product separation and could significantly reduce the
number of steps required in the conversion process. Furthermore, the enzymatic reaction occurs at
mild reaction conditions, and could thus cut back on costs associated with heating, pressurization,
and other feedstock conditioning steps.

6.4. Food and Beverage Industry

The use of biocatalysts in food and beverage processes dates back thousands of years to the advents of
culinary practices (wine and cheese making). In modern times, the widespread use of enzymes
in food and beverage industries for food quality preservation or modification is one of the earliest
successful industrial applications of biocatalysis, observed in beer fermentation, juice debittering,
and bread baking . The replacement of conventional chemical treatment with enzyme-catalyzed
pathways for conversion of starch to glucose and fructose first took place several decades ago.
The conventional production route requires temperatures up to 175 0C and considerable pressurization,
whereas biocatalytic processes can be carried out at temperatures near 100 _C and at ambient
pressure
via sequential _-amylase-catalyzed reactions encompassing both liquefaction and saccharification
steps. In addition to milder reaction conditions, the multi-enzymatic process resulted in higher
product selectivity and therefore allowed for better defined production routes for varying sugar
products like maltose, fructose syrup, and crystalline sugar, as dictated by biocatalyst selection.
An emerging trend is the use of enzyme catalysis for commercial-scale production of probiotics,
artificial sweeteners, and rare sugars. Probiotics, such as oligosaccharides, lactulose, lactilol
hydrolysates, and inulin, are non-digestible food additives that stimulate growth of gut bacteria and
can
reportedly improve human health. Dietary supplement producers have become particularly
interested in simple, efficient enzyme-catalyzed synthesis routes of probiotics
The
enzyme-catalyzed production of galacto-oligosaccharides (GOS), a lucrative probiotic with digestive
health benefits and use as low-calorie sweeteners. GOS are produced by transgalactosylation
simultaneous to hydrolysis of lactose via _-galactosidase; lab-scale results have shown GOS yields
near 40% for free enzyme, while immobilized enzymes show the potential for larger yields of up to
50% through implementation in a continuous system resulting in decreased product inhibition.
The enzymatic production of protein hydrosylates for use as nutritional supplements and flavor
enhancers has been developed due to milder reaction conditions and increased control over product
formation relative to traditional chemical routes. When hydrolyzed, a parent protein forms
biofunctional peptides exhibiting antioxidant, antimicrobial, and antihypertensive properties, among
other therapeutic effects. The production of fish protein hydrolysates from seafood processing
waste via papain, a proteolytic enzyme derived from papaya that has found widespread industrial
application, has garnered attention recently because the process is a potential solution for minimizing
pollution from fishing industries
Flavors and Aromas Industry
Biocatalytic processes economically and environmentally advantageous to conventional chemical
processes are in development for commercial-scale production of fragrance compounds, flavor
compounds, and aromatics. The chemical structures of such substances are often characterized
by regio-, chemo-, or stereoselective positioning of functional groups like alcohols, aldehydes,
ketones, and esters; thus, scale-up of efficient enzyme-catalyzed processes for production of aromatic
compounds is a potentially lucrative endeavour.
Lipase enzymes play an integral role in the development of biocatalytic fragrance and flavor
production due to its capability to transfer acyl groups from esters to other nucleophiles.
Though most progress has been in bench-scale synthesis of aroma esters, the results suggest that
a combination of protein engineering and process engineering can facilitate scale-up to profitable
industrial processes.

Detergents Industry
Successful employment of biocatalysts is cited as the driving force of production of cost effective,
environmentally benign detergents.
enzymes are a product rather than a chemical process-specific catalyst. Nonetheless, favorable market
trends in the detergents’ industry reinforce the underlying view that biocatalytic products are
inherently
safer and more sustainable than traditional chemical products that pose health and safety risks.
Alkaline proteases—which are effective in the removal of protein stains and the cleaving of damaged
cotton
fibers—isolated from microbial sources comprise significant portions of multiple detergents
produced
and sold at commercial scale.

Sector Enzymes Applications

Pharmaceuticals Nitrile hydratase, transaminase, monoamine oxidase, lipase, penicillin
acylase Synthesis of intermediates for production of active pharmaceutical
ingredients
Food Processing Trypsin, amylase, glucose isomerase, papain, pectinase
Conversion of starch to glucose, production
of high fructose corn syrup, production of
prebiotics, debittering of fruit juice
Detergent
Protease, lipase, amylase,
cellulase Stain removal,
removal of fats and oils,
color retention,
Biofuels
Lipase, cellulase, xylanase
Production of fatty acid methyl esters,
decomposition of lignocellulotic material
for bioethanol production
Paper and Pulp
Lipase, cellulase, xylanase
Removal of lignin for improved
bleaching,
improvement in fiber properties
Pharmaceuticals Industry
Enzyme catalysis has been successfully used for the production of pharmaceutically active
chemicals at the industrial scale. The most significant advantages enzyme catalysis holds over
conventional catalysis are the high regio-, chemo-, and stereo selectivity at which enzymes convert
substrate to product. A high degree of product specificity is largely desirable in such
pharmaceutical processes due to the streamlining of product synthesis routes and subsequent
improvement in process economics.
The use of appropriate enzymes not
only obviates such steps, but has also been shown to yield higher enantiomeric excesses of desired
stereoisomers. Furthermore, enzyme-catalyzed synthesis routes often reduced or eliminated the
need for chemically harsh substances or high-temperature conditions that can otherwise require
intense

Medical applications
Diagnostic applications of enzymes
Enzymes have been used widely in diagnostic applications varying from immunoassays
to biosensors. Enzyme immunoassay methods hold great promise for
application under a wide variety of conditions. Under laboratory conditions they
can be as sensitive as radio-immunoassays, but they can also be adapted as simple
field screening procedures. The examination of enzyme quantity in the
extracellular body fluids (blood plasma and serum, urine, digestive juices, amniotic
fluid and cerebrospinal fluid) are vital aids to the clinical diagnosis and management
of disease. Most enzyme-catalyzed reactions occur within living cells, however, when
an energy imbalance occurs in the cells because of exposure to infective agents,
bacterial toxins, etc, enzymes ‘leak’ through the membranes into the circulatory
system. This causes their fluid level to be raised above the normal cell level.
Estimation of the type, extent and duration of these raised enzyme activities can
then furnish information on the identity of the damaged cell and indicate the extent
of injury. Enzyme assays can make an important contribution to the diagnosis of
diseases, as a minute change in enzyme concentration can easily be measured.
Determination of the changes in enzyme level thus offers a greater degree of organ
and disease differentiation in comparison to other possible clinico-chemical parameters,
e.g. albumin or gamma globulin. Currently, the diagnostic specificity of
enzyme tests is such that they are limited primarily to confirming diagnosis, offering
data to be weighed alongside other clinical reports, owing to lack of disease specific
enzymes.

Enzyme examinations in diseases of the liver and biliary

The diseases of the liver and gastrointestinal tract were among the first to which
serum enzyme tests were applied. They have proved to be most effective owing to the
large size of the organs and the wide range and abundance of enzymes. The
liver-based enzymes GOT, GPT and AP are examined to evaluate the site and
nature of liver disease. LD, GGT, OCT and CHE are also examined.

Enzyme applications in heart disease

The discovery of serum glutamine oxalacetic acid transaminase
determination (GOT) in 1954 was considered a significant step forward in the
diagnosis of acute myocardial infarction. A mixture of results from assays of CPK
(creatine phosphokinase), HBD (α-hydroxybutyrate dehydrogenase) and GOT
(glutamine oxalacetic acid transaminase)—each of which has been shown to be
elevated in more than 90% of cases—is used for diagnostic purposes.

Diagnosis of muscle disease

Skeletal muscle disorders include diseases of the muscle fibers (myopathies) or of the
muscle nerves (neurogenic disorders). In myopathies CPJ, LD, ALD, GOT and
GPT levels are raised. In the case of neurogenic diseases and hereditary diseases,
CPK is occasionally raised (2–3 fold) . Damage to the muscle may be due to
extensive muscular exercise, drugs, physical trauma, inflammatory diseases, microbial
infection or metabolic dysfunction, or it may be genetically predisposed. In
muscular disorders the level of CPK is elevated in serum with the highest frequency
and is assayed in the diagnosis of these disorders. An additional useful assayed
enzyme is acetyl cholinesterase (AChE), which is significant in regulating certain
nerve impulses

Enzymes in therapeutics
Enzymes have two significant features that differentiate them from all other types of
drugs. First, enzymes frequently bind and act on their targeted sites with high
affinity and specificity. Second, enzymes are catalytic and convert numerous target
molecules to the desired products. These two important features make enzymes
specific and potent drugs that can achieve therapeutic biochemistry in the body that
small molecules cannot. These features have resulted in the development of many
enzyme-based drugs for a wide range of disorders. Currently, numerous
enzymes are used as therapeutic agents, owing to the following features:
• High specificity to their substrates.
• Proficient in producing the desired effect without provoking any side effects.
• Water soluble.
• Extremely effective in a biological environment.
Enzymes as therapeutic agents also have some serious disadvantages which
restrict their application. Their bulky structure, due to their large molecular weight,
excludes them from the intracellular domain. Owing to their high proteinaceous
nature they are highly antigenic and are rapidly cleared from blood plasma.
Extensive purification from pyrogens and toxins is essential for parenteral enzymes,
which increases the cost.
Enzyme therapy of cancer
In traditional medicine, proteolytic enzymes derived from plant extracts have been
used for a long time In addition to proteolytic enzymes from natural resources such
as plants, ‘modern’ enzyme therapy includes pancreatic enzymes.

the use of proteolytic enzymes is partly based on scientific reports and is partly
empirical. Clinical evidence of the use of proteolytic enzymes in cancer studies
has typically been obtained with an enzyme preparation comprising a combination
of papain, trypsin and chymotrypsin. Earlier reports proved that enzyme therapy
can reduce the adverse effects caused by radiotherapy and chemotherapy. There is
also a report available that, in some types of tumors, survival may be sustained. The
positive effects of systemic enzyme therapy appear to be based on its antiinflammatory
potential. Nevertheless, the exact mechanism of action of systemic
enzyme therapy remains unsolved.
A number of enzymes have been examined and evidenced as antitumor
agents. L-serine dehydratase, L-arginase, carboxypeptidase G (folate depletion),
L-asparaginase, L-methioninase, L-phenylalanine ammonia lyase, L-glutaminase,
L-tyrosinase and xanthine oxidase have been studied for their anticancer activity.
Enzyme preparations such as asparaginase (amidase), bromelain (protease) and
chymotrypsin (protease) have also been studied as cancer treatments
The prospects of enzyme-based treatment against cancer are very bright, but the
difficulties of antigenicity and short circulation time remain to be overcome.
Enzymes In Digestive Disorders And Inflammations
Enzymes play an essential role in the management of various digestive disorders,
such as exocrine pancreatic insufficiency. Supplementation with enzymes may
also be advantageous for other conditions associated with poor digestion, such as
lactose intolerance
Utilization of microbe-derived lipase has presented promise with reports
showing benefits alike to pancreatic enzymes, but with a lower dosage concentration
and a broader pH range. The safety and efficacy of enzymes derived from microbial
species in the treatment of conditions such as malabsorption and lactose intolerance
is promising. Plant-derived enzymes, e.g. bromelain from pineapple, serve as active
digestive aids in the breakdown of proteins.
Buccal administration of pancreatin (derived from an
alcoholic extract of animal pancreas) enhances the enzymatic digestion of starch
and proteins in patients with pancreatic cysts and pancreatitis. Pancreatin in combination
with lipase is used to treat patients with fatty stools. Hydrolytic enzymes such as
papain and fungal extracts (Aspergillus niger and Aspergillus otyzae) are used to
enhance absorption from the small intestine . These fungal extracts comprise
amylases and proteases along with cellulases, which support the breakdown of the
otherwise indigestible fibers of cabbages, etc, and thus reduce dyspepsia and flatulence