Enzymes are produced by cellular anabolism, the naturally occurring biological
process of making more complex molecules from simpler ones. Source organisms
include bacteria, fungi, higher plants and animals (White
and White, 1997). Enzymes may be extracted from a given source organism
by a number of different methods (Nielsen et al.,
1996). Most of the organisms that produce commercial enzymes are fungi.
These organisms are molds Rhizopus oryzae, Aspergillus niger,
Rhizomucor meihei, blights such as Endothia parasitica and yeasts
such as Saccharomyces and Candida sp.
In cells and organisms most reactions are catalyzed by enzymes, which are regenerated
during the course of a reaction. These biological catalysts are physiologically
important because they speed up the rates of reactions that would otherwise
be too slow to support life (Porcelli et al., 2010).
Enzymes increase reaction rates, sometimes by as much as one million-fold, but
more typically by about one thousand fold. Catalysts speed up the forward and
reverse reactions proportionately so that, although the magnitude of the rate
constants of the forward and reverse reactions is are increased, the ratio of
the rate constants remains the same in the presence or absence of enzyme (Qijun
et al., 2010). Since the equilibrium constant is equal to a ratio
of rate constants, it is apparent that enzymes and other catalysts have no effect
on the equilibrium constant of the reactions they catalyze.
Enzymes increase reaction rates by decreasing the amount of energy required
to form a complex of reactants that is competent to produce reaction products.
This complex is known as the activated state or transition state complex for
the reaction. Enzymes and other catalysts accelerate reactions by lowering the
energy of the transition state. The free energy required to form an activated
complex is much lower in the catalyzed reaction (Jiang et
al., 2007). The amount of energy required to achieve the transition
state is lowered; consequently, at any instant a greater proportion of the molecules
in the population can achieve the transition state. The result is that the reaction
rate is increased.
While it is clear that enzymes are responsible for the catalysis of almost
all biochemical reactions, it is important to also recognize that rarely, if
ever, do enzymatic reactions proceed in isolation. The most common scenario
is that enzymes catalyze individual steps of multi-step metabolic pathways,
as is the case with glycolysis, gluconeogenesis or the synthesis of fatty acids.
As a consequence of these lock-step sequences of reactions, any given enzyme
is dependent on the activity of preceding reaction steps for its substrate (Abir
et al., 2005). In humans, substrate concentration is dependent on
food supply and is not usually a physiologically important mechanism for the
routine regulation of enzyme activity. Enzyme concentration, by contrast, is
continually modulated in response to physiological needs. Three principal mechanisms
are known to regulate the concentration of active enzyme in tissues: (1) Regulation
of gene expression controls the quantity and rate of enzyme synthesis. (2) Proteolytic
enzyme activity determines the rate of enzyme degradation. (3) Covalent modification
of preexisting pools of inactive proenzymes produces active enzymes.
Enzyme synthesis and proteolytic degradation are comparatively slow mechanisms
for regulating enzyme concentration (Griffith et al.,
2004) with response times of hours, days or even weeks. Proenzyme activation
is a more rapid method of increasing enzyme activity but, as a regulatory mechanism,
it has the disadvantage of not being a reversible process. Proenzymes are generally
synthesized in abundance, stored in secretory granules and covalently activated
upon release from their storage sites. Examples of important proenzymes include
pepsinogen, trypsinogen and chymotrypsinogen, which give rise to the proteolytic
digestive enzymes. Likewise, many of the proteins involved in the cascade of
chemical reactions responsible for blood clotting are synthesized as proenzymes.
Other important proteins, such as peptide hormones and collagen, are also derived
by covalent modification of precursors.
Another mechanism of regulating enzyme activity is to sequester enzymes in compartments where access to their substrates is limited. For example, sequestering these enzymes within the lysosome controls the proteolysis of cell proteins and glycolipids by enzymes responsible for their degradation.
In contrast to regulatory mechanisms that alter enzyme concentration, there
is an important group of regulatory mechanisms that do not affect enzyme concentration,
are reversible and rapid in action and actually carry out most of the moment
to moment physiological regulation of enzyme activity. These mechanisms include
allosteric regulation, regulation by reversible covalent modification and regulation
by control proteins such as calmodulin. Reversible covalent modification is
a major mechanism for the rapid and transient regulation of enzyme activity.
The best examples, again, come from studies on the regulation of glycogen metabolism
where phosphorylation of glycogen synthase and glycogen phosphorylase kinase
results in the stimulation of glycogen degradation while glycogen synthesis
is coordinately inhibited. Numerous other enzymes of intermediary metabolism
are affected by phosphorylation, either positively or negatively (Shinoda
and Itoyama, 2003). These covalent phosphorylations can be reversed by a
separate sub-subclass of enzymes known as phosphatases. Recent research has
indicated that the aberrant phosphorylation of growth factor and hormone receptors,
as well as of proteins that regulate cell division, often leads to unregulated
cell growth or cancer. The usual sites for phosphate addition to proteins are
the serine, threonine and tyrosine R group hydroxyl residues.
CLASSIFICATION OF ENZYME AND CO ENZYMES
This is a special commission of the International Union of Biochemistry (IUB)
that made recommendations for the classification and naming of enzymes and for
the definitions of the mathematical constants used in enzymology. The recommendations
were first published in 1964 and were published in revised form in 1972, 1978
and 1984. This is the systematic arrangement and the naming of enzymes that
is based on the 1972 recommendations of the Enzyme Commission of the International
Union of Biochemistry (Stenesh, 1989). Reactions and the
enzymes that catalyze them form 6 classes, each having 4-13 subclasses. The
enzyme name has 2 parts. The first Soetan et al. 383 names the substrate
or substrates. The second, ending in-ase, indicates the type of reaction catalysed.
A number composed of four figures denotes each enzyme. The first figure denotes
one of the six main divisions: oxidoreductases, transferases, hydrolases, lyases,
isomerases and ligases. The second figure denotes the subclass and the third
figure denotes the sub-subclass. The last figure denotes the serial number of
the enzyme in its sub-subclass. The enzyme number is preceded by the abbreviation
E.C. For example, E.C.126.96.36.199 denotes class 2 (a transferase), subclass 7 (transfer
of phosphate), subsubclass 1 (an alcohol is the phosphate acceptor). The final
digit denotes hexokinase, or ATP: D-hexose 6 -Phos-photransferase, an enzyme
catalyzing phosphate transfer from ATP to the hydroxyl group on carbon 6 of
glucose. There are 6 classes of enzymes as follows:
Oxidoreductases: These enzymes are involved in oxidations and reductions of their substrates e.g., alcohol dehydrogenase, lactate dehydrogenase, xanthine oxidase, glutathione reductase, glucose-6-phosphate dehydrogenase.
Transferases: These enzymes catalyze the transfer of a particular group from one substrate to another e.g., aspartate amino transferase (AST), alanine aminotransferase (ALT), hexokinase, phosphoglucomutase, hexose- 1-phosphate uridyltransferase, ornithine carbamoyl transferase etc.
Hydrolases: These enzymes bring about hydrolysis e.g., glucose- 6 -phosphatase, pepsin, trypsin, esterases, glycoside hydrolases etc.
Lyases: These are enzymes that facilitate the removal of small molecule from a large substrate e.g., fumarase, argino succinase, histidine decarboxylase.
Isomerases: These enzymes are involved in isomerization of substrate e.g., UDP-glucose, epimerase, retinal isomerase, racemases, triose phosphate isomerase.
Ligases: These enzymes are involved in joining together 2 substrates e.g., alanyl-t-RNA synthetase, glutamine synthetase, DNA ligases.
Many enzymes require a coenzyme which functions as group transfer reagents. Many enzymes that catalyze group transfer and other reactions require, in addition to their substrate, a second organic molecule known as a coenzyme, without which they are inactive. Coenzymes expand the repertoire of the catalytic capabilities of an enzyme far beyond those offered by the functional groups alone of the amino acids that constitute the bulk of the enzyme. Coenzymes that are tightly associated with an enzyme through either covalent bonding or non covalent forces are often referred to as prosthetic groups. Coenzymes that are freely diffusible generally serve as continually recycled carriers of hydrogen, flavin adenine dinucleotide (reduced) (FADH), hydride nicotinamide adenine dinucleotide (reduced) (NADH) and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), or chemical units such as acyl groups (coenzyme A) or methyl groups (folates), shuttling them between their points of generation and consumption. These latter coenzymes can thus be considered as second substrates. Enzymes that require coenzymes include those which catalyze oxidoreductions, group transfer and isomerization reactions and reactions that form covalent bonds (IUB classes 1, 2, 5 and 6). Lytic reactions, including the hydrolytic reactions catalyzed by the digestive enzymes, do not require coenzymes.
Coenzymes can be classified according to the group whose transfer they facilitate
based on the above concept, coenzymes may be classified as follows: Coenzymes
involved in transfer of groups other than hydrogen: Biotin, CoA-SH, cobamide
(B12) coenzymes, folate coenzymes, pyridoxal phosphate, lipoic acid, sugar phosphates,
thiamine pyrophosphate and Coenzymes involved in transfer of hydrogen. Nicotinamide
adenine dinucleotide (oxidized) (NAD+), Nicotinamide adenine dinucleotide phosphate
(oxidized) (NADP+). Flavin mononucleotide (FMN), Flavin Adenine Dinucleotide
(FAD), lipoic acid, coenzyme Q (Murray et al., 2000).
ENZYME APPLICATION IN MICROBIAL TECHNOLOGY
There are many enzymes employed in biotechnological operations. These include:
SI Endonuclease: This enzyme is isolated from Aspergilus cryzae and it acts exclusively on ssDNA or RNA. It can break supercoiled DNA because it contains ss bubbles. It can also be used to distinguish supercoiled from both non-supercoiled, covalent circles and nicked circular DNA, both of which are resistant to the enzyme.
Restriction endonucleases: Restriction endonucleases are enzymes that cut DNA molecules at specific positions. It recognizes foreign (unmodified) DNA at a specific site, and degrades it by internal cleavage. Most commonly used are the type II restriction enzymes, which cut within the recognition site. Some examples are EcoRI from E. coli, HindIII from Haemophilus influenzae, BamHI from Bacillus amyloliquefaciens, PstI from Providencia stuartii, SmaI from Serratia marcescens, Sau3A from Staphylococcus aureus, AluI from Arthrobacter luteus, TagI from Thermus aquaticus and HpaII from Haemophilus parainfluenza.
DNA polymerase I: It is primarily known as a repair polymerase, which fills in single-stranded gaps. It is also involved in repair of the gaps formed on the lagging strand during replication. It also possesses both 5 I --- 3 I and 3 I --- 5 I exonuclease activity.
Klenow fragment of E. coli DNA polymerase 1: This enzyme is used for sequencing DNA using the Sanger Dideoxy System, filling the 3 recessed termini of restriction enzyme treated DNA and also used for labeling the termini of DNA fragments. The enzyme is also used for second strand cDNA synthesis in the cDNA procedures. Klenow fragment is a proteolytic cleavage of DNA polymerase I. It leaves a fragment that is devoid of 5 I ---3 I exonuclease activity.
DNA polymerase III: This is the main replication polymerase.
Helicase: It unwinds DNA, for example in conjugal plasmid transfer
S1 nuclease: This enzyme degrades single-stranded DNA
T4 DNA polymerase: This enzyme is used in the labeling of DNA fragments for use as hybridization probes.
RNA Polymerase: This enzyme synthesizes RNA, using a DNA template.
Primase: This is a special RNA polymerase, which makes a short primer required for DNA synthesis.
Replicase: This is RNA-directed RNA polymerase used in replication of some RNA viruses.
Ribonuclease (RNase): This enzyme degrades RNA molecules.
Rnase H: This is a specific RNAse which cuts RNA--- DNA hybrids. It is involved in replication of Col EI-like plasmid
Exonuclease: This is an enzyme that removes nucleotides from the ends
of DNA fragments. A 5I ----3I exonuclease removes nucleotides
from the 5I end, while a 3I ----5I exonuclease
removes nucleotides from the 3Iend. This enzyme recognizes the terminal 5
I -phosphate of dsDNA for its exonuclease activity. Its primary use is
the removal of protruding 5I terminus from dsDNA which is needed
for the terminal transferase tailing of DNA (Daini, 2000).
Exonuclease III: This enzyme is used for generating linear template DNA for the dideoxysequencing technique and generating staggered ends on dsDNA due to its 3I ---- 5I exonuclease activity.
Ligases: It seals single-stranded gaps (nicks) in double-stranded DNA. It is also used for the formation of recombinant DNA molecules in gene cloning. T4 DNA Ligase: This enzyme catalysis the formation of a Phosphodiester bond between 3I-OH and 5I- phosphate ends in DNA using DNA molecules with cohesive ends as substrate.
Alkaline phosphatase: In gene cloning, this enzyme is used to remove phosphate groups from the 5I end of DNA molecules and also used as a reporter gene for identification of secretion signals.
Polynucleotide kinase: It transfers a phosphate group from ATP to the 5I OH end of DNA or RNA. T4 Polynucleotide kinase is an enzyme is isolated from T4 infected E. coli and catalyzes the transfer of-phosphate of ATP to a 5I -OH end in DNA or RNA. It is also used for the labeling of 5I termini of DNA for Maxam and Gilbert DNA sequencing and the phosphorylation of DNA lacking 5I P termini.
Reverse transcriptase: This is RNA-directed DNA polymerase. It synthesizes DNA (complementary DNA) using mRNA template. For example Reverse transcriptase is an enzyme coded for by avian myeloblastosis virus which catalyses the synthesis of cDNA from an RNA template. It can also be used for the labeling of termini of DNA with extended 5I ends.
Topoisomerase: This is a class of enzymes that alters the conformation of DNA, for example by changing the degree of winding or super coiling.
Transposase: This enzyme catalysis the initial steps in transpositions.
Terminal transferase: This adds nucleotides to the 3I end of DNA, without requiring a template strand.
Terminal deoxynucleotidyl transferase: This enzyme is isolated from calf thymus and catalyses the addition of dNTP to the 3 I -OH of DNA molecules. One of the primary uses of terminal transferase is the tailing of vectors and cDNA with complementary bases, thus permitting the cloning of the cDNA fragments. It can also be used for labeling of 3I ends of DNA fragments.
Since the second half of the last century, numerous efforts have been devoted
to the development of insoluble immobilized enzymes for a variety of applications
these applications can clearly benefit from use of the immobilized enzymes rather
than the soluble counterparts, for instance as reusable heterogeneous biocatalysts,
with the aim of reducing production costs by efficient recycling and control
of the process (Reetz et al., 2000) as stable
and reusable devices for analytical and medical applications as selective adsorbents
for purification of proteins and enzymes as fundamental tools for solid-phase
protein chemistry and as effective micro devices for controlled release of protein
drugs (Fig. 1).
This is an enzyme that is physically confined while it carries out its catalytic
function. This may occur naturally, as in the case of particulate enzymes, or
itmay be produced artificially by chemical or by physical methods (Stenesh,
||Range of application of immobilized enzymes
In the chemical methods, the enzyme is linked covalently to a support. These
methods include attachment of the enzyme to a water-insoluble support, incorporation
of the enzyme into a growing polymer chain, or cross linking of the enzyme with
a multifunctional low molecular weight reagent (Van Roon
et al., 2002). In the physical methods, the enzyme is not linked
covalently to a support. These methods include adsorption of the enzyme to a
water-insoluble matrix, entrapment of the enzyme within either a water-insoluble
gel or a microcapsule, or containment of the enzyme within special devices equipped
with semi permeable membrane (Soares et al., 2001)
. Expensive enzymes can be recovered and used again. The enzyme can also be
used in a variety of configurations of bioreactors that permit continuous operation
ENZYME IMMUNE SYSTEM
Enzymes become part of the immune system (Alpha-2-macroglobulin), working with it and facilitating its function. The modulation of growth factor binding properties of alpha 2-macroglobulin by enzyme therapy. Ingestion of proteinases was found to trigger the formation of intermediate forms of alpha-2- macroglobulin displaying high affinity of transforming growth factor- beta (TGF-β). They observed maximum binding of TGF- β 1 - 2 h after bolus ingestion and steadily leveled off with time. They concluded that intestinal absorption of proteinase triggers the formation of TGF-β binding species of alpha 2 macroglobulin in blood mediated by this process of high concentrations of TGF- β might be reduced via enhanced clearance of alpha 2-macroglobulin TGF- β complexes. Thus, proteinase therapy may have beneficial effects in treatment of fibrosis and certain cancers accompanied by excessively high TGF- β concentrations. The oral therapy with proteolytic enzymes decreases excessive TGF-beta levels in human blood. In the study, they stated that therapy with oral proteolytic enzymes (OET) with combination drug products containing papain, bromelain, trypsin and chymotrypsin have been shown to be beneficial in clinical settings such as radiotherapyinduced fibrosis, bleomycin pneumotoxicity and immunosuppression in cancer, all of which are nowadays knowned to be accompanied by excessive transforming growth factor-beta (TGF- β) production. They concluded that their results support the concept that OET is beneficial in diseases characterized in part by TGF- β1 overproduction.
ENZYMES AS MARKERS FOR DISEASE
Some enzymes are found only in specific tissues or in a limited number of such
tissues. For example, lactase dehydrogenase (LDH) has 2 different forms, called
isozymes, in heart and skeletal muscle. Two forms differ slightly in amino acid
composition and Can be separated on the basis of charge as a result. Since LDH
is a tetramer of four subunits, it too can exist in 5 different forms depending
on the source of the subunits. An increase of any form of LDH in the blood indicates
some kind of tissue damage. A heart attack can usually be diagnosed with certainty
if there is an increase of LDH from heart. Also, there are different forms of
Creatine Kinase (CK), an enzyme that occurs in the brain, heart and skeletal
muscle. Appearance of the brain type can indicate a stroke or a brain tumour,
whereas the heart type indicates a heart attack. After a heart attack, CK shows
up more rapidly in the blood than LDH. Monitoring the presence of both enzymes
extends the possibility of diagnosis, which is useful, since a very mild heart
attack might be difficult to diagnose. An elevated level of the isozyme from
heart in blood is a definite indication of damage to the heart tissue (Drolet
et al., 2007). Another useful enzyme assayed is acetyl cholinesterase
(AChE), which is important in controlling certain nerve impulses. Many pesticides
affect this enzyme, so farm workers are often tested to be sure that they have
not received inappropriate exposure to these important agricultural toxins.
There are several enzymes that are typically used in the clinical laboratory
to diagnose diseases. There are highly specific markers for enzymes active in
the pancrease, red blood cells, liver, heart, brain, prostate gland and many
of the endocrine glands. Since these enzymes are relatively easy to assay using
automated techniques, they are part of the standard blood test veterinary and
medical doctors are likely to need in the diagnosis and treatment/management
ENZYME AS DRUG OR ANTIBIOTICS
New antibiotics that are active against resistant bacteria (Raja
et al., 2010) have lived on earth for several billion years. During
this time, they encountered in nature a wide range naturally occurring antibiotics
or drugs. To survive, bacteria developed antibiotic resistance mechanism (Hoskeri
et al., 2010). Enzymes as drugs have two important features that
distinguish them from all other types of drugs. First, enzymes often bind and
act on their targets with great affinity and specificity. Second, enzymes are
catalytic and convert multiple target molecules to the desired products. These
two features make enzymes specific and potent drugs that can accomplish therapeutic
biochemistry in the body that small molecules cannot. These characteristics
have resulted in the development of many enzyme drugs for a wide range of disorders
ENZYMES IN THE DIAGNOSIS OF PATHOLOGY
The measurement of the serum levels of numerous enzymes has been shown to be
of diagnostic significance. This is because the presence of these enzymes in
the serum indicates that tissue or cellular damage has occurred resulting in
the release of intracellular components into the blood (Machetti
et al., 1998). Hence, when a physician indicates that he/she is going
to assay for liver enzymes, the purpose is to ascertain the potential for liver
cell damage. Commonly assayed enzymes are the amino transferases: alanine transaminase,
ALT (sometimes still referred to as serum glutamate-pyruvate aminotransferase,
SGPT) and aspartate aminotransferase, AST (also referred to as serum glutamate-oxaloacetate
aminotransferase, SGOT); lactate dehydrogenase, LDH; creatine kinase, CK (also
called creatine phosphokinase, CPK); gamma-glutamyl transpeptidase, GGT. Other
enzymes are assayed under a variety of different clinical situations. Many enzymes
are involved in the clinical diagnoses of various diseases in human and veterinary
medicine (Nielsen et al., 1996). These enzymes
facilitate or enhance rapid diagnoses of these diseases. These enzymes could
be classified into many classes. They are:
Alkaline phosphatase: Alkaline phosphatases were the earliest serum
enzymes to be recognized to have clinical significance, when in the 1920s, it
was discovered that they increase in bone and liver diseases . Since then, they
have been the subject of more publications than any other enzyme (Lone
et al., 2003). Alkaline phosphates are a group of isoforms which
hydrolyse many types of phosphate esters, whose natural substrate or substrates
are unknown. The term >alkaline= refers to the optimal alkaline
pH of this class of phosphatases in vitro. In both humans and animals,
the major sources of ALPs are the liver, bone, kidney and placenta. In humans,
it is involved in bone and hepatobiliary diseases. ALPs are also of diagnostic
importance in animal diseases. Total serum ALP activity has diagnostic value
in the hepatic and bone diseases in dogs and cats. It is of little value in
hepatic diseases of horses and ruminants because of the broad range of reference
values against which the patients values must be compared. The range of
serum ALP value in goats may be 10-fold with no evidence of hepatic damage.
Values within the individual are fairly constant for sequential evaluation.
Creatine kinase: Creatine kinase isozymes are the most organ-specific
serum enzymes in clinical use. They catalyse the reversible phosphorylation
of creatine by ATP to form creatine phosphate, the major storage form of high-energy
phosphate required by muscle. Creatine kinases are found in many parts of the
body like the heart, brain, skeletal muscle and smooth muscle but they have
their highest specific activity in the skeletal muscle (Aksenova
et al., 2000) . In humans, Creatine kinase is associated with myocardial
infarction and muscle diseases. Increase in Creatine kinase in cerebrospinal
fluid has been associated with a number of disorders in dogs, cats, cattle and
horses. The Creatine kinase are such sensitive indicators of muscle damage that,
generally, only large increases in serum activity are of clinical significance.
Alanine aminotransferase: It was formerly known as Glutamic Puruvate
Transaminase; (GPT). It catalyses the reversible transamination of Lalanine
and 2-oxoglutarate to pyruvate and glutamate in the cytoplasm of the cell. ALT
can be found in the liver, skeletal muscle and heart. The greatest specific
activity of ALT in primates, dogs, cats, rabbits and rats is in the liver. It
is a well established, sensitive liver-specific indicator of damage. However,
ALT in the tissues of pigs, horses, cattle, sheep or goats is too low to be
of diagnostic value ( Kikuchi et al., 1999).
It is used as an indicator of hepatopathy in toxicological studies which use
small laboratory rodents as well as dogs.
Aspartate aminotransferase: It wasformerly called Glutamic Oxaloacetic
Transaminase; (GOT). It catalyses the transamination of L-aspartate and 2-oxoglutarate
to oxaloacetate and glutamate. AST is found in skeletal muscle, heart, liver,
kidney and erythrocytes and is associated with myocardial, hepatic parenchymal
and muscle diseases in humans and animals. The pre-sence of AST in so many tissues
make their serum level a good marker of soft tissue but precludes its use as
an organspecific enzyme (Bittinger et al., 2003).
Red blood cells contain a large amount of AST which leaks into plasma before
haemolysis is seen.
Sorbitol dehydrogenase (SDH): It is also called L-iditol dehydrogenase;
(IDH). It catalyses the reversible oxidation of D-sorbitol to D-fructose with
the cofactor NAD. The plasma activity is low in dog and horse plasma but appreciably
greater in cattle, sheep, and goat serum. Aside from the testes, it is found
in appreciable amounts only in hepatocytes (El-Kabbani et
al., 2004). As a result of this, an increase in plasma SDH is consistent
with hepatocyte damage. SDH is liver specific in humans and all species of animals
and hepatic injury appears to be the only source of increased SDH activity.
Although SDH is liver specific in all species, the already established usage
of ALT in dogs and cats has limited SDH as a diagnostic indicator of hepatocellular
damage to horses, cattle, sheep and goats.
Lactate dehydrogenases(LDH): It catalyses the reversible oxidation of
pyruvate to L(+) lactate with the cofactor NAD. The equilibrium favours lactate
formation, but the preferred assay method is in the direction of pyruvate because
pyruvate has an inhibitory effect on LDH. Lactate dehydrogenase has isoenzymes.
LDH can be found in the heart, liver, erythrocyte, skeletal muscle, platelets
and lymph nodes. In humans, it is involved in myocardial infarction, haemolysis
and liver disease. LDH isoenzyme profiles were the first isoenzyme profiles
used in clinical veterinary medicine in an attempt to detect specific organ
damage. The introduction of more highly organ-specific procedures has resulted
in LDH no longer being in common use in veterinary medicine (Murray
et al., 2000)
Cholinesterase (ChE): Serum cholinesterase (ChE) activity is composed
of two distinct cholinesterases. The major substrate is acetylcholine, the neurotransmitter
found at the myoneural junction. Acetylcholinesterase (AChE; EC 188.8.131.52) found
at the myoneural junction is the true ChE and is essential in hydrolyzing acetylcholine
so that the junction can be reestablished and prepared for additional signals
(Ellis, 2005). The myoneural junction AChE is also found
in Red Blood Cells (RBC), mouse, pig, brain and rat liver. Only a small amount
of AChE is found in plasma. The ChE of plasma is a pseudocholine sterase, butylcholinesterase
(ButChE; EC 184.108.40.206), which hydrolyses butyrylcholine four times faster than
acetylcholine and is also located in white matter of the brain, liver, pancrease
and intestinal mucosa . Decreases in ButChE have been reported in humans with
acute infection, muscular dystrophy, chronic renal disease and pregnancy, as
well as insecticide intoxification.
Lipase: Serum pancreatic lipases (EC 220.127.116.11; triacylglycerol lipase)
catalyse the hydrolysis of triglycerides preferentially at the 1 and 3 positions,
releasing two fatty acids and a 2-monoglyceride. Lipase can be found in the
pancrease and hepatobiliary tract and is involved in pancreatitis and hepatobiliary
disease (Nduka, 1999).
Amylase: Amylases are calcium-dependent metalloenzymes that randomly
catalyze the hydrolysis of complex carbohydrates, e.g., glycogen at the -1-4
linkages. The products of this action are maltose and limit dextrins. The enzyme
is a Ca2+ metalloenzyme which requires one of a number
of activator ions such as Cl-or Br-. Amylase can be found in the salivary glands,
pancrease and ovaries and is used as a diagnostic aid for pancreatitis (Gupta
et al., 2001).
Glutamyltransferase: This is a carboxypeptidase which cleaves C-terminal
glutamyl groups and transfers them to peptides and other suitable acceptors.
It is speculated that GGT is associated with glutathione metabolism (Kaneko,
1989). The major sources are the liver and kidney and are involved in hepatobiliary
disease and alcoholism. Cholestatic disorders of all species examined result
in increased serum GGT activity.
Trypsin: Trypsins are serum proteases which hydrolyse the peptide bonds formed by lysine or arginine with other amino acids. The pancreas as the zymogen trypsinogen, which is converted to tyrosine by intestinal enterokinase or trypsin itself, secretes them.
Glutathione peroxidases: These are metalloenzymes containing four atoms
of selenium per molecule of enzyme. They catalyze the oxidation of reduced glutathione
by peroxide to form water and oxidized glutathione. Because of the high concentration
of selenium in glutathione peroxidases, there is a good direct correlation between
the amount of red blood cell GPx activity and the selenium concentration of
other organs (Chatterjea and Shinde, 2002). Other enzymes
with disease diagnosis applications are acid phosphatase (ACP), found in prostate
and erythrocytes and are used in diagnosis of prostate carcinoma. Aldolase (ALD),
found in skeletal muscle and heart and involved in muscle disease. Glutamate
dehydrogenase (GLDH), found in the liver is used to diagnose hepatic parenchymal
disease. Hydroxybutyrate dehydrogenase (HBD), which is the heart form of lactate
dehydrogenase is involved in myocardial infarctionJust as enzyme assay is used
to diagnose diseases in humans and animals, it may also be applied to the investigation
of diseases in plants. For example, it has been found that an injury (either
mechanical or pathogenic) results in a marked, localized increase in the activity
of glucose-6-phosphate dehydrogenase, but not of glucose phosphate isomerase,
indicating diversion of glucose breakdown from glycolysis to the pentose phosphate
ENZYMES USED IN IMMUNOASSAYS
Enzymes may also be used as an alternative to radioisotopes as markers in immunoassays have been used for the determination of a variety of proteins and hormones. The role of enzymes in immunoassay used to replace radioisotopes as markers, since they are not hazardous to health and can be detected by techniques which are more generally available. Any enzyme with a sensitive and convenient assay procedure can be used for this purpose. Two common examples of enzyme immunoassay (EIA) procedures are enzyme-linked immunosorbent assay (ELISA) and Enzyme-Multiplied Immunoassay Test (EMIT). ELISA is a highly sensitive assay that can be used to detect either antigen or antibody. Applications of ELISA include diagnostics for noninfectious diseases involving hormones, drugs, serum components, oncofetal proteins, or autoimmune diseases, as well as diagnostics for infectious diseases caused by bacterial, viral, mycotic or parasitic organisms. The enzymes frequently used in ELISA are Horseradish peroxidase, alkaline phosphatase and βgalactosidase. In EMIT, the activity of malate dehydrogenase is assayed by standard enzyme methodology for the detection of thyroxine by Enzyme-labelled immunoassay.
ENZYMES ACTED AS THERAPEUTIC AGENTS
In a few cases enzymes have been used as drugs in the therapy of specific medical
problems (Devlin, 1986). Streptokinase is an enzyme mixture
prepared from streptococcus. It is usefull in clearing blood clots that occur
in the lower extremities. Streptokinase activates the fibrinolytic proenzyme
plasminogen that is normally present in plasma. The activated enzyme is plasmin
is a serine protease like trypsin that attacks fibrin, cleaving it into several
soluble components. Another enzyme of therapeutic importance is asparaginase.
Asparaginase therapy is used for some types of adult leukemia. Tumor cells have
a nutritional requirement for asparagine and must scavenge it from the hosts
plasma. By administering asparaginase i.e., the hosts plasma level of
asparagine is markedly depressed, which results in depressing the viability
of the tumor. Enzyme replacement in individuals that are genetically deficient
in a particular enzyme are also applications of enzymes as therapeutic agents.
Also, enzymes such as u-plasminogen activator, formerly known as urokinase,
extracted from human urine, can be infused into the blood stream of patients
at risk from a pulmonary embolism (a fragment of a blood-clot lodging in the
pulmonary artery): these enzymes stimulate a cascade system responsible for
the production of active plasmin, a proteolytic enzyme which digests fibrin,
the main structural component of blood-clots. Some enzymes may also be used
to restrict the growth of cancer cells by depriving them of essential nutrients:
for example, Lasaparaginase may be used in the treatment of several types of
leukaemia, since the tumour cells, in contrast to normal cells, have a requirement
for exogenous Lasaparagine. Another example of therapeutic application of enzymes
is the use of immobilized enzymes as components of artificial kidney machines,
which are used to remove urea and other waste products from the body, where
kidney disease prevents this being done by natural processes (Palmer,
2001). Urea enters the machine from the blood, by dialysis (termed haemodialysis)
and is converted to CO2 and NH4+ by immobilized
urease; toxic NH4+ is then either trapped on ion exchange
resins or incorporated into glutamate by the action of immobilized glutamate
dehydrogenase linked to alcohol dehydrogenase to ensure coenzyme recycling,
before the fluid is returned to the blood stream.
We thank the Department of Microbiology, Jamal Mohamed College (Autonomous), Tiruchirappalli-620 020 for supporting and fulfilling all the needs to carry out this work.