Enzyme Immobilization is a process where enzymes are attached to an insoluble carrier or support to facilitate their reuse. There are several advantages to immobilizing enzymes including increased stability, continuous processability, and easier product separation. Common immobilization methods include adsorption, covalent binding, entrapment, and membrane confinement. Adsorption involves weak physical binding of enzymes to a carrier, while covalent binding uses chemical bonds to strongly attach enzymes. Entrapment traps enzymes within a gel or fiber matrix. Immobilized enzymes have various applications in food production, industrial processes, and biotechnology.

1. Enzyme Immobilization
By: Bijaya Kumar Uprety

2. WHAT ARE ENZYMES AND WHAT DO THEY DO?
•Enzymesareproteinswithhighlyspecializedcatalyticfunctions,producedbyalllivingorganisms.OR“Enzymesareproteins,whichcatalysespecificbiochemicalreactionsinaveryefficientmanner.”
•Enzymesareresponsibleformanyessentialbiochemicalreactionsinmicro-organisms,plants,animals,andhumanbeings.
•Althoughlikeallotherproteins,enzymesarecomposedofaminoacids,theydifferinfunctioninthattheyhavetheuniqueabilitytofacilitatebiochemicalreactionswithoutundergoingchangethemselves.Thiscatalyticcapabilityiswhatmakesenzymesunique.

3. •Enzymesarenaturalproteinmoleculesthatactashighlyefficientcatalysts.
•Enzymesnotonlyworkefficientlyandrapidly,theyarealsobiodegradable.
•Enzymesarehighlyefficientinincreasingthereactionrateofbiochemicalprocessesthatotherwiseproceedveryslowly,orinsomecases,notatall.
•Enzymesaregenerallycategorizedaccordingtothecompoundstheyactupon.Someofthemostcommonenzymesinclude;
1.Proteaseswhichbreakdownproteins,
2.Cellulaseswhichbreakdowncellulose,
3.Lipaseswhichsplitfats(lipids)intoglycerolandfattyacids,
4.Amylasewhichbreakdownstarchintosimplesugars.
•However,EnzymeCommision’ssystemofclassificationdividedenzymesintosixgroups.

4. Enzyme Class
Type of reaction catalysed
Example
Oxidoreductases
Oxidation/reductionreactions-Catalysethe transfer of H atoms, or O atoms or electrons from one substrate to another
(S)-lactate, isocitrate, D-amino acid, etc.
Transferases
Transferof an atom or group between two molecules (excluding reactions in other classes)
Methyltransferase, hydroxymethyltransferase, phosphotransferase, etc.
Hydrolases
Hydrolysisreactions
Alkalinephosphatase(catalyses inorganic phosphate into organic phosphatase)
Lyases
Removal of a group from substrate (not by hydrolysis)
L-histidinecarboxy-lyasecatalyses histidineinto histamine.
Isomerase
Isomerizationreactions
Alanineracemasewhich catalyses
L-alanineD-alanine
Ligases
The synthetic joining of two molecules, coupledwith the breakdown of pyrophosphate bond in a nucleoside triphosphate
Acid-ammonia ligases, acid-aminoligases, ammonia ligasescatalyses
L-glutamteL-glutamine

5. Uses of Enzyme
•Enzymesplayadiversifiedroleinmanyaspectsofeverydaylifeincludingaidingindigestion,theproductionoffoodandseveralindustrialapplication.Enzymesarenature’scatalyst.
1.Humanbodyusesvariousenzymestocarryoutvariousbiochemicalprocesses.Oneofthebestexampleofenzymebasedprocessisdigestion.Enzymeshelpbreakdowncarbohydrate,proteinsandfatsintosimplecompoundsthatcanbeabsorbedbythebody.Amylaseandlipaseinsalivabreaksdowncarbohydrateandfatsrespectively.Proteasereleasedinstomachhelpsindigestionofproteins&Lipase, amylases,andproteasesaresecretedinsmallintestineandplayanimportantroleincompletingdigestiveprocess.

6. 2. Food Production and Industrial Applications:
•Sinceancienttimes,enzymeshaveplayedanimportantpartinfoodproduction.Oneoftheearliestexamplesofindustrialenzymeusewasintheproductionofwhiskey.
•Todaynearlyallcommerciallypreparedfoodscontainatleastoneingredientsthathasbeenmadewithenzymes.Someofthetypicalapplicationsincludeenzymeuseintheproductionofsweetners,chocolate,syrups,bakeryproducts,alcoholicbeverages,precookedcereals,infantsfoods,vegetableoilandpuree,candy,spiceandflavorextracts,andliquidcoffee,aswellasfordoughconditioning,flavordevelopmentandmeattenderizing.
•Enzymesalsoplayasignificantroleinnon-foodapplications. Industrialenzymesareusedinlaundryanddishwashingdetergents,stonewashingjeans,pulpandpapermanufacture, leatherdehairingandtanning,desizingoftextiles,deinkingofpaper,anddegreasingofhides.

7. How enzymes are made?
•Commercialsourcesofenzymesareobtainedfromthreeprimarysourcesi.e.animaltissue,plantsandmicrobes.
•Thesenaturallyoccurringenzymesarequiteoftennotreadilyavailableinsufficientquantitiesforfoodapplicationsorindustrialuse.However,byisolatingmicrobialstrainsthatproducethedesiredenzymeandoptimizingtheconditionsforgrowthcommercialquantitiescanbeobtained.Thistechniqueiswellknownformorethan3000yearsandisknownasfermentation.
•Todaythisfermentationprocessiscarriedoutinacontainedvessel.Oncefermentationiscompleted,themicroorganismsaredestroyed,theenzymesareisolated,andfurtherprocessedforcommercialuse.

8. •Enzymemanufacturersproduceenzymesinaccordancewithallapplicablegovernmentalregulations,includingtheappropriatefederalagencies(e.g.FoodandDrugAdministration,UnitedStatesDepartmentofAgriculture,EnvironmentalProtectionAgency,etc).
•Regardlessofsource,enzymesintendedforfooduseareproducedinstrictadherencetoFDA’scurrentGoodManufacturingPractices(cGMP)andmeetcompositionalandpurityrequirementsasdefinedintheFoodChemicalsCodex(acompendiumoffoodingredientsspecificationsdevelopedinco-operationwithFDA).

9. Advantages of using enzymes
•Enzymescanoftenreplacechemicalsorprocessesthatpresentsafetyorenvironmentalissuese.g.replacingacidsinstarchprocessingandalkalisinfabricdesizing,reduceuseofsulfideintanneries,removingstainsfromfabrics(clothescanbewashedatlowertemperaturethussavingenergy).
•Contributetosaferworkingconditionbyeliminatingtheuseofchemicaltreatmentsduringproductionprocesses.

10. Enzyme immobilization
•Microbialenzymesaremostextensivelyemployedinthefoodandbeverageindustriesacrosstheworldtomeettheincreasingdemandfornutritionallysuperbandhigh-valueproducts.
•However,thepredominantuseoftheenzymesinindustrialenvironmenthasbeenlimitedbythefactthatlargenumberoftheseenzymesareunstableandthecostofisolation,purificationandrecoveryoftheactiveenzymefromthereactionmixtureishigh.
•Inactualpractice,thesolubleenzymesengagedinbatchoperationsisfoundtobeuneconomicalastheactiveenzymeisvirtuallylost(notrecovered)aftereachviablereaction.
•Therefore,inordertoovercomesuchnon-productive,economicallynotfeasible, anddeleteriouseffectstheenzymeshavebeenultimatelyimmobilizedandthisprocessofimmobilizationofenzymeistermedasenzymeimmobilization.
“Enzymeimmobilizationmaybedefinedasconfiningtheenzymemoleculestoadistinctphasefromtheonewhereinthesubstrateandproductarepresent.”

11. •Animmobilizedenzymeisanenzymethatisattachedtoaninert,insolublematerialsuchascalciumalginate(producedbyreactingamixtureofsodiumalginatesolutionandenzymesolutionwithcalciumchloride).
•ThiscanprovideincreasedresistancetochangesinconditionssuchaspHortemperature.
•Italsoallowsenzymestobeheldinplacethroughoutthereaction,followingwhichtheyareeasilyseparatedfromtheproductsandmaybeusedagain-afarmoreefficientprocessandsoiswidelyusedinindustryforenzymecatalysedreactions.Analternativetoenzymeimmobilizationiswholecellimmobilization.

12. Standard defination of enzyme immobilization.
•Animmobilizedenzymeistheonewhichhasbeenattachedtoorenclosedbyaninsolublesupportmedium(termedascarrier)oronewheretheenzymemoleculeshavebeencross-linkedtoeachother,withoutlossofcatalyticactivity.

13. Salient feature of enzyme immobilization
1.Enzymesaremoreorlessphysicallyconfinedinthecourseofadefinitecontinuouscatalyticprocess.Theymaybesuitablyrecoveredfromthereactionmixtureandusedoverandoveragaintherebygainfullyimprovingtheeconomicviabilityoftheentire process.
2.Itmaybeaccomplishedbyfixingtheenzymemoleculestoorwithincertainappropriatesubstance.
3.Itshouldbeabsolutelycriticalthatboththesubstrateandtheproductsmigratequitefreelyinandoutofthephasetowhichthespecificmoleculesareactuallyconfined.
4.Certainenzymeswhicharethermolabileinnaturecouldbemadeheat-stablebyattachmentintoinertpolymericsupports.
5.Thesemayberecycled,rapidlycontrolled,immobilizedenzymesmayberecycled,rapidlycontrolled,operatedcontinuously, product(s)easilyseparable,abovealltheenzyme(iestability, pH)arealteredfavourably.

14. Carrier Matrices
•Thesubstancesthataresolelyemployedfortheimmobilizationofenzymesareknownascarriermatricese.g.,inorganicmaterials(salts),andinertpolymers.
•Anidealcarriermatrixhasthefollowingcharacteristicfeatures,namely:
(a)costeffectiveness,
(b)inertness,
(c)reasonablephysicalstrength,
(d)adequatestability,
(e)regenerabilityafterthegainfullifespanoftheimmobilizedenzyme,
(f)enhancementinspecificityofenzyme,
(g)reductioninproductinhibition,
(h)apossibleshiftinoptimumpHforenzymeactivitytothedesiredvaluefortheprocess,and
(i)appreciablereductioninnon-specificadsorptionandmicrobialcontamination.

15. Method of immobilization
•There are four distinct type of immobilization methods.
Adsorption
Covalent bonding
Entrapment
Membrane confinement

16. 1. Physical adsorption
•Physicaladsorptionontoaninertcarrierisaverysimpleprocedureforimmobilizinganenzyme,foritrequiresjustthemixingoftheenzymesolutionwiththecarrier.Adsorptionofanenzymemaybeaccomplishedbyallowingthecontactoftheenzymeandthepolymersupporteitherbypercolatingtheenzymeviaapackedbed,tube,membraneformedfromasupportmaterialorinastirredbioreactor.Eventuallytheenzymesgetadheredtothesurfaceofcarriermatrix.
•In1916,NelsonandGriffinshowedtheinvertasecouldbeadsorbedontoactivatedcharcoalwithoutanychangeinenzymaticactivity,thusproducingthefirstimmobilizedenzyme,althoughtheymadenosubsequentuseofit.
•Otherinorganicmaterialswhichcouldbeusedascarriersincludeclay,aluminaandsilica.
•Theweaklinkagesestablishedbetweenenzymeandcarrier(mainlyVanderWaalsandhydrogenbonds)havelittleeffectoncatalyticactivity.
•However,becausethebondsaresoweak,theenzymecaneasilybedesorbedfromthecarrier.ThiscanbebroughtaboutbychangeinpH,ionicstrength,orsubstrateconcentration.
•Also,theadsorptionprocessisnon-specific,somanyothersubstanceswillbecomeattachedtothecarrierastheimmobilizedenzymeisused.

17. Methodology
•Theactualmethodologyinvolvedintheadsorptionofenzymestothematricesisquitesimple,easy,andemployedlargely.
Theappropriateenzymeisadequatelymixedwitharight
adsorbentusuallyunderappropriatepHparametersaswellasthedesiredionicstrengthAfterincubation
forastipulateddurationthecarriermatrixiswashedthoroughlytogetridoftheentireunabsorbed
enzymemoleculeswherebythe‘immobilizedenzyme’isreadyforactualusage.Interestingly,this
specificmethodinvariablygivesrisetoahighloading(nearly1genzymepergmatrix)oftheenzyme.

20. Covalent bonding
•Theenzymemoleculesareattachedtocarriermatrixbytheformationofcovalentbonds.Duetothistheactualstrengthofbondagehappenstobequitestrong,andhencenolossofenzymestakeplaceduringitsusage.
•Theformationofcovalentbondusuallytakesplaceparticularlywiththesidechainsofaminoacidspresentintheenzyme.However, theiractualstrengthofreactivitybeingexclusivelylinkedtothestatusof‘charge’presentinthem.
•Thevariousfunctionalmoitiesmostlypresentinenzymewhichactivelytakepartintheformationofnumerousviablechemicalbondsaresulphide,oxide,carboxyl,hydroxyl,amino,imidazole, guanidyl,etc.

21. Methodology
•Thecovalentbondingofanenzymemaybeaccomplishedeitherbyactivatingthepolymerwithareactivemoietyorbyeffectivelyemployingthebifunctionalreagenttoserveasabridgebetweenthetwoentities: enzymeandpolymer.
•Where3-Dnetworkmaybeobtainedbycross-linkingwithlowmolecularweightbifunctionalagent.
•Indoingso,theenzymemaygetinactivatedbecausethereactionsnormallyengageafunctionalmoietystrategicallylocatedattheactivesiteoftheenzyme.(Insimplewords,Immobilizationbythismethodmayleadtoultimatelossintheextentofenzymeactivityastheactivesiteisinvolvedduringtheimmobilizationprocess.)
•Thustheoverallneteffectbeingthesubstantiallossofenzymaticactivity. Thislossofenzymaticactivitycouldbeovercomebycarryingoutenzymeimmobilizationeitherinpresenceofcompetitiveinhibitororanenzymesubstrate.

23. Fig: Showing the various enzyme immobilized by Covalent bonding

24. Entrapment
•2.3.Entrapment
•Entrapmentreferstothephenomenonwherebytheenzymemoleculesareeitherheldorentrappedwithintheappropriatefibresorgels(mostcommonlyusedispolyacrylamidegel).
•Entrapmentmayormaynotbeaccomplishedviacovalentbondingbetweenenzymeandcarriermatrix.
•Incasewherecovalentbondingisrequiredtheenzymesneedtobetreatedwithsyntheticreagentssuchasacryloylchloride,celluloseacetate,calciumalginate,etc.
•Example:Celluloseacetatefibresfinditsapplicationforenzymeentrapment.Enzymeandcelluloseacetateisblendedtogethertoobtainanemulsionpreferablyinanorganicsolvent,methylenechloride.Theresultingemulsionissubjectedtotheprocessof‘extrusion’toobtainfibresintoasolutionofanaqueousprecipitant.
Calciumalginateisthematerialofchoicefortheentrapmentofmicrobial,plantcellsandanimalcells.

25. •Inthismethodofimmobilization,practicallynegligibleamountoflossofbiologicalactivityofenzymetakesplacecomparedtocouplingmethodasthismethoddoesn’tallowthebindingofenzymeitselfeithertothegelmatrixorthemembrane.

26. Methodology
Methodology:Thevariousstepsinvolvedin‘entrapment’areasbelow
(1)Theenzyme(s)maybedissolvedinasolutionofthepolymer’sprecursors.
(2)Polymersmaybeselectedfromavarietyofmaterialse.g., naturalgels(e.g.,cellulosetriacetate,alginate,agar, gelatin);syntheticgelse.g.,polyacrylamidegels.
(3)Inordertocheckandpreventthepossibleleakageofthelowmolecularweightenzymesfromthebodyofthegel, theaverageporesizeofthegelmustbemaintainedaslargeaspossible.
(4)Effortsshouldbegearedintoactiontopracticallycontaintwoimportantaspectsin‘entrapment’process,namely:
(a)excessivediffusionlimitation,and
(b)variabilityofporesize.

27. •Example:Penicillinacylaserepresentsthecategoryoffibre- entrappedenzymesthatessentiallyaffordsimmobilizationviaentrapmentinthemicrocavitiesofthesyntheticfibres.
•Liposomeentrapmentreferstothephysicalphenomenonwherebyentrapmentmaybeaccomplishedbycarryingoutthedissolutionofa‘fibre’formingpolymere.g.,cellulosetriacetateinanorganicsolventwhichbeingimmiscibleinaqueousmedium,andsubsequentlyemulsifyingtheresultingsolutionwiththeaqueoussolutionofenzymecarefully.
•Theemulsionthusobtainedisextrudedviaaspinneretintoliquidcoagulant(e.g.,toluene,petroleumether)whichspecificallyprecipitatethepolymerinitsdesiredfilamentousformhavingaprecisemicrodropletofthe‘enzyme’solutionmeticulouslyentrappedinthefibre.Thistechnique,obviouslypossesstworemarkableadvantages,namely:
(a)minimisesthediffusionlimitation,and
(b)relativesurfacetovolumeratioisappreciablyhigh.

28. •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.

30. Encapsulation/microencapsulation/membrane confinement
•Inthismethod,enzymemoleculesinaqueousmediumareconfinedwithinasemipermeablemembranethatideallypermitsanalmostabsolute‘freemovement’oftheenzymesineitherdirectiontotheproductsandsubstratebutdoesn’tallowtheenzymetoescapefromtheconfinedmembrane.
•Theenzymeimmobilizationprevailinginencapsulatingmethodpredominatlyoccurswellwithinthemicrocapsules(preparedfromorganicpolymersinorderthatenzymesarepreventedfromthegreatescape.
•Inaddition,lowmolecularwtproductsandsubstratecaneitherenterorleavethecapsulebydiffusionviamembrane.

32. •Twowellknownmethodsforpreparingmembranouscapsuleforencapsulation.
1.Phaseseparation:membranesusuallymadebyadoptingtheprocessofphase-separationwhichresembletothehomogenizationofwaterinoil.Inthisinstance,onephaseisnotmisciblewithothergivingrisetodropletwithinwhichenzymesgetentrapped.
1.Chemicalpolymerization:Thechemicalpolymerisationaidsinthepreparationofthespecificwaterinsolublemembrane,andthustheenzymeinquestiongetdulyentrappedduringthisongoingphenomenonofpolymerization.E.gsemipermeablenylonorcollodionintheshapeofspheresareutilizedformicroencapsulationofenzyme.

34. Advantage of enzyme immobilization
(1)Enzymesbeingquiteexpensiveandalsohavingtheuniqueabilitytobeusedrepeatedlyonlyinasituationwhenthesemayberecoveredcompletelyfromtheaccomplishedreactionmixtures.Intruesense, immobilizationdistinctlyandspecificallyallowstheirrepeatedusagebyvirtueofthefactthatsuchenzymepreparationmaybeseparatedconvenientlyfromthereactionsysteminvolved.
(2)Importantly,thefinaldesiredproductshouldbereadilyseparatedfromtheenzyme.Itgoesalongwayinaffectingreductionandsavinguponthecostof‘downstreamprocessing’oftheensuingend-product.
(3)Non-aqueoussystems(i.e.,usingorganicsolventsexclusively)arefoundtobefairlycompatiblewiththeimmobilizedenzymesparticularly,andthismayberegardedtobeextremelydesirableincertaintypicalandspecificinstances.
4)Immobilizedenzymesmaybeusedpredominentlyinmostcontinuousproductionsystems;and,ofcourse,thisnotabsolutelyfeasibleandpossiblewiththe‘free-enzymes’.

35. (5)Immobilizedenzymes,afewselectedones,mayexhibitthermostabilityofthehighestorder,viz.,thefree-enzymeglucoseisomeraseusuallygetsdenaturedonlyat45°Cinsolution;however,whenimmobilizedsuitablytheenzymeisfoundtobestableenoughupto1yearat65°C.
(6)Importantly,theultimaterecoveryof‘immobilizedenzyme’woulddrasticallyminimisethehigheffluentdisposableproblems(whichisquiteacuteinseveralfermentationindustries).
(7)Immobilizedenzymesmaybeemployedatamuchhigherconcentrationrangeincomparisontothecorrespondingfreeenzyme.

36. Disadvantages of Enzyme Immobilization
Immobilizedenzymesdoofferseveraldisadvantageswhicharebrieflydiscussedinthesection
thatfollows:
(1)Enzymeimmobilizationevidentlygivesrisetoanadditionalbearingoncost.Hence,this
improvedtechniqueisgottobeusedonlyinsuchaneventwhenthereprevailsasound
economicviability,feasibility,safety,andaboveallapositiveedgeoverthecorresponding
‘solubleenzymes’.
(2)Immobilizationofenzymesinvariablyaffectsthestabilityand/oractivityadversely.In
ordertocircumventsuchtypicalinstancesonemayhavetoadherestrictlytothelaiddown
developedimmobilizationprotocols.
(3)Practicalutilizationofthe‘immobilizedenzymes’maynotprovetobeofanyuseoradvantage
whenoneofthesubstratesisfoundtobeinsoluble.
(4)Certainimmobilizationprotocolsdoofferagoodnumberofseriousproblemswithrespectto
thediffusionoftheensuingsubstratetohaveanaccesstothecorrespondingenzyme.

37. 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.

38. •Ithasbeendulyobservedthattherateofreactioncatalyzedbyanenzymeparticularlyenhances‘linearity’withthecorrespondingincreaseinthesubstrateconcentrationgenerallyuptoacertainpoint.
•However,itsoonapproachesthemaximumvalue,usuallytermedasVmax;andbeyondwhichthereisabsolutelynofurtherenhancementintherateofreactionasshowninfigureinnextslide.Itisknownassaturation.

39. •Ontheotherhand,therateofanonenzymaticallycatalyzedreactionthatenhanceslinearlyverymuchacrosstheentirerangeofattainablesubstrateconcentrations.
•Importantly,theprevailing‘immobilizationphenomenon’doeshelpintheactualconversionofthecatalystfromhomogeneous(i.e.,solubleenzyme) naturetotheheterogeneousone,wherebytheenzymeisintimatelyassociatedeitherwithaparticularenvelopingmatrixorasupportingmatrix.Nevertheless,inthecourseofimmobilizationphenomenon,theactivityofensuingenzymeisvirtuallylostbyvirtueoftwovitalreasons, namely:(a)variousreactionsinvolvedintheprocess;and(b)effectiveocclusionofactivesitesintheenzymesupportcomplex.
•Examples:
(1)Hem-containingproteins:Haemoglobin:IthasbeenobservedthathaemoglobingetsboundtoO2;andindoingsoseveralO2-moleculesmaybindandreleaseduringoneminute,whileatanymaterialtimeonlyoneO2moleculebecomesintimatelyassociatedwithonehemcentre.

41. Enzyme Activity
•Enzymeactivityisthemeasureoftheabilityofanenzymetocatalyzeaspecificreaction.OrEnzymeactivityisthecatalyticeffectexertedbyanenzyme, expressedasunitspermilligramofenzyme(specificactivity)orasmoleculesofsubstratetransformedperminutepermoleculeofenzyme(molecularactivity).
•Therateofenzymecatalysedreactionisoftencalleditsvelocity.
•Enzymevelocityiscommonlyexpressedbytheinitialrate(Vo)ofthereactionbeingcatalyzed.TheunitsofVoisμmol/min,whichcanberepresentedbytheenzymeunit(U)orthekatal(kat),
1μmol=1U=16.67nanokat.
•ExperimentallyV0ismeasuredbeforemorethanapproximately10%ofthesubstratehasbeenconvertedtoproductinordertominimizesuchcomplicatingfactors.Atypicalplotofproductformedagainsttimeforanenzyme-catalyzedreactionshowsaninitialperiodofrapidproductformationwhichgivesthelinearportionoftheplot(Fig.1).Thisisfollowedbyaslowingdownoftheenzymerateassubstrateisusedupand/orastheenzymelosesactivity.V0isobtainedbydrawingastraightlinethroughthelinearpartofthecurve,startingatthezerotime-point(Fig.1).TheslopeofthisstraightlineisequaltoV0.

43. •Enzymereferstoanorganiccatalystinvariablyproducedby‘livingcells’butcapableofactingeitheroutsidecellsoreveninvitro.
•Enzymesare‘proteins’thatchangetherateofchemicalreactionswithoutneedinganexternalenergysourceorbeingchangedthemselves;anenzymesmaycatalyzeareactionnumeroustimes.
•Enzymesarehighlyreactionspecificinthattheyactonlyoncertainsubstancesusuallyknownassubstrates.Nevertheless,theenzymeanditssubstrateorsubstratesinvariablygiverisetoatemporaryconfiguration,calledanenzyme- substratecomplexthatessentiallyinvolvesbothphysicalshapeandchemicalbonding.
•Theenzymeusuallypromotestheformationofbondsbetweenseparatesubstrates, orinducesthebreakingofbondsinasinglesubstratetoformtheproductorproductsofreaction.Thehumanbodycontainsthousandsofenzymes,eachcatalyzingoneofthemanyreactionsthateventuallyoccuraspartofmetabolism.
•Thetermactivity(ortotalactivity)referstothetotalunitsofenzymeinasample,whereasspecificactivityisthenumberofunitspermilligramofprotein(unitsmg-1)

44. Substrate concentration
•Thenormalpatternofdependenceofenzymerateonsubstrateconcentration([S])isthatatlowsubstrateconcentrationsadoublingof[S]willleadtoadoublingoftheinitialvelocity(V0).
•However,athighersubstrateconcentrationstheenzymebecomessaturated,andfurtherincreasesin[S]leadtoverysmallchangesinV0.Thisoccursbecauseatsaturatingsubstrateconcentrationseffectivelyalloftheenzymemoleculeshaveboundsubstrate.
•Theoverallenzymerateisnowdependentontherateatwhichtheproductcandissociatefromtheenzyme,andaddingfurthersubstratewillnotaffectthis.TheshapeoftheresultinggraphwhenV0isplottedagainst[S]iscalledahyperboliccurve.

46. Enzyme concentration
•Insituationswherethesubstrateconcentrationissaturating(i.e.alltheenzymeconcentrationmoleculesareboundtosubstrate),adoublingoftheenzymeconcentrationwillleadtoadoublingofV0.
•ThisgivesastraightlinegraphwhenV0isplottedagainstenzymeconcentration.

48. pH
•EachenzymehasanoptimumpHatwhichtherateofthereactionthatitcatalyzesisatitsmaximum.
•SmalldeviationsinpHfromtheoptimumvalueleadtodecreasedactivityduetochangesintheionizationofgroupsattheactivesiteoftheenzyme.
•LargerdeviationsinpHleadtothedenaturationoftheenzymeproteinitself,duetointerferencewiththemanyweaknoncovalentbondsmaintainingitsthree-dimensionalstructure.
•AgraphofV0plottedagainstpHwillusuallygiveabellshapedcurve. ManyenzymeshaveapHoptimumofaround6.8,butthereisgreatdiversityinthepHoptimaofenzymes,duetothedifferentenvironmentsinwhichtheyareadaptedtowork.
•Forexample,thedigestiveenzymepepsinisadaptedtoworkattheacidicpHofthestomach(aroundpH2.0).

50. Michaelis-MentenModel

53. Significance of Km Values
ThevariousimportantsignificanceofKmvaluesareasfollows:
(1)Indicativeofsubstrateconcentration(S),
(2)Affinityofenzymewithcorrespondingsubstrate,
(3)Indicativepartiallyofenzyme-substrateconcentrationprevailinginthecellularcompartmenti.e.,thetargetwheremostofthereactioninvariablytakesplace.
(4)Km-valuesarefoundtobeinverselyproportionaltotheensuingaffinityoftheenzymeforitssubstratei.e.,higherKm-valuesgiverisetolowerstabilityoftheenzymesubstrate(ES)-complexapparently.

54. Determination of Km

56. Some important enzymes
•Enzymeplaysasignificantroleinbiochemicalreactions. Fewofthetypicalhumanailments(persistentbodilydisorderordiseasecouldbeattributedtothepartialdeficiencyorcompleteabsenceofoneormorethanoneenzymespresentinthetissueorgans.
•Ithasbeenamplyobservedthatincertainextremeabnormalconditionstheunnaturalandtwomuchinherentactivityofaparticularenzymeinvivocouldbeadequatelymanagedandcontrolledattimesbya‘specificdrugsubstance’designedtocontrolaswellasinhibititsoverallcatalyticactivity.[Insimplewords,incertaindiseaseswhereenzymeactivityismorevariousenzymespecificdrugscouldbeusedtocontroltheactivityofenzymeortoinhibititsactivity

57. •Enzymesarealsorequiredforcarryingoutvarioussynthesisreactionsinthelivingbody.
•Synthesisofproteins,nucleicacids,phospholipidsforcellmembranes, hormones,andglycogenallessentiallyneedatleastoneifnotmanyenzymes. Forinstance:DNApolymeraseIsextremelyneededforcarryingoutthephenomenonofDNAreplication,thatprecedesmitosis.Evenbloodclotting, theformationofangiotensinsIItoboostupbloodpressure,andthetransportofCO2inthebloodalsorequirespecificenzymes.
•Enzymesmaybeemployedinthereplacementtherapywherebyeitherthe‘missingenzyme’ormalfunctioningofcertainimpairedorgansarecorrectedtoprolongthelife-expectencyofpatients.Numeroushereditarydiseasesarecausedduetolackofoneortwoenzymesinthebodywhichgiverisetotheaccumulationofundesired,abnormalsubstanceswhichotherwisewouldhavebrokendowninpresenceofenzyme.Eg.Fabry’sdiseasei.e.,aninheritedmetabolicdiseaseinwhichthereisagalactosidase(responsibleforhydrolysinggalactosidesintomonosaccharides)deficiency,whichleadstoaccumulationofglycosphinogolipidsthroughoutthebody.

58. •Adenosinedeaminasedeficiencyinvariablycausesseverecombinedimmunodeficiency(SCID)whichwouldcategoricallyrespondtothedesiredandmuchrequiredenzymereplacementtherapy,wherein
thesaidpurifiedenzymeisfirstdulystabilizedinpolyethyleneglycol(PEG),andthenadministeredparenterally.
•Few of the vital and important enzymes are listed below:
•(i) Hyaluronidase
•(ii) Penicillinase
•(iii) Streptokinase
•(iv) Streptodornase
•(v) Amylases
•(vi) Proteases

59. Hyaluronidase
•Itisanenzymefoundinthetestesandsemen.Itdepolymerizeshyaluronicacid,therebyenhancingthepermeabilityofconnectivetissuesbydissolvingthesubstancesthatessentiallyholdbodycellstogether.
•Itactstodispersethecellsofthecoronaradiataaboutthenewlyovulatedovum,thusfacilitatingentryofthesperm.
•Theenzymeacceleratesspecificallythesubcutaneousspreadoftheensuing(resulting)particulatematter.
•Hyaluronidasefindsitsabundantutilityasadispersionagentalongwiththeotherinjecteddrugsbeingemployedasatherapeuticmeasure.Itisalsousedasapotentialadjunctparticularlyinsubcutaneousurographyfornotonlyaugmenting(intensifying)butalsomarkedlyimprovingtheresorption(lysisandassimilationprocess)ofradiopaqueagents.
•Besides,italsohelpsintheenhancementof‘adsorptionofdrugs’particularlyintransudates,tissuespaces,andoedemas.

60. •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 mucopolysaccharidesof the hyaluronicacid type.
•It usually contains not more than 0.25 μgof tyrosine for each Hyaluronidase Unit.
Therapeutic Applications : The various therapeutic applications are as follows :
(1)Bycatalyzingthehydrolysisofhyaluronicacid, a constituent of theextracellular matrix(ECM), hyaluronidase lowers theviscosityof hyaluronicacid, thereby increasingtissuepermeability. It is, therefore, used in medicine in conjunction with otherdrugsto speed their dispersion and delivery. Common applications areophthalmicsurgery, in combination withlocal anesthetics.
(2) The most prominent clinical usage of hyaluronidase is to distinctly facilitate the administration of fluids by the aid of hypodermoclysis.

61. Penicillinase
•Penicillinaseisabacterialenzymethatinvariablyinactivatesmostbutnotallpenicillins.
•Thisisregardedasanextracellulartypeenzymeproducedadaptivelybymembersofthecoliformgroupofbacteria,bymostBacillusspecies,andcertainstrainsofStaphylococcus.Theenzymeexclusivelycarriesoutthehydrolysisofpenicillintopenicilloicacidi.e.,adicarboxylicacidasdepictedinfigure.
•Ithasbeendulyobservedthataratherlargesegmentofpenicillin- resistantpathogenicstrainsofStaphylococcusaureusinvariablycompriseofthisspecificenzyme;andperhapsitoverwhelminglycontributesamajorfactorofpenicillinresistanceduringinfection.
•Importantly,thisenzymecausesanextremelyrapiddegradationofpenicillinparticularlyinpenicillinfermentationsincaseaspecificcontaminantwhichproducestheenzymeincidentallygainsanaccesstoandbeabletogrowsimultaneouslyinthefermentationbroth.
•PenicillinaseobtainedfromB.subtilisandB.cereusrepresenttheindustriallyproducedenzymeswhichexertactiontosomeextentintheremovalofpenicillinviaspecificinactivation.

63. Streptokinase
•Streptokinaseisasingle-chaincoenzymeobtainedfromculturesoftheGroupCβstrainofStreptococcushaemolyticusthatiscapableofsolelyconvertingplasminogentoplasmin.
•Itisusedextensivelyasapredominantfibrinolyticagenttohelpinabigwayforthespecificremovaloffibrinthrombi(bloodclot)fromarteries.
•TheGlobalutilizationofStreptokinaseandtPA(tissuetypeplasmogenactivator)ofOccludedArteries(GUSTO)trialwasdesignedmeticulouslytoinvestigateandestablishthebenefitsofa‘frontloaded’doseofalteplase(alargeinitialbolusfollowedbyaninfusionofthetotaldoseoveraspanof90minutes)whencomparedwiththeusualconventionalalteplaseadministration.
Note:alteplaseistheFDAapprovedrecombinanttissueplasminogenactivator.

64. •Besides,anintensiveandextensivestudywascarriedoutwithrespecttothesafetyandimprovementinmortalityofcombiningalteplaseandstreptokinasewitheitheragentalone.Interestingly,theoutcomeofresultsfromtheseextendedinvestigationsappeartoexhibitanappreciablyfavourablemortalityrateinpatientshavingbeentreatedwithalteplasecomparisontothestreptokinasetreatedsubjects.
•Amixtureofstreptokinaseandstreptodornase,asproducedbyahemolyticstreptococcusgrowninaspecificenvironmentofaerated-submergedculture,isusedmeticulouslytocleanupthedebrisfromwoundsandburnseffectively.

65. TherapeuticApplications:
Thetherapeuticapplicationsofstreptokinaseareasstatedbelow:
(1)Itisrecommendedforthemanagementandcontrolofmyocardialinfarction(AMI)inadultstobringaboutvarioustherapeuticbenefits,suchas:
•specificlysisofintracoronarythrombi
•improvementinventricularfunction
•remarkablereductionofmortalityassociatedwithAMI.
•reductionofinfarctsizeandcongestiveheartfailureassociatedwithAMIwhenadministeredbytheIVroute.
(2)Itisalsoindicatedfortheadequatelysisofobjectivelydiagnosedpulmonaryemboli,involvingobstructionofbloodflowtoalobeormultiplesegments, withorwithoutunstablehaemodynamics.
(3)Besides,the‘drug’isabundantlyrecommendedforthelysisofobjectivelydiagnosed,acute,andextensivethrombiofthedeepveins,emboli,andarterialthrombirespectively.
(4)Individualshavingquiterecentstreptococcalinfectionsmaypossessanappreciablequantumofcirculatingantisteptokinaseantibodies;andtocounteractthissituationa‘loadingdose’sufficienttoneutralizetheprevailingantibodiesisrequiredurgently.

66. Streptodornase
•Streptodornasereferstooneoftheenzymesproducedbycertainstrainsofhaemolyticstreptococci.Itiscapableofliquefyingfibrinousandpurulentexudates.
•Itisalsoemployedsometimesasawounddebridementi.e.,removalofdamagedtissue.
•StreptodornaseisactuallyobtainedfromStreptococcushaemolyticusthataffordsdepolymerizationofpolymerizeddeoxyribonucleoproteins.
•Itisusedextensivelyinconjunctionwithstreptokinaseasde-sloughingagenttocleanseulcersandpromotethehealingprocessprogressively.

67. Definition
1.Enzyme: Any of numerous proteins orconjugated proteinsproduced 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:Anonproteinaceousorganicsubstancethatusuallycontainsavitaminormineralandcombineswithaspecificprotein,theapoenzyme,toformanactiveenzymesystem.