Enzyme

Immobilization

By: Bijaya Kumar Uprety

WHAT ARE ENZYMES AND WHAT DO THEY DO?

Enzymes are proteins with highly specialized catalyticfunctions, produced by all living organisms. OR Enzymesare proteins, which catalyse specific biochemical reactions ina very efficient manner.

Enzymes are responsible for many essential biochemicalreactions in micro-organisms, plants, animals, and humanbeings.

Although like all other proteins, enzymes are composed ofamino acids, they differ in function in that they have theunique ability to facilitate biochemical reactions withoutundergoing change themselves. This catalytic capability iswhat makes enzymes unique.

Enzymes are natural protein molecules that act as highly efficient

catalysts.

Enzymes not only work efficiently and rapidly, they are alsobiodegradable.

Enzymes are highly efficient in increasing the reaction rate ofbiochemical processes that otherwise proceed very slowly, or insome cases, not at all.

Enzymes are generally categorized according to the compoundsthey 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 dividedenzymes into six groups.

Enzyme Class Type of reaction catalysed Example

Oxidoreductases

Oxidation/reduction reactions- Catalysethe transfer of H atoms, or O atoms or electrons from one substrate to another

(S)-lactate, isocitrate, D-amino acid, etc.

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 catalysesL-alanine D-alanine

Ligases The synthetic joining of two molecules, coupled with the breakdown of pyrophosphate bond in a nucleoside triphosphate

Acid-ammonia ligases, acid-aminoligases, 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 andseveral industrial application. Enzymes are natures catalyst.

1. Human body uses various enzymes to carry out variousbiochemical processes. One of the best example of enzymebased process is digestion. Enzymes help break downcarbohydrate, proteins and fats into simple compounds thatcan be absorbed by the body. Amylase and lipase in salivabreaks down carbohydrate and fats respectively. Proteasereleased in stomach helps in digestion of proteins & Lipase,amylases, and proteases are secreted in small intestine andplay 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 industrialenzyme use was in the production of whiskey.

Today nearly all commercially prepared foods contain at least oneingredients that has been made with enzymes. Some of thetypical applications include enzyme use in the production ofsweetners, chocolate, syrups, bakery products, alcoholicbeverages, precooked cereals, infants foods, vegetable oil andpuree, candy, spice and flavor extracts, and liquid coffee, as wellas for dough conditioning, flavor development and meattenderizing.

Enzymes also play a significant role in non-food applications.Industrial enzymes are used in laundry and dishwashingdetergents, stonewashing jeans, pulp and paper manufacture,leather dehairing and tanning, desizing of textiles, deinking ofpaper, and degreasing of hides.

How enzymes are made? Commercial sources of enzymes are obtained from

three primary sources i.e. animal tissue, plants andmicrobes.

These naturally occurring enzymes are quite often notreadily available in sufficient quantities for foodapplications or industrial use. However, by isolatingmicrobial strains that produce the desired enzyme andoptimizing the conditions for growth commercialquantities can be obtained. This technique is wellknown for more than 3000 years and is known asfermentation.

Today this fermentation process is carried out in acontained vessel. Once fermentation is completed, themicroorganisms are destroyed, the enzymes areisolated, and further processed for commercial use.

Enzyme manufacturers produce enzymes in accordancewith all applicable governmental regulations, includingthe appropriate federal agencies (e.g. Food and DrugAdministration, United States Department ofAgriculture, Environmental Protection Agency, etc).

Regardless of source, enzymes intended for food useare produced in strict adherence to FDAs current GoodManufacturing Practices (cGMP) and meetcompositional and purity requirements as defined inthe Food Chemicals Codex ( a compendium of foodingredients specifications developed in co-operationwith FDA).

Advantages of using enzymes Enzymes can often replace chemicals or

processes that present safety or environmentalissues e.g. replacing acids in starch processingand alkalis in fabric desizing, reduce use of sulfidein tanneries, removing stains from fabrics (clothescan be washed at lower temperature thus savingenergy).

Contribute to safer working condition byeliminating the use of chemical treatments duringproduction 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 superband high-value products.

However, the predominant use of the enzymes in industrial environment has beenlimited by the fact that large number of these enzymes are unstable and the costof isolation, purification and recovery of the active enzyme from the reactionmixture is high.

In actual practice, the soluble enzymes engaged in batch operations is found to beuneconomical as the active enzyme is virtually lost (not recovered) after eachviable reaction.

Therefore, in order to overcome such non-productive, economically not feasible,and deleterious effects the enzymes have been ultimately immobilized and thisprocess of immobilization of enzyme is termed as enzyme immobilization.

Enzyme immobilization may be defined as confining the enzyme molecules toa distinct phase from the one wherein the substrate and product are present.

An immobilized enzyme is an enzyme that is attachedto an inert, insoluble material such as calcium alginate(produced by reacting a mixture of sodiumalginate solution and enzyme solution with calciumchloride).

This can provide increased resistance to changes inconditions such as pH or temperature.

It also allows enzymes to be held in place throughoutthe reaction, following which they are easily separatedfrom the products and may be used again - a far moreefficient process and so is widely used in industryfor enzyme catalysed reactions. An alternative toenzyme immobilization is whole cell immobilization.

Standard defination of enzyme immobilization.

An immobilized enzyme is the one which hasbeen attached to or enclosed by an insolublesupport medium (termed as carrier) or onewhere the enzyme molecules have beencross-linked to each other, without loss ofcatalytic activity.

Salient feature of enzyme immobilization1. Enzymes are more or less physically confined in the course of a

definite continuous catalytic process. They may be suitablyrecovered from the reaction mixture and used over and over againthereby gainfully improving the economic viability of the entireprocess.

2. It may be accomplished by fixing the enzyme molecules to orwithin certain appropriate substance.

3. It should be absolutely critical that both the substrate and theproducts migrate quite freely in and out of the phase to which thespecific molecules are actually confined.

4. Certain enzymes which are thermolabile in nature could be madeheat-stable by attachment into inert polymeric supports.

5. These may be recycled, rapidly controlled, immobilized enzymesmay 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 areknown 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 theprocess, 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 withthe carrier. Adsorption of an enzyme may be accomplished by allowing the contactof the enzyme and the polymer support either by percolating the enzyme via apacked bed, tube, membrane formed from a support material or in a stirredbioreactor. Eventually the enzymes get adhered to the surface of carrier matrix.

In 1916, Nelson and Griffin showed the invertase could be adsorbed onto activatedcharcoal without any change in enzymatic activity, thus producing the firstimmobilized enzyme, although they made no subsequent use of it.

Other inorganic materials which could be used as carriers include clay, alumina andsilica.

The weak linkages established between enzyme and carrier (mainly Van der Waalsand hydrogen bonds) have little effect on catalytic activity.

However, because the bonds are so weak, the enzyme can easily be desorbed fromthe carrier. This can be brought about by change in pH, ionic strength, or substrateconcentration.

Also, the adsorption process is non-specific, so many other substances will becomeattached to the carrier as the immobilized enzyme is used.

Methodology

The actual methodology involved in the adsorption ofenzymes to the matrices is quite simple, easy, andemployed largely.

The appropriate enzyme is adequately mixed with a rightadsorbent usually under appropriate pH parameters aswell as the desired ionic strength After incubationfor a stipulated duration the carrier matrix is washedthoroughly to get rid of the entire unabsorbedenzyme molecules whereby the immobilized enzyme isready for actual usage. Interestingly, thisspecific 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 theformation of covalent bonds. Due to this the actual strength ofbondage happens to be quite strong, and hence no loss of enzymestake place during its usage.

The formation of covalent bond usually takes place particularly withthe side chains of amino acids present in the enzyme. However,their actual strength of reactivity being exclusively linked to thestatus of charge present in them.

The various functional moities mostly present in enzyme whichactively take part in the formation of numerous viable chemicalbonds 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 employingthe 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 molecularweight bifunctional agent.

In doing so, the enzyme may get inactivated because the reactionsnormally engage a functional moiety strategically located at the active siteof the enzyme. (In simple words, Immobilization by this method may leadto ultimate loss in the extent of enzyme activity as the active site isinvolved 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 enzymeimmobilization either in presence of competitive inhibitor or an enzymesubstrate.

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 appropriatefibres or gels (most commonly used is polyacrylamide gel).

Entrapment may or may not be accomplished via covalent bondingbetween enzyme and carrier matrix.

In case where covalent bonding is required the enzymes need to betreated with synthetic reagents such as acryloyl chloride, celluloseacetate, calcium alginate, etc.

Example: Cellulose acetate fibres find its application for enzymeentrapment. Enzyme and cellulose acetate is blended together toobtain an emulsion preferably in an organic solvent, methylenechloride. The resulting emulsion is subjected to the process ofextrusion to obtain fibres into a solution of an aqueous precipitant.Calcium alginate is the material of choice for the entrapment ofmicrobial, plant cells and animal cells.

In this method of immobilization, practicallynegligible amount of loss of biological activityof enzyme takes place compared to couplingmethod as this method doesnt allow thebinding of enzyme itself either to the gelmatrix or the membrane.

MethodologyMethodology : 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 thelow molecular weight enzymes from the body of the gel,the average pore size of the gel must be maintained aslarge as possible.

(4) Efforts should be geared into action to practically containtwo important aspects in entrapment process, namely :

(a) excessive diffusion limitation, and(b) variability of pore size.

Example : Penicillin acylase represents the category of fibre-entrapped enzymes that essentially affords immobilization viaentrapment in the microcavities of the synthetic fibres.

Liposome entrapment refers to the physical phenomenon wherebyentrapment may be accomplished by carrying out the dissolution ofa fibre forming polymer e.g., cellulose triacetate in an organicsolvent which being immiscible in aqueous medium, andsubsequently emulsifying the resulting solution with the aqueoussolution of enzyme carefully.

The emulsion thus obtained is extruded via a spinneret into liquidcoagulant (e.g., toluene, petroleum ether) which specificallyprecipitate the polymer in its desired filamentous form having aprecise microdroplet of the enzyme solution meticulouslyentrapped in the fibre. This technique, obviously possess tworemarkable 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 areconfined within a semipermeable membrane that ideallypermits an almost absolute free movement of the enzymesin either direction to the products and substrate but doesntallow the enzyme to escape from the confined membrane.

The enzyme immobilization prevailing in encapsulatingmethod predominatly occurs well within the microcapsules(prepared from organic polymers in order that enzymes areprevented from the great escape.

In addition , low molecular wt products and substrate caneither enter or leave the capsule by diffusion via membrane.

Two well known methods for preparing membranouscapsule for encapsulation.

1. Phase separation: membranes usually made by adoptingthe process of phase-separation which resemble to thehomogenization of water in oil. In this instance, one phaseis not miscible with other giving rise to droplet withinwhich enzymes get entrapped.

1. Chemical polymerization: The chemical polymerisationaids in the preparation of the specific water insolublemembrane, and thus the enzyme in question get dulyentrapped during this on going phenomenon ofpolymerization. E.g semipermeable nylon or collodionin the shape of spheres are utilized formicroencapsulation of enzyme.

Advantage of enzyme immobilization

(1) Enzymes being quite expensive and also having the unique ability to beused repeatedly only in a situation when these may be recoveredcompletely from the accomplished reaction mixtures. In true sense,immobilization distinctly and specifically allows their repeated usage byvirtue of the fact that such enzyme preparation may be separatedconveniently from the reaction system involved.

(2) Importantly, the final desired product should be readily separated fromthe enzyme. It goes a long way in affecting reduction and saving upon thecost of downstream processing of the ensuing end-product.

(3) Non-aqueous systems (i.e., using organic solvents exclusively) are found tobe fairly compatible with the immobilized enzymes particularly, and thismay be regarded to be extremely desirable in certain typical and specificinstances.

4) Immobilized enzymes may be used predominently in most continuousproduction systems ; and, of course, this not absolutely feasible andpossible with the free-enzymes.

(5) Immobilized enzymes, a few selected ones, mayexhibit thermostability of the highest order, viz., thefree-enzyme glucose isomerase usually gets denaturedonly at 45C in solution ; however, when immobilizedsuitably the enzyme is found to be stable enough upto1 year at 65C.

(6) Importantly, the ultimate recovery of immobilizedenzyme would drastically minimise the high effluentdisposable problems (which is quite acute in severalfermentation industries).

(7) Immobilized enzymes may be employed at a muchhigher concentration range in comparison to thecorresponding free enzyme.

Disadvantages of Enzyme ImmobilizationImmobilized 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 Michaelis-Menten equation:

Various factors affecting enzyme kinetics include:

pH

temperature

Substrate cocentration.

It has been duly observed that the rate ofreaction catalyzed by an enzyme particularlyenhances linearity with the correspondingincrease in the substrate concentration generallyupto a certain point.

However, it soon approaches the maximumvalue, usually termed as Vmax ; and beyondwhich there is absolutely no furtherenhancement in the rate of reaction as shown infigure in next slide. It is known as saturation.

On the other hand, the rate of a nonenzymatically catalyzed reaction thatenhances linearly very much across the entire range of attainablesubstrate concentrations.

Importantly, the prevailing immobilization phenomenon does help in theactual conversion of the catalyst from homogeneous (i.e., soluble enzyme)nature to the heterogeneous one, whereby the enzyme is intimatelyassociated either with a particular enveloping matrix or a supportingmatrix. Nevertheless, in the course of immobilization phenomenon, theactivity of ensuing enzyme is virtually lost by virtue of two vital reasons,namely : (a) various reactions involved in the process ; and (b) effectiveocclusion of active sites in the enzyme support complex.

Examples :

(1) Hem-containing proteins : Haemoglobin : It has been observed thathaemoglobin gets bound to O2 ; and in doing so several O2-molecules maybind and release during one minute, while at any material time only oneO2 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 ofsubstrate 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 reactionbeing catalyzed. The units of Vo is mol/min, which can be represented by theenzyme unit( U) or the katal (kat),1mol= 1U= 16.67 nanokat.

Experimentally V0 is measured before more than approximately 10% of thesubstrate has been converted to product in order to minimize such complicatingfactors. A typical plot of product formed against time for an enzyme-catalyzedreaction shows an initial period of rapid product formation which gives the linearportion of the plot (Fig. 1). This is followed by a slowing down of the enzyme rateas substrate is used up and/or as the enzyme loses activity. V0 is obtained bydrawing a straight line through the linear part of the curve, starting at the zerotime-point (Fig. 1). The slope of this straight line is equal to V0.

Enzyme refers to an organic catalyst invariably produced by living cells butcapable of acting either outside cells or even in vitro.

Enzymes are proteins that change the rate of chemical reactions without needingan external energy source or being changed themselves ; an enzymes may catalyzea reaction numerous times.

Enzymes are highly reaction specific in that they act only on certain substancesusually known as substrates. Nevertheless, the enzyme and its substrate orsubstrates invariably give rise to a temporary configuration, called an enzyme-substrate complex that essentially involves both physical shape and chemicalbonding.

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 orproducts of reaction. The human body contains thousands of enzymes, eachcatalyzing 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 asample, 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 adoubling of [S] will lead to a doubling of the initial velocity (V0).

However, at higher substrate concentrations the enzyme becomessaturated, and further increases in [S] lead to very small changesin V0. This occurs because at saturating substrate concentrationseffectively all of the enzyme molecules have bound substrate.

The overall enzyme rate is now dependent on the rate at which theproduct can dissociate from the enzyme, and adding furthersubstrate will not affect this. The shape of the resulting graph whenV0 is plotted against [S] is called a hyperbolic curve.

Enzyme concentration In situations where the substrate

concentration is saturating (i.e. all the enzymeconcentration molecules are bound tosubstrate), a doubling of the enzymeconcentration will lead to a doubling of V0.

This gives a straight line graph when V0 isplotted 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 activitydue 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 proteinitself, due to interference with the many weak noncovalent bondsmaintaining 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 greatdiversity in the pH optima of enzymes, due to the different environmentsin which they are adapted to work.

For example, the digestive enzyme pepsin is adapted to work at the acidicpH 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 thecellular compartment i.e., the target where most of the reaction invariablytakes place.

(4) Km-values are found to be inversely proportional to the ensuing affinityof the enzyme for its substrate i.e., higher Km-values give rise to lowerstability 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 bodilydisorder or disease could be attributed to the partialdeficiency or complete absence of one or more than oneenzymes present in the tissue organs.

It has been amply observed that in certain extremeabnormal conditions the unnatural and two much inherentactivity of a particular enzyme in vivo could be adequatelymanaged and controlled at times by a specific drugsubstance designed to control as well as inhibit its overallcatalytic activity. [In simple words, in certain diseaseswhere enzyme activity is morevarious enzyme specific drugs could be used to control theactivity of enzyme or to inhibit its activity

Enzymes are also required for carrying out various synthesis reactions in theliving 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 thephenomenon of DNA replication, that precedes mitosis. Even blood clotting,the formation of angiotensins II to boost up blood pressure, and the transportof CO2 in the blood also require specific enzymes.

Enzymes may be employed in the replacement therapy whereby either themissing enzyme or malfunctioning of certain impaired organs are correctedto prolong the life-expectency of patients. Numerous hereditary diseases arecaused due to lack of one or two enzymes in the body which give rise to theaccumulation of undesired, abnormal substances which otherwise would havebroken down in presence of enzyme. Eg. Fabrys disease i.e., an inheritedmetabolic disease in which there is a galactosidase (responsible forhydrolysing galactosides into monosaccharides) deficiency, which leads toaccumulation of glycosphinogolipids throughout the body.

Adenosine deaminase deficiency invariably causes severe combinedimmunodeficiency (SCID) which would categorically respond to thedesired 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 bydissolving the substances that essentially hold body cells together.

It acts to disperse the cells of the corona radiata about the newly ovulatedovum, thus facilitating entry of the sperm.

The enzyme accelerates specifically the subcutaneous spread of theensuing (resulting) particulate matter.

Hyaluronidase finds its abundant utility as a dispersion agent along withthe other injected drugs being employed as a therapeutic measure. It isalso used as a potential adjunct particularly in subcutaneous urography fornot only augmenting (intensifying) but also markedly improving theresorption (lysis and assimilation process) of radiopaque agents.

Besides, it also helps in the enhancement of adsorption of drugsparticularly 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 bymembers of the coliform group of bacteria, by most Bacillus species, andcertain strains of Staphylococcus. The enzyme exclusively carries out thehydrolysis of penicillin to penicilloic acid i.e., a dicarboxylic acid asdepicted in figure.

It has been duly observed that a rather large segment of penicillin-resistant pathogenic strains of Staphylococcus aureus invariably compriseof this specific enzyme ; and perhaps it overwhelminglycontributes a major factor of penicillin resistance during infection.

Importantly, this enzyme causes an extremely rapid degradation ofpenicillin particularly in penicillin fermentations in case a specificcontaminant which produces the enzyme incidentally gains an access toand be able to grow simultaneously in the fermentation broth.

Penicillinase obtained from B. subtilis and B. cereus represent theindustrially produced enzymes which exert action to some extent in theremoval of penicillin via specific inactivation.

Streptokinase Streptokinase is a single-chain coenzyme obtained from

cultures of the Group C strain of Streptococcus haemolyticusthat is capable of solely converting plasminogen to plasmin.

It is used extensively as a predominant fibrinolytic agent tohelp in a big way for the specific removal of fibrin thrombi(blood clot) from arteries.

The Global utilization of Streptokinase and tPA (tissue typeplasmogen activator) of Occluded Arteries (GUSTO) trial wasdesigned meticulously to investigate and establish thebenefits of a front loaded dose of alteplase (a large initialbolus followed by an infusion of the total dose over a span of90 minutes) when compared with the usual conventionalalteplase administration.

Note: alteplase is the FDA approved recombinant tissueplasminogen activator.

Besides, an intensive and extensive study was carried out withrespect to the safety and improvement in mortality ofcombining alteplase and streptokinase with either agentalone. Interestingly, the outcome of results from theseextended investigations appear to exhibit an appreciablyfavourable mortality rate in patients having been treated withalteplase comparison to the streptokinase treated subjects.

A mixture of streptokinase and streptodornase, as producedby a hemolytic streptococcus grown in a specific environmentof aerated-submerged culture, is used meticulously to cleanup 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 pulmonaryemboli, 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 objectivelydiagnosed, acute, and extensive thrombi of the deep veins, emboli, andarterial thrombi respectively.

(4) Individuals having quite recent streptococcal infections may possess anappreciable quantum of circulating antisteptokinase antibodies ; and tocounteract this situation a loading dose sufficient to neutralize the prevailingantibodies is required urgently.

Streptodornase Streptodornase refers to one of the enzymes produced by

certain strains of haemolytic streptococci. It is capable ofliquefying fibrinous and purulent exudates.

It is also employed sometimes as a wound debridementi.e., removal of damaged tissue.

Streptodornase is actually obtained from Streptococcushaemolyticus that affords depolymerization of polymerizeddeoxyribonucleoproteins.

It is used extensively in conjunction with streptokinase asde-sloughing agent to cleanse ulcers and promote thehealing 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 containsa vitamin or mineral and combines with a specific protein, theapoenzyme, to form an active enzyme system.