Enzyme
Immobilization
By: Bijaya Kumar Uprety
WHAT ARE ENZYMES AND WHAT DO THEY DO?
Enzymes are proteins with highly specialized catalytic
functions, produced by all living organisms. OR Enzymes
are proteins, which catalyse specific biochemical reactions in
a very efficient manner.
Enzymes are responsible for many essential biochemical
reactions in micro-organisms, plants, animals, and human
beings.
Although like all other proteins, enzymes are composed of
amino acids, they differ in function in that they have the
unique ability to facilitate biochemical reactions without
undergoing change themselves. This catalytic capability is
what makes enzymes unique.
Enzymes are natural protein molecules that act as highly efficient
catalysts.
Enzymes not only work efficiently and rapidly, they are also
biodegradable.
Enzymes are highly efficient in increasing the reaction rate of
biochemical processes that otherwise proceed very slowly, or in
some cases, not at all.
Enzymes are generally categorized according to the compounds
they act upon. Some of the most common enzymes include;
1. Proteases which break down proteins,
2. Cellulases which break down cellulose,
3. Lipases which split fats (lipids) into glycerol and fatty acids,
4. Amylase which break down starch into simple sugars.
However, Enzyme Commisions system of classification divided
enzymes into six groups.
Enzyme Class
Type of reaction catalysed
Example
Oxidoreducta
ses
Oxidation/reduction reactions- Catalyse (S)-lactate, isocitrate, D-amino acid,
the transfer of H atoms, or O atoms or
etc.
electrons from one substrate to another
Transferases
Transfer of an atom or group between
two molecules (excluding reactions in
other classes)
Methyltransferase,
hydroxymethyltransferase,
phosphotransferase, etc.
Hydrolases
Hydrolysis reactions
Alkaline phosphatase (catalyses
inorganic phosphate into organic
phosphatase)
Lyases
Removal of a group from substrate (not
by hydrolysis)
L-histidine carboxy-lyase catalyses
histidine into histamine.
Isomerase
Isomerization reactions
Alanine racemase which catalyses
L-alanine
D-alanine
Ligases
The synthetic joining of two molecules,
coupled with the breakdown of
pyrophosphate bond in a nucleoside
Acid-ammonia ligases, acid-amino
ligases, ammonia ligases catalyses
L-glutamte
L-glutamine
Uses of Enzyme
Enzymes play a diversified role in many aspects of everyday
life including aiding in digestion, the production of food and
several industrial application. Enzymes are natures catalyst.
1. Human body uses various enzymes to carry out various
biochemical processes. One of the best example of enzyme
based process is digestion. Enzymes help break down
carbohydrate, proteins and fats into simple compounds that
can be absorbed by the body. Amylase and lipase in saliva
breaks down carbohydrate and fats respectively. Protease
released in stomach helps in digestion of proteins & Lipase,
amylases, and proteases are secreted in small intestine and
play an important role in completing digestive process.
2. Food Production and Industrial Applications:
Since ancient times, enzymes have played an important part in
food production. One of the earliest examples of industrial
enzyme use was in the production of whiskey.
Today nearly all commercially prepared foods contain at least one
ingredients that has been made with enzymes. Some of the
typical applications include enzyme use in the production of
sweetners, chocolate, syrups, bakery products, alcoholic
beverages, precooked cereals, infants foods, vegetable oil and
puree, candy, spice and flavor extracts, and liquid coffee, as well
as for dough conditioning, flavor development and meat
tenderizing.
Enzymes also play a significant role in non-food applications.
Industrial enzymes are used in laundry and dishwashing
detergents, stonewashing jeans, pulp and paper manufacture,
leather dehairing and tanning, desizing of textiles, deinking of
paper, and degreasing of hides.
How enzymes are made?
Commercial sources of enzymes are obtained from
three primary sources i.e. animal tissue, plants and
microbes.
These naturally occurring enzymes are quite often not
readily available in sufficient quantities for food
applications or industrial use. However, by isolating
microbial strains that produce the desired enzyme and
optimizing the conditions for growth
commercial
quantities can be obtained. This technique is well
known for more than 3000 years and is known as
fermentation.
Today this fermentation process is carried out in a
contained vessel. Once fermentation is completed, the
microorganisms are destroyed, the enzymes are
isolated, and further processed for commercial use.
Enzyme manufacturers produce enzymes in accordance
with all applicable governmental regulations, including
the appropriate federal agencies (e.g. Food and Drug
Administration, United States Department of
Agriculture, Environmental Protection Agency, etc).
Regardless of source, enzymes intended for food use
are produced in strict adherence to FDAs current Good
Manufacturing
Practices
(cGMP)
and
meet
compositional and purity requirements as defined in
the Food Chemicals Codex ( a compendium of food
ingredients specifications developed in co-operation
with FDA).
Advantages of using enzymes
Enzymes can often replace chemicals or
processes that present safety or environmental
issues e.g. replacing acids in starch processing
and alkalis in fabric desizing, reduce use of sulfide
in tanneries, removing stains from fabrics (clothes
can be washed at lower temperature thus saving
energy).
Contribute to safer working condition by
eliminating the use of chemical treatments during
production processes.
Enzyme immobilization
Microbial enzymes are most extensively employed in the food and beverage
industries across the world to meet the increasing demand for nutritionally superb
and high-value products.
However, the predominant use of the enzymes in industrial environment has been
limited by the fact that large number of these enzymes are unstable and the cost
of isolation, purification and recovery of the active enzyme from the reaction
mixture is high.
In actual practice, the soluble enzymes engaged in batch operations is found to be
uneconomical as the active enzyme is virtually lost (not recovered) after each
viable reaction.
Therefore, in order to overcome such non-productive, economically not feasible,
and deleterious effects the enzymes have been ultimately immobilized and this
process of immobilization of enzyme is termed as enzyme immobilization.
Enzyme immobilization may be defined as confining the enzyme molecules to
a distinct phase from the one wherein the substrate and product are present.
An immobilized enzyme is an enzyme that is 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 allows enzymes to 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.
Standard defination of enzyme immobilization.
An immobilized enzyme is the one which has
been attached to or enclosed by an insoluble
support medium (termed as carrier) or one
where the enzyme molecules have been
cross-linked to each other, without loss of
catalytic activity.
Salient feature of enzyme immobilization
1.
2.
3.
4.
5.
Enzymes are more or less physically confined in the course of a
definite continuous catalytic process. They may be suitably
recovered from the reaction mixture and used over and over again
thereby gainfully improving the economic viability of the entire
process.
It may be accomplished by fixing the enzyme molecules to or
within certain appropriate substance.
It should be absolutely critical that both the substrate and the
products migrate quite freely in and out of the phase to which the
specific molecules are actually confined.
Certain enzymes which are thermolabile in nature could be made
heat-stable by attachment into inert polymeric supports.
These may be recycled, rapidly controlled, immobilized enzymes
may be recycled, rapidly controlled, operated continuously,
product (s) easily separable, above all the enzyme (ie stability,
pH) are altered favourably.
Carrier Matrices
The substances that are solely employed for the immobilization of enzymes are
known as carrier matrices e.g., inorganic materials (salts), and inert polymers.
An ideal carrier matrix has the following characteristic features, namely :
(a) cost effectiveness,
(b) inertness,
(c) reasonable physical strength,
(d) adequate stability,
(e) regenerability after the gainful lifespan of the immobilized enzyme,
(f) enhancement in specificity of enzyme,
(g) reduction in product inhibition,
(h) a possible shift in optimum pH for enzyme activity to the desired value for the
process, and
(i) appreciable reduction in non-specific adsorption and microbial contamination.
Method of immobilization
There are four distinct type of immobilization
methods.
Adsorption
Covalent bonding
Entrapment
Membrane confinement
1. Physical adsorption
Physical adsorption on to an inert carrier is a very simple procedure for
immobilizing an enzyme, for it requires just the mixing of the enzyme solution with
the carrier. Adsorption of an enzyme may be accomplished by allowing the contact
of the enzyme and the polymer support either by percolating the enzyme via a
packed bed, tube, membrane formed from a support material or in a stirred
bioreactor. Eventually the enzymes get adhered to the surface of carrier matrix.
In 1916, Nelson and Griffin showed the invertase could be adsorbed onto activated
charcoal without any change in enzymatic activity, thus producing the first
immobilized enzyme, although they made no subsequent use of it.
Other inorganic materials which could be used as carriers include clay, alumina and
silica.
The weak linkages established between enzyme and carrier (mainly Van der Waals
and hydrogen bonds) have little effect on catalytic activity.
However, because the bonds are so weak, the enzyme can easily be desorbed from
the carrier. This can be brought about by change in pH, ionic strength, or substrate
concentration.
Also, the adsorption process is non-specific, so many other substances will become
attached to the carrier as the immobilized enzyme is used.
Methodology
The actual methodology involved in the adsorption of
enzymes to the matrices is quite simple, easy, and
employed largely.
The appropriate enzyme is adequately mixed with a right
adsorbent usually under appropriate pH parameters as
well as the desired ionic strength After incubation
for a stipulated duration the carrier matrix is washed
thoroughly to get rid of the entire unabsorbed
enzyme molecules whereby the immobilized enzyme is
ready for actual usage. Interestingly, this
specific method invariably gives rise to a high loading
(nearly 1 g enzyme per g matrix) of the enzyme.
Covalent bonding
The enzyme molecules are attached to carrier matrix by the
formation of covalent bonds. Due to this the actual strength of
bondage happens to be quite strong, and hence no loss of enzymes
take place during its usage.
The formation of covalent bond usually takes place particularly with
the side chains of amino acids present in the enzyme. However,
their actual strength of reactivity being exclusively linked to the
status of charge present in them.
The various functional moities mostly present in enzyme which
actively take part in the formation of numerous viable chemical
bonds are sulphide, oxide, carboxyl, hydroxyl, amino, imidazole,
guanidyl, etc.
Methodology
The covalent bonding of an enzyme may be accomplished either by
activating the polymer with a reactive moiety or by effectively employing
the bifunctional reagent to serve as a bridge between the two entities:
enzyme and polymer.
Where 3-D network may be obtained by cross-linking with low molecular
weight bifunctional agent.
In doing so, the enzyme may get inactivated because the reactions
normally engage a functional moiety strategically located at the active site
of the enzyme. (In simple words, Immobilization by this method may lead
to ultimate loss in the extent of enzyme activity as the active site is
involved during the immobilization process.)
Thus the overall net effect being the substantial loss of enzymatic activity.
This loss of enzymatic activity could be overcome by carrying out enzyme
immobilization either in presence of competitive inhibitor or an enzyme
substrate.
Fig: Showing the various enzyme immobilized by Covalent bonding
Entrapment
2.3. Entrapment
Entrapment refers to the phenomenon whereby the enzyme
molecules are either held or entrapped within the appropriate
fibres or gels (most commonly used is polyacrylamide gel).
Entrapment may or may not be accomplished via covalent bonding
between enzyme and carrier matrix.
In case where covalent bonding is required the enzymes need to be
treated with synthetic reagents such as acryloyl chloride, cellulose
acetate, calcium alginate, etc.
Example: Cellulose acetate fibres find its application for enzyme
entrapment. Enzyme and cellulose acetate is blended together to
obtain an emulsion preferably in an organic solvent, methylene
chloride. The resulting emulsion is subjected to the process of
extrusion to obtain fibres into a solution of an aqueous precipitant.
Calcium alginate is the material of choice for the entrapment of
microbial, plant cells and animal cells.
In this method of immobilization, practically
negligible amount of loss of biological activity
of enzyme takes place compared to coupling
method as this method doesnt allow the
binding of enzyme itself either to the gel
matrix or the membrane.
Methodology
Methodology : The various steps involved in entrapment
are as below
(1) The enzyme(s) may be dissolved in a solution of the
polymers precursors.
(2) Polymers may be selected from a variety of materials e.g.,
natural gels (e.g., cellulose triacetate, alginate, agar,
gelatin) ; synthetic gels e.g., polyacrylamide gels.
(3) In order to check and prevent the possible leakage of the
low molecular weight enzymes from the body of the gel,
the average pore size of the gel must be maintained as
large as possible.
(4) Efforts should be geared into action to practically contain
two important aspects in entrapment process, namely :
(a) excessive diffusion limitation, and
(b) variability of pore size.
Example : Penicillin acylase represents the category of fibreentrapped enzymes that essentially affords immobilization via
entrapment in the microcavities of the synthetic fibres.
Liposome entrapment refers to the physical phenomenon whereby
entrapment may be accomplished by carrying out the dissolution of
a fibre forming polymer e.g., cellulose triacetate in an organic
solvent which being immiscible in aqueous medium, and
subsequently emulsifying the resulting solution with the aqueous
solution of enzyme carefully.
The emulsion thus obtained is extruded via a spinneret into liquid
coagulant (e.g., toluene, petroleum ether) which specifically
precipitate the polymer in its desired filamentous form having a
precise microdroplet of the enzyme solution meticulously
entrapped in the fibre. This technique, obviously possess two
remarkable advantages, namely :
(a) minimises the diffusion limitation, and
(b) relative surface to volume ratio is appreciably high.
Another way is to entrap enzyme (s) inside the
polymerised gel.
Enzyme containing gel is made in aqueous
medium this gel is polymerized enzymes
entrapped.
See picture.
Encapsulation/microencapsulation/membrane
confinement
In this method, enzyme molecules in aqueous medium are
confined within a semipermeable membrane that ideally
permits an almost absolute free movement of the enzymes
in either direction to the products and substrate but doesnt
allow the enzyme to escape from the confined membrane.
The enzyme immobilization prevailing in encapsulating
method predominatly occurs well within the microcapsules
(prepared from organic polymers in order that enzymes are
prevented from the great escape.
In addition , low molecular wt products and substrate can
either enter or leave the capsule by diffusion via membrane.
Two well known methods for preparing membranous
capsule for encapsulation.
1. Phase separation: membranes usually made by adopting
the process of phase-separation which resemble to the
homogenization of water in oil. In this instance, one phase
is not miscible with other giving rise to droplet within
which enzymes get entrapped.
1. Chemical polymerization: The chemical polymerisation
aids in the preparation of the specific water insoluble
membrane, and thus the enzyme in question get duly
entrapped during this on going phenomenon of
polymerization. E.g semipermeable nylon or collodion
in the shape of spheres are utilized for
microencapsulation of enzyme.
Advantage of enzyme immobilization
(1) Enzymes being quite expensive and also having the unique ability to be
used repeatedly only in a situation when these may be recovered
completely from the accomplished reaction mixtures. In true sense,
immobilization distinctly and specifically allows their repeated usage by
virtue of the fact that such enzyme preparation may be separated
conveniently from the reaction system involved.
(2) Importantly, the final desired product should be readily separated from
the enzyme. It goes a long way in affecting reduction and saving upon the
cost of downstream processing of the ensuing end-product.
(3) Non-aqueous systems (i.e., using organic solvents exclusively) are found to
be fairly compatible with the immobilized enzymes particularly, and this
may be regarded to be extremely desirable in certain typical and specific
instances.
4) Immobilized enzymes may be used predominently in most continuous
production systems ; and, of course, this not absolutely feasible and
possible with the free-enzymes.
(5) Immobilized enzymes, a few selected ones, may
exhibit thermostability of the highest order, viz., the
free-enzyme glucose isomerase usually gets denatured
only at 45C in solution ; however, when immobilized
suitably the enzyme is found to be stable enough upto
1 year at 65C.
(6) Importantly, the ultimate recovery of immobilized
enzyme would drastically minimise the high effluent
disposable problems (which is quite acute in several
fermentation industries).
(7) Immobilized enzymes may be employed at a much
higher concentration range in comparison to the
corresponding free enzyme.
Disadvantages of Enzyme Immobilization
Immobilized enzymes do offer several disadvantages which are briefly discussed in the section
that follows :
(1) Enzyme immobilization evidently gives rise to an additional bearing on cost. Hence, this
improved technique is got to be used only in such an event when there prevails a sound
economic viability, feasibility, safety, and above all a positive edge over the corresponding
soluble enzymes.
(2) Immobilization of enzymes invariably affects the stability and/or activity adversely. In
order to circumvent such typical instances one may have to adhere strictly to the laid down
developed immobilization protocols.
(3) Practical utilization of the immobilized enzymes may not prove to be of any use or advantage
when one of the substrates is found to be insoluble.
(4) Certain immobilization protocols do offer a good number of serious problems with respect to
the diffusion of the ensuing substrate to have an access to the corresponding enzyme.
Factors affecting enzyme kinetics
Enzyme kinetics refers to the indepth study of enzyme in action.
Kinetics is the study of reaction rates (velocities).
Study of enzyme kinetics is useful for measuring
concentration of an enzyme in a mixture (by its catalytic activity),
its purity (specific activity),
its catalytic efficiency and/or specificity for different substrates
comparison of different forms of the same enzyme in different tissues or
organisms,
effects of inhibitors (which can give information about catalytic mechanism, structure of active
site, potential therapeutic agents...)
Dependence of velocity on [substrate] is described for many enzymes by the MichaelisMenten equation:
Various factors affecting enzyme kinetics include:
pH
temperature
Substrate cocentration.
It has been duly observed that the rate of
reaction catalyzed by an enzyme particularly
enhances linearity with the corresponding
increase in the substrate concentration generally
upto a certain point.
However, it soon approaches the maximum
value, usually termed as Vmax ; and beyond
which there is absolutely no further
enhancement in the rate of reaction as shown in
figure in next slide. It is known as saturation.
On the other hand, the rate of a nonenzymatically catalyzed reaction that
enhances linearly very much across the entire range of attainable
substrate concentrations.
Importantly, the prevailing immobilization phenomenon does help in the
actual conversion of the catalyst from homogeneous (i.e., soluble enzyme)
nature to the heterogeneous one, whereby the enzyme is intimately
associated either with a particular enveloping matrix or a supporting
matrix. Nevertheless, in the course of immobilization phenomenon, the
activity of ensuing enzyme is virtually lost by virtue of two vital reasons,
namely : (a) various reactions involved in the process ; and (b) effective
occlusion of active sites in the enzyme support complex.
Examples :
(1) Hem-containing proteins : Haemoglobin : It has been observed that
haemoglobin gets bound to O2 ; and in doing so several O2-molecules may
bind and release during one minute, while at any material time only one
O2 molecule becomes intimately associated with one hem centre.
Enzyme Activity
Enzyme activity is the measure of the ability of an enzyme to catalyze a specific
reaction. Or Enzyme activity is the catalytic effect exerted by an enzyme,
expressed as units per milligram of enzyme (specific activity) or as molecules of
substrate transformed per minute per molecule of enzyme (molecular activity).
The rate of enzyme catalysed reaction is often called its velocity.
Enzyme velocity is commonly expressed by the initial rate (Vo) of the reaction
being catalyzed. The units of Vo is mol/min, which can be represented by the
enzyme unit( U) or the katal (kat),
1mol= 1U= 16.67 nanokat.
Experimentally V0 is measured before more than approximately 10% of the
substrate has been converted to product in order to minimize such complicating
factors. A typical plot of product formed against time for an enzyme-catalyzed
reaction shows an initial period of rapid product formation which gives the linear
portion of the plot (Fig. 1). This is followed by a slowing down of the enzyme rate
as substrate is used up and/or as the enzyme loses activity. V0 is obtained by
drawing a straight line through the linear part of the curve, starting at the zero
time-point (Fig. 1). The slope of this straight line is equal to V0.
Enzyme refers to an organic catalyst invariably produced by living cells but
capable of acting either outside cells or even in vitro.
Enzymes are proteins that change the rate of chemical reactions without needing
an external energy source or being changed themselves ; an enzymes may catalyze
a reaction numerous times.
Enzymes are highly reaction specific in that they act only on certain substances
usually known as substrates. Nevertheless, the enzyme and its substrate or
substrates invariably give rise to a temporary configuration, called an enzymesubstrate complex that essentially involves both physical shape and chemical
bonding.
The enzyme usually promotes the formation of bonds between separate substrates,
or induces the breaking of bonds in a single substrate to form the product or
products of reaction. The human body contains thousands of enzymes, each
catalyzing one of the many reactions that eventually occur as part of metabolism.
The term activity (or total activity) refers to the total units of enzyme in a
sample, whereas specific activity is the number of units per milligram of protein
(units mg-1)
Substrate concentration
The normal pattern of dependence of enzyme rate on substrate
concentration ([S]) is that at low substrate concentrations a
doubling of [S] will lead to a doubling of the initial velocity (V0).
However, at higher substrate concentrations the enzyme becomes
saturated, and further increases in [S] lead to very small changes
in V0. This occurs because at saturating substrate concentrations
effectively all of the enzyme molecules have bound substrate.
The overall enzyme rate is now dependent on the rate at which the
product can dissociate from the enzyme, and adding further
substrate will not affect this. The shape of the resulting graph when
V0 is plotted against [S] is called a hyperbolic curve.
Enzyme concentration
In
situations
where
the
substrate
concentration is saturating (i.e. all the enzyme
concentration molecules are bound to
substrate), a doubling of the enzyme
concentration will lead to a doubling of V0.
This gives a straight line graph when V0 is
plotted against enzyme concentration.
pH
Each enzyme has an optimum pH at which the rate of the reaction that it
catalyzes is at its maximum.
Small deviations in pH from the optimum value lead to decreased activity
due to changes in the ionization of groups at the active site of the enzyme.
Larger deviations in pH lead to the denaturation of the enzyme protein
itself, due to interference with the many weak noncovalent bonds
maintaining its three-dimensional structure.
A graph of V0 plotted against pH will usually give a bell shaped curve.
Many enzymes have a pH optimum of around 6.8, but there is great
diversity in the pH optima of enzymes, due to the different environments
in which they are adapted to work.
For example, the digestive enzyme pepsin is adapted to work at the acidic
pH of the stomach (around pH 2.0).
Michaelis-Menten Model
Significance of Km Values
The various important significance of Km values are as follows :
(1) Indicative of substrate concentration (S),
(2) Affinity of enzyme with corresponding substrate,
(3) Indicative partially of enzyme-substrate concentration prevailing in the
cellular compartment i.e., the target where most of the reaction invariably
takes place.
(4) Km-values are found to be inversely proportional to the ensuing affinity
of the enzyme for its substrate i.e., higher Km-values give rise to lower
stability of the enzyme substrate (ES)-complex apparently.
Determination of Km
Some important enzymes
Enzyme plays a significant role in biochemical reactions.
Few of the typical human ailments (persistent bodily
disorder or disease could be attributed to the partial
deficiency or complete absence of one or more than one
enzymes present in the tissue organs.
It has been amply observed that in certain extreme
abnormal conditions the unnatural and two much inherent
activity of a particular enzyme in vivo could be adequately
managed and controlled at times by a specific drug
substance designed to control as well as inhibit its overall
catalytic activity. [In simple words, in certain diseases
where
enzyme
activity
is
more
various enzyme specific drugs could be used to control the
activity of enzyme or to inhibit its activity
Enzymes are also required for carrying out various synthesis reactions in the
living body.
Synthesis of proteins, nucleic acids, phospholipids for cell membranes,
hormones, and glycogen all essentially need at least one if not many enzymes.
For instance : DNA polymerase Is extremely needed for carrying out the
phenomenon of DNA replication, that precedes mitosis. Even blood clotting,
the formation of angiotensins II to boost up blood pressure, and the transport
of CO2 in the blood also require specific enzymes.
Enzymes may be employed in the replacement therapy whereby either the
missing enzyme or malfunctioning of certain impaired organs are corrected
to prolong the life-expectency of patients. Numerous hereditary diseases are
caused due to lack of one or two enzymes in the body which give rise to the
accumulation of undesired, abnormal substances which otherwise would have
broken down in presence of enzyme. Eg. Fabrys disease i.e., an inherited
metabolic disease in which there is a galactosidase (responsible for
hydrolysing galactosides into monosaccharides) deficiency, which leads to
accumulation of glycosphinogolipids throughout the body.
Adenosine deaminase deficiency invariably causes severe combined
immunodeficiency (SCID) which would categorically respond to the
desired and much required enzyme replacement therapy, wherein
the said purified enzyme is first duly stabilized in polyethylene glycol
(PEG), and then administered parenterally.
Few of the vital and important enzymes are listed below:
(i) Hyaluronidase
(ii) Penicillinase
(iii) Streptokinase
(iv) Streptodornase
(v) Amylases
(vi) Proteases
Hyaluronidase
It is an enzyme found in the testes and semen. It depolymerizes hyaluronic
acid, thereby enhancing the permeability of connective tissues by
dissolving the substances that essentially hold body cells together.
It acts to disperse the cells of the corona radiata about the newly ovulated
ovum, thus facilitating entry of the sperm.
The enzyme accelerates specifically the subcutaneous spread of the
ensuing (resulting) particulate matter.
Hyaluronidase finds its abundant utility as a dispersion agent along with
the other injected drugs being employed as a therapeutic measure. It is
also used as a potential adjunct particularly in subcutaneous urography for
not only augmenting (intensifying) but also markedly improving the
resorption (lysis and assimilation process) of radiopaque agents.
Besides, it also helps in the enhancement of adsorption of drugs
particularly in transudates, tissue spaces, and oedemas.
Hyaluronidase For Injection [Wydase(R)] : It is obtained as a sterile dry,
soluble enzyme product obtained from the mammalian (bovine) testes and
capable of hydrolyzing mucopolysaccharides of the hyaluronic acid type.
It usually contains not more than 0.25 g of tyrosine for each Hyaluronidase
Unit.
Therapeutic Applications : The various therapeutic applications are as follows :
(1) By catalyzing the hydrolysis of hyaluronic acid, a constituent of
the extracellular matrix (ECM), hyaluronidase lowers the viscosity of
hyaluronic acid, thereby increasing tissue permeability. It is, therefore,
used in medicine in conjunction with other drugs to speed their dispersion
and delivery. Common applications are ophthalmic surgery, in combination
with local anesthetics.
(2) The most prominent clinical usage of hyaluronidase is to distinctly facilitate
the administration of fluids by the aid of hypodermoclysis.
Penicillinase
Penicillinase is a bacterial enzyme that invariably inactivates most but not
all penicillins.
This is regarded as an extracellular type enzyme produced adaptively by
members of the coliform group of bacteria, by most Bacillus species, and
certain strains of Staphylococcus. The enzyme exclusively carries out the
hydrolysis of penicillin to penicilloic acid i.e., a dicarboxylic acid as
depicted in figure.
It has been duly observed that a rather large segment of penicillinresistant pathogenic strains of Staphylococcus aureus invariably comprise
of this specific enzyme ; and perhaps it overwhelmingly
contributes a major factor of penicillin resistance during infection.
Importantly, this enzyme causes an extremely rapid degradation of
penicillin particularly in penicillin fermentations in case a specific
contaminant which produces the enzyme incidentally gains an access to
and be able to grow simultaneously in the fermentation broth.
Penicillinase obtained from B. subtilis and B. cereus represent the
industrially produced enzymes which exert action to some extent in the
removal of penicillin via specific inactivation.
Streptokinase
Streptokinase is a single-chain coenzyme obtained from
cultures of the Group C strain of Streptococcus haemolyticus
that is capable of solely converting plasminogen to plasmin.
It is used extensively as a predominant fibrinolytic agent to
help in a big way for the specific removal of fibrin thrombi
(blood clot) from arteries.
The Global utilization of Streptokinase and tPA (tissue type
plasmogen activator) of Occluded Arteries (GUSTO) trial was
designed meticulously to investigate and establish the
benefits of a front loaded dose of alteplase (a large initial
bolus followed by an infusion of the total dose over a span of
90 minutes) when compared with the usual conventional
alteplase administration.
Note: alteplase is the FDA approved recombinant tissue
plasminogen activator.
Besides, an intensive and extensive study was carried out with
respect to the safety and improvement in mortality of
combining alteplase and streptokinase with either agent
alone. Interestingly, the outcome of results from these
extended investigations appear to exhibit an appreciably
favourable mortality rate in patients having been treated with
alteplase comparison to the streptokinase treated subjects.
A mixture of streptokinase and streptodornase, as produced
by a hemolytic streptococcus grown in a specific environment
of aerated-submerged culture, is used meticulously to clean
up the debris from wounds and burns effectively.
Therapeutic Applications :
The therapeutic applications of streptokinase are as stated below :
(1) It is recommended for the management and control of myocardial infarction
(AMI) in adults to bring about various therapeutic benefits, such as :
specific lysis of intracoronary thrombi
improvement in ventricular function
remarkable reduction of mortality associated with AMI.
reduction of infarct size and congestive heart failure associated with AMI when
administered by the IV route.
(2) It is also indicated for the adequate lysis of objectively diagnosed pulmonary
emboli, involving obstruction of blood flow to a lobe or multiple segments,
with or without unstable haemodynamics.
(3) Besides, the drug is abundantly recommended for the lysis of objectively
diagnosed, acute, and extensive thrombi of the deep veins, emboli, and
arterial thrombi respectively.
(4) Individuals having quite recent streptococcal infections may possess an
appreciable quantum of circulating antisteptokinase antibodies ; and to
counteract this situation a loading dose sufficient to neutralize the prevailing
antibodies is required urgently.
Streptodornase
Streptodornase refers to one of the enzymes produced by
certain strains of haemolytic streptococci. It is capable of
liquefying fibrinous and purulent exudates.
It is also employed sometimes as a wound debridement
i.e., removal of damaged tissue.
Streptodornase is actually obtained from Streptococcus
haemolyticus that affords depolymerization of polymerized
deoxyribonucleoproteins.
It is used extensively in conjunction with streptokinase as
de-sloughing agent to cleanse ulcers and promote the
healing process progressively.
Definition
1.
Enzyme: Any of numerous proteins or conjugated proteins produced by
living organisms and functioning as biochemical catalysts.
2.
Apoenzyme: The protein component of an enzyme, to which the
coenzyme attaches to form an active enzyme.
3.
Co-enzyme: A nonproteinaceous organic substance that usually contains
a vitamin or mineral and combines with a specific protein, the
apoenzyme, to form an active enzyme system.
