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Enzyme Immobilization 
By: Bijaya Kumar Uprety
WHAT ARE ENZYMES AND WHAT DO THEY DO? 
•Enzymesareproteinswithhighlyspecializedcatalyticfunctions,producedbyalllivingorgan...
•Enzymesarenaturalproteinmoleculesthatactashighlyefficientcatalysts. 
•Enzymesnotonlyworkefficientlyandrapidly,theyarealso...
Enzyme Class 
Type of reaction catalysed 
Example 
Oxidoreductases 
Oxidation/reductionreactions-Catalysethe transfer of H...
Uses of Enzyme 
•Enzymesplayadiversifiedroleinmanyaspectsofeverydaylifeincludingaidingindigestion,theproductionoffoodandse...
2. Food Production and Industrial Applications: 
•Sinceancienttimes,enzymeshaveplayedanimportantpartinfoodproduction.Oneof...
How enzymes are made? 
•Commercialsourcesofenzymesareobtainedfromthreeprimarysourcesi.e.animaltissue,plantsandmicrobes. 
•...
•Enzymemanufacturersproduceenzymesinaccordancewithallapplicablegovernmentalregulations,includingtheappropriatefederalagenc...
Advantages of using enzymes 
•Enzymescanoftenreplacechemicalsorprocessesthatpresentsafetyorenvironmentalissuese.g.replacin...
Enzyme immobilization 
•Microbialenzymesaremostextensivelyemployedinthefoodandbeverageindustriesacrosstheworldtomeettheinc...
•Animmobilizedenzymeisanenzymethatisattachedtoaninert,insolublematerialsuchascalciumalginate(producedbyreactingamixtureofs...
Standard defination of enzyme immobilization. 
•Animmobilizedenzymeistheonewhichhasbeenattachedtoorenclosedbyaninsolublesu...
Salient feature of enzyme immobilization 
1.Enzymesaremoreorlessphysicallyconfinedinthecourseofadefinitecontinuouscatalyti...
Carrier Matrices 
•Thesubstancesthataresolelyemployedfortheimmobilizationofenzymesareknownascarriermatricese.g.,inorganicm...
Method of immobilization 
•There are four distinct type of immobilization methods. 
Adsorption 
Covalent bonding 
Entra...
1. Physical adsorption 
•Physicaladsorptionontoaninertcarrierisaverysimpleprocedureforimmobilizinganenzyme,foritrequiresju...
Methodology 
•Theactualmethodologyinvolvedintheadsorptionofenzymestothematricesisquitesimple,easy,andemployedlargely. 
The...
Covalent bonding 
•Theenzymemoleculesareattachedtocarriermatrixbytheformationofcovalentbonds.Duetothistheactualstrengthofb...
Methodology 
•Thecovalentbondingofanenzymemaybeaccomplishedeitherbyactivatingthepolymerwithareactivemoietyorbyeffectivelye...
Fig: Showing the various enzyme immobilized by Covalent bonding
Entrapment 
•2.3.Entrapment 
•Entrapmentreferstothephenomenonwherebytheenzymemoleculesareeitherheldorentrappedwithintheapp...
•Inthismethodofimmobilization,practicallynegligibleamountoflossofbiologicalactivityofenzymetakesplacecomparedtocouplingmet...
Methodology 
Methodology:Thevariousstepsinvolvedin‘entrapment’areasbelow 
(1)Theenzyme(s)maybedissolvedinasolutionofthepol...
•Example:Penicillinacylaserepresentsthecategoryoffibre- entrappedenzymesthatessentiallyaffordsimmobilizationviaentrapmenti...
•Another way is to entrap enzyme (s) inside the polymerised gel. 
Enzyme containing gel is made in aqueous mediumthis gel...
Encapsulation/microencapsulation/membrane confinement 
•Inthismethod,enzymemoleculesinaqueousmediumareconfinedwithinasemip...
•Twowellknownmethodsforpreparingmembranouscapsuleforencapsulation. 
1.Phaseseparation:membranesusuallymadebyadoptingthepro...
Advantage of enzyme immobilization 
(1)Enzymesbeingquiteexpensiveandalsohavingtheuniqueabilitytobeusedrepeatedlyonlyinasit...
(5)Immobilizedenzymes,afewselectedones,mayexhibitthermostabilityofthehighestorder,viz.,thefree-enzymeglucoseisomeraseusual...
Disadvantages of Enzyme Immobilization 
Immobilizedenzymesdoofferseveraldisadvantageswhicharebrieflydiscussedinthesection ...
Factors affecting enzyme kinetics 
•Enzyme kinetics refers to the indepth study of enzyme in action. 
•Kinetics is the stu...
•Ithasbeendulyobservedthattherateofreactioncatalyzedbyanenzymeparticularlyenhances‘linearity’withthecorrespondingincreasei...
•Ontheotherhand,therateofanonenzymaticallycatalyzedreactionthatenhanceslinearlyverymuchacrosstheentirerangeofattainablesub...
Enzyme Activity 
•Enzymeactivityisthemeasureoftheabilityofanenzymetocatalyzeaspecificreaction.OrEnzymeactivityisthecatalyt...
•Enzymereferstoanorganiccatalystinvariablyproducedby‘livingcells’butcapableofactingeitheroutsidecellsoreveninvitro. 
•Enzy...
Substrate concentration 
•Thenormalpatternofdependenceofenzymerateonsubstrateconcentration([S])isthatatlowsubstrateconcent...
Enzyme concentration 
•Insituationswherethesubstrateconcentrationissaturating(i.e.alltheenzymeconcentrationmoleculesarebou...
pH 
•EachenzymehasanoptimumpHatwhichtherateofthereactionthatitcatalyzesisatitsmaximum. 
•SmalldeviationsinpHfromtheoptimum...
Michaelis-MentenModel
Significance of Km Values 
ThevariousimportantsignificanceofKmvaluesareasfollows: 
(1)Indicativeofsubstrateconcentration(S...
Determination of Km
Some important enzymes 
•Enzymeplaysasignificantroleinbiochemicalreactions. Fewofthetypicalhumanailments(persistentbodilyd...
•Enzymesarealsorequiredforcarryingoutvarioussynthesisreactionsinthelivingbody. 
•Synthesisofproteins,nucleicacids,phosphol...
•Adenosinedeaminasedeficiencyinvariablycausesseverecombinedimmunodeficiency(SCID)whichwouldcategoricallyrespondtothedesire...
Hyaluronidase 
•Itisanenzymefoundinthetestesandsemen.Itdepolymerizeshyaluronicacid,therebyenhancingthepermeabilityofconnec...
•Hyaluronidase For Injection [Wydase(R)] : It is obtained as a sterile dry, soluble enzyme product obtained from the mamma...
Penicillinase 
•Penicillinaseisabacterialenzymethatinvariablyinactivatesmostbutnotallpenicillins. 
•Thisisregardedasanextr...
Streptokinase 
•Streptokinaseisasingle-chaincoenzymeobtainedfromculturesoftheGroupCβstrainofStreptococcushaemolyticusthati...
•Besides,anintensiveandextensivestudywascarriedoutwithrespecttothesafetyandimprovementinmortalityofcombiningalteplaseandst...
TherapeuticApplications: 
Thetherapeuticapplicationsofstreptokinaseareasstatedbelow: 
(1)Itisrecommendedforthemanagementan...
Streptodornase 
•Streptodornasereferstooneoftheenzymesproducedbycertainstrainsofhaemolyticstreptococci.Itiscapableofliquef...
Definition 
1.Enzyme: Any of numerous proteins orconjugated proteinsproduced by living organisms and functioning as bioche...
Enzyme immobilization
Enzyme immobilization
Enzyme immobilization
Enzyme immobilization
Enzyme immobilization
Enzyme immobilization
Enzyme immobilization
Enzyme immobilization
Enzyme immobilization
Enzyme immobilization
Enzyme immobilization
Enzyme immobilization
Enzyme immobilization
Enzyme immobilization
Enzyme immobilization
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Enzyme immobilization

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Enzyme immobilization

  1. 1. Enzyme Immobilization By: Bijaya Kumar Uprety
  2. 2. WHAT ARE ENZYMES AND WHAT DO THEY DO? •Enzymesareproteinswithhighlyspecializedcatalyticfunctions,producedbyalllivingorganisms.OR“Enzymesareproteins,whichcatalysespecificbiochemicalreactionsinaveryefficientmanner.” •Enzymesareresponsibleformanyessentialbiochemicalreactionsinmicro-organisms,plants,animals,andhumanbeings. •Althoughlikeallotherproteins,enzymesarecomposedofaminoacids,theydifferinfunctioninthattheyhavetheuniqueabilitytofacilitatebiochemicalreactionswithoutundergoingchangethemselves.Thiscatalyticcapabilityiswhatmakesenzymesunique.
  3. 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. 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. 5. Uses of Enzyme •Enzymesplayadiversifiedroleinmanyaspectsofeverydaylifeincludingaidingindigestion,theproductionoffoodandseveralindustrialapplication.Enzymesarenature’scatalyst. 1.Humanbodyusesvariousenzymestocarryoutvariousbiochemicalprocesses.Oneofthebestexampleofenzymebasedprocessisdigestion.Enzymeshelpbreakdowncarbohydrate,proteinsandfatsintosimplecompoundsthatcanbeabsorbedbythebody.Amylaseandlipaseinsalivabreaksdowncarbohydrateandfatsrespectively.Proteasereleasedinstomachhelpsindigestionofproteins&Lipase, amylases,andproteasesaresecretedinsmallintestineandplayanimportantroleincompletingdigestiveprocess.
  6. 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. 7. How enzymes are made? •Commercialsourcesofenzymesareobtainedfromthreeprimarysourcesi.e.animaltissue,plantsandmicrobes. •Thesenaturallyoccurringenzymesarequiteoftennotreadilyavailableinsufficientquantitiesforfoodapplicationsorindustrialuse.However,byisolatingmicrobialstrainsthatproducethedesiredenzymeandoptimizingtheconditionsforgrowthcommercialquantitiescanbeobtained.Thistechniqueiswellknownformorethan3000yearsandisknownasfermentation. •Todaythisfermentationprocessiscarriedoutinacontainedvessel.Oncefermentationiscompleted,themicroorganismsaredestroyed,theenzymesareisolated,andfurtherprocessedforcommercialuse.
  8. 8. •Enzymemanufacturersproduceenzymesinaccordancewithallapplicablegovernmentalregulations,includingtheappropriatefederalagencies(e.g.FoodandDrugAdministration,UnitedStatesDepartmentofAgriculture,EnvironmentalProtectionAgency,etc). •Regardlessofsource,enzymesintendedforfooduseareproducedinstrictadherencetoFDA’scurrentGoodManufacturingPractices(cGMP)andmeetcompositionalandpurityrequirementsasdefinedintheFoodChemicalsCodex(acompendiumoffoodingredientsspecificationsdevelopedinco-operationwithFDA).
  9. 9. Advantages of using enzymes •Enzymescanoftenreplacechemicalsorprocessesthatpresentsafetyorenvironmentalissuese.g.replacingacidsinstarchprocessingandalkalisinfabricdesizing,reduceuseofsulfideintanneries,removingstainsfromfabrics(clothescanbewashedatlowertemperaturethussavingenergy). •Contributetosaferworkingconditionbyeliminatingtheuseofchemicaltreatmentsduringproductionprocesses.
  10. 10. Enzyme immobilization •Microbialenzymesaremostextensivelyemployedinthefoodandbeverageindustriesacrosstheworldtomeettheincreasingdemandfornutritionallysuperbandhigh-valueproducts. •However,thepredominantuseoftheenzymesinindustrialenvironmenthasbeenlimitedbythefactthatlargenumberoftheseenzymesareunstableandthecostofisolation,purificationandrecoveryoftheactiveenzymefromthereactionmixtureishigh. •Inactualpractice,thesolubleenzymesengagedinbatchoperationsisfoundtobeuneconomicalastheactiveenzymeisvirtuallylost(notrecovered)aftereachviablereaction. •Therefore,inordertoovercomesuchnon-productive,economicallynotfeasible, anddeleteriouseffectstheenzymeshavebeenultimatelyimmobilizedandthisprocessofimmobilizationofenzymeistermedasenzymeimmobilization. “Enzymeimmobilizationmaybedefinedasconfiningtheenzymemoleculestoadistinctphasefromtheonewhereinthesubstrateandproductarepresent.”
  11. 11. •Animmobilizedenzymeisanenzymethatisattachedtoaninert,insolublematerialsuchascalciumalginate(producedbyreactingamixtureofsodiumalginatesolutionandenzymesolutionwithcalciumchloride). •ThiscanprovideincreasedresistancetochangesinconditionssuchaspHortemperature. •Italsoallowsenzymestobeheldinplacethroughoutthereaction,followingwhichtheyareeasilyseparatedfromtheproductsandmaybeusedagain-afarmoreefficientprocessandsoiswidelyusedinindustryforenzymecatalysedreactions.Analternativetoenzymeimmobilizationiswholecellimmobilization.
  12. 12. Standard defination of enzyme immobilization. •Animmobilizedenzymeistheonewhichhasbeenattachedtoorenclosedbyaninsolublesupportmedium(termedascarrier)oronewheretheenzymemoleculeshavebeencross-linkedtoeachother,withoutlossofcatalyticactivity.
  13. 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. 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. 15. Method of immobilization •There are four distinct type of immobilization methods. Adsorption Covalent bonding Entrapment Membrane confinement
  16. 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. 17. Methodology •Theactualmethodologyinvolvedintheadsorptionofenzymestothematricesisquitesimple,easy,andemployedlargely. Theappropriateenzymeisadequatelymixedwitharight adsorbentusuallyunderappropriatepHparametersaswellasthedesiredionicstrengthAfterincubation forastipulateddurationthecarriermatrixiswashedthoroughlytogetridoftheentireunabsorbed enzymemoleculeswherebythe‘immobilizedenzyme’isreadyforactualusage.Interestingly,this specificmethodinvariablygivesrisetoahighloading(nearly1genzymepergmatrix)oftheenzyme.
  18. 18. Covalent bonding •Theenzymemoleculesareattachedtocarriermatrixbytheformationofcovalentbonds.Duetothistheactualstrengthofbondagehappenstobequitestrong,andhencenolossofenzymestakeplaceduringitsusage. •Theformationofcovalentbondusuallytakesplaceparticularlywiththesidechainsofaminoacidspresentintheenzyme.However, theiractualstrengthofreactivitybeingexclusivelylinkedtothestatusof‘charge’presentinthem. •Thevariousfunctionalmoitiesmostlypresentinenzymewhichactivelytakepartintheformationofnumerousviablechemicalbondsaresulphide,oxide,carboxyl,hydroxyl,amino,imidazole, guanidyl,etc.
  19. 19. Methodology •Thecovalentbondingofanenzymemaybeaccomplishedeitherbyactivatingthepolymerwithareactivemoietyorbyeffectivelyemployingthebifunctionalreagenttoserveasabridgebetweenthetwoentities: enzymeandpolymer. •Where3-Dnetworkmaybeobtainedbycross-linkingwithlowmolecularweightbifunctionalagent. •Indoingso,theenzymemaygetinactivatedbecausethereactionsnormallyengageafunctionalmoietystrategicallylocatedattheactivesiteoftheenzyme.(Insimplewords,Immobilizationbythismethodmayleadtoultimatelossintheextentofenzymeactivityastheactivesiteisinvolvedduringtheimmobilizationprocess.) •Thustheoverallneteffectbeingthesubstantiallossofenzymaticactivity. Thislossofenzymaticactivitycouldbeovercomebycarryingoutenzymeimmobilizationeitherinpresenceofcompetitiveinhibitororanenzymesubstrate.
  20. 20. Fig: Showing the various enzyme immobilized by Covalent bonding
  21. 21. Entrapment •2.3.Entrapment •Entrapmentreferstothephenomenonwherebytheenzymemoleculesareeitherheldorentrappedwithintheappropriatefibresorgels(mostcommonlyusedispolyacrylamidegel). •Entrapmentmayormaynotbeaccomplishedviacovalentbondingbetweenenzymeandcarriermatrix. •Incasewherecovalentbondingisrequiredtheenzymesneedtobetreatedwithsyntheticreagentssuchasacryloylchloride,celluloseacetate,calciumalginate,etc. •Example:Celluloseacetatefibresfinditsapplicationforenzymeentrapment.Enzymeandcelluloseacetateisblendedtogethertoobtainanemulsionpreferablyinanorganicsolvent,methylenechloride.Theresultingemulsionissubjectedtotheprocessof‘extrusion’toobtainfibresintoasolutionofanaqueousprecipitant. Calciumalginateisthematerialofchoicefortheentrapmentofmicrobial,plantcellsandanimalcells.
  22. 22. •Inthismethodofimmobilization,practicallynegligibleamountoflossofbiologicalactivityofenzymetakesplacecomparedtocouplingmethodasthismethoddoesn’tallowthebindingofenzymeitselfeithertothegelmatrixorthemembrane.
  23. 23. 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.
  24. 24. •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.
  25. 25. •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.
  26. 26. Encapsulation/microencapsulation/membrane confinement •Inthismethod,enzymemoleculesinaqueousmediumareconfinedwithinasemipermeablemembranethatideallypermitsanalmostabsolute‘freemovement’oftheenzymesineitherdirectiontotheproductsandsubstratebutdoesn’tallowtheenzymetoescapefromtheconfinedmembrane. •Theenzymeimmobilizationprevailinginencapsulatingmethodpredominatlyoccurswellwithinthemicrocapsules(preparedfromorganicpolymersinorderthatenzymesarepreventedfromthegreatescape. •Inaddition,lowmolecularwtproductsandsubstratecaneitherenterorleavethecapsulebydiffusionviamembrane.
  27. 27. •Twowellknownmethodsforpreparingmembranouscapsuleforencapsulation. 1.Phaseseparation:membranesusuallymadebyadoptingtheprocessofphase-separationwhichresembletothehomogenizationofwaterinoil.Inthisinstance,onephaseisnotmisciblewithothergivingrisetodropletwithinwhichenzymesgetentrapped. 1.Chemicalpolymerization:Thechemicalpolymerisationaidsinthepreparationofthespecificwaterinsolublemembrane,andthustheenzymeinquestiongetdulyentrappedduringthisongoingphenomenonofpolymerization.E.gsemipermeablenylonorcollodionintheshapeofspheresareutilizedformicroencapsulationofenzyme.
  28. 28. 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’.
  29. 29. (5)Immobilizedenzymes,afewselectedones,mayexhibitthermostabilityofthehighestorder,viz.,thefree-enzymeglucoseisomeraseusuallygetsdenaturedonlyat45°Cinsolution;however,whenimmobilizedsuitablytheenzymeisfoundtobestableenoughupto1yearat65°C. (6)Importantly,theultimaterecoveryof‘immobilizedenzyme’woulddrasticallyminimisethehigheffluentdisposableproblems(whichisquiteacuteinseveralfermentationindustries). (7)Immobilizedenzymesmaybeemployedatamuchhigherconcentrationrangeincomparisontothecorrespondingfreeenzyme.
  30. 30. 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.
  31. 31. 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.
  32. 32. •Ithasbeendulyobservedthattherateofreactioncatalyzedbyanenzymeparticularlyenhances‘linearity’withthecorrespondingincreaseinthesubstrateconcentrationgenerallyuptoacertainpoint. •However,itsoonapproachesthemaximumvalue,usuallytermedasVmax;andbeyondwhichthereisabsolutelynofurtherenhancementintherateofreactionasshowninfigureinnextslide.Itisknownassaturation.
  33. 33. •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.
  34. 34. 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.
  35. 35. •Enzymereferstoanorganiccatalystinvariablyproducedby‘livingcells’butcapableofactingeitheroutsidecellsoreveninvitro. •Enzymesare‘proteins’thatchangetherateofchemicalreactionswithoutneedinganexternalenergysourceorbeingchangedthemselves;anenzymesmaycatalyzeareactionnumeroustimes. •Enzymesarehighlyreactionspecificinthattheyactonlyoncertainsubstancesusuallyknownassubstrates.Nevertheless,theenzymeanditssubstrateorsubstratesinvariablygiverisetoatemporaryconfiguration,calledanenzyme- substratecomplexthatessentiallyinvolvesbothphysicalshapeandchemicalbonding. •Theenzymeusuallypromotestheformationofbondsbetweenseparatesubstrates, orinducesthebreakingofbondsinasinglesubstratetoformtheproductorproductsofreaction.Thehumanbodycontainsthousandsofenzymes,eachcatalyzingoneofthemanyreactionsthateventuallyoccuraspartofmetabolism. •Thetermactivity(ortotalactivity)referstothetotalunitsofenzymeinasample,whereasspecificactivityisthenumberofunitspermilligramofprotein(unitsmg-1)
  36. 36. Substrate concentration •Thenormalpatternofdependenceofenzymerateonsubstrateconcentration([S])isthatatlowsubstrateconcentrationsadoublingof[S]willleadtoadoublingoftheinitialvelocity(V0). •However,athighersubstrateconcentrationstheenzymebecomessaturated,andfurtherincreasesin[S]leadtoverysmallchangesinV0.Thisoccursbecauseatsaturatingsubstrateconcentrationseffectivelyalloftheenzymemoleculeshaveboundsubstrate. •Theoverallenzymerateisnowdependentontherateatwhichtheproductcandissociatefromtheenzyme,andaddingfurthersubstratewillnotaffectthis.TheshapeoftheresultinggraphwhenV0isplottedagainst[S]iscalledahyperboliccurve.
  37. 37. Enzyme concentration •Insituationswherethesubstrateconcentrationissaturating(i.e.alltheenzymeconcentrationmoleculesareboundtosubstrate),adoublingoftheenzymeconcentrationwillleadtoadoublingofV0. •ThisgivesastraightlinegraphwhenV0isplottedagainstenzymeconcentration.
  38. 38. pH •EachenzymehasanoptimumpHatwhichtherateofthereactionthatitcatalyzesisatitsmaximum. •SmalldeviationsinpHfromtheoptimumvalueleadtodecreasedactivityduetochangesintheionizationofgroupsattheactivesiteoftheenzyme. •LargerdeviationsinpHleadtothedenaturationoftheenzymeproteinitself,duetointerferencewiththemanyweaknoncovalentbondsmaintainingitsthree-dimensionalstructure. •AgraphofV0plottedagainstpHwillusuallygiveabellshapedcurve. ManyenzymeshaveapHoptimumofaround6.8,butthereisgreatdiversityinthepHoptimaofenzymes,duetothedifferentenvironmentsinwhichtheyareadaptedtowork. •Forexample,thedigestiveenzymepepsinisadaptedtoworkattheacidicpHofthestomach(aroundpH2.0).
  39. 39. Michaelis-MentenModel
  40. 40. Significance of Km Values ThevariousimportantsignificanceofKmvaluesareasfollows: (1)Indicativeofsubstrateconcentration(S), (2)Affinityofenzymewithcorrespondingsubstrate, (3)Indicativepartiallyofenzyme-substrateconcentrationprevailinginthecellularcompartmenti.e.,thetargetwheremostofthereactioninvariablytakesplace. (4)Km-valuesarefoundtobeinverselyproportionaltotheensuingaffinityoftheenzymeforitssubstratei.e.,higherKm-valuesgiverisetolowerstabilityoftheenzymesubstrate(ES)-complexapparently.
  41. 41. Determination of Km
  42. 42. Some important enzymes •Enzymeplaysasignificantroleinbiochemicalreactions. Fewofthetypicalhumanailments(persistentbodilydisorderordiseasecouldbeattributedtothepartialdeficiencyorcompleteabsenceofoneormorethanoneenzymespresentinthetissueorgans. •Ithasbeenamplyobservedthatincertainextremeabnormalconditionstheunnaturalandtwomuchinherentactivityofaparticularenzymeinvivocouldbeadequatelymanagedandcontrolledattimesbya‘specificdrugsubstance’designedtocontrolaswellasinhibititsoverallcatalyticactivity.[Insimplewords,incertaindiseaseswhereenzymeactivityismorevariousenzymespecificdrugscouldbeusedtocontroltheactivityofenzymeortoinhibititsactivity
  43. 43. •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.
  44. 44. •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
  45. 45. Hyaluronidase •Itisanenzymefoundinthetestesandsemen.Itdepolymerizeshyaluronicacid,therebyenhancingthepermeabilityofconnectivetissuesbydissolvingthesubstancesthatessentiallyholdbodycellstogether. •Itactstodispersethecellsofthecoronaradiataaboutthenewlyovulatedovum,thusfacilitatingentryofthesperm. •Theenzymeacceleratesspecificallythesubcutaneousspreadoftheensuing(resulting)particulatematter. •Hyaluronidasefindsitsabundantutilityasadispersionagentalongwiththeotherinjecteddrugsbeingemployedasatherapeuticmeasure.Itisalsousedasapotentialadjunctparticularlyinsubcutaneousurographyfornotonlyaugmenting(intensifying)butalsomarkedlyimprovingtheresorption(lysisandassimilationprocess)ofradiopaqueagents. •Besides,italsohelpsintheenhancementof‘adsorptionofdrugs’particularlyintransudates,tissuespaces,andoedemas.
  46. 46. •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.
  47. 47. Penicillinase •Penicillinaseisabacterialenzymethatinvariablyinactivatesmostbutnotallpenicillins. •Thisisregardedasanextracellulartypeenzymeproducedadaptivelybymembersofthecoliformgroupofbacteria,bymostBacillusspecies,andcertainstrainsofStaphylococcus.Theenzymeexclusivelycarriesoutthehydrolysisofpenicillintopenicilloicacidi.e.,adicarboxylicacidasdepictedinfigure. •Ithasbeendulyobservedthataratherlargesegmentofpenicillin- resistantpathogenicstrainsofStaphylococcusaureusinvariablycompriseofthisspecificenzyme;andperhapsitoverwhelminglycontributesamajorfactorofpenicillinresistanceduringinfection. •Importantly,thisenzymecausesanextremelyrapiddegradationofpenicillinparticularlyinpenicillinfermentationsincaseaspecificcontaminantwhichproducestheenzymeincidentallygainsanaccesstoandbeabletogrowsimultaneouslyinthefermentationbroth. •PenicillinaseobtainedfromB.subtilisandB.cereusrepresenttheindustriallyproducedenzymeswhichexertactiontosomeextentintheremovalofpenicillinviaspecificinactivation.
  48. 48. Streptokinase •Streptokinaseisasingle-chaincoenzymeobtainedfromculturesoftheGroupCβstrainofStreptococcushaemolyticusthatiscapableofsolelyconvertingplasminogentoplasmin. •Itisusedextensivelyasapredominantfibrinolyticagenttohelpinabigwayforthespecificremovaloffibrinthrombi(bloodclot)fromarteries. •TheGlobalutilizationofStreptokinaseandtPA(tissuetypeplasmogenactivator)ofOccludedArteries(GUSTO)trialwasdesignedmeticulouslytoinvestigateandestablishthebenefitsofa‘frontloaded’doseofalteplase(alargeinitialbolusfollowedbyaninfusionofthetotaldoseoveraspanof90minutes)whencomparedwiththeusualconventionalalteplaseadministration. Note:alteplaseistheFDAapprovedrecombinanttissueplasminogenactivator.
  49. 49. •Besides,anintensiveandextensivestudywascarriedoutwithrespecttothesafetyandimprovementinmortalityofcombiningalteplaseandstreptokinasewitheitheragentalone.Interestingly,theoutcomeofresultsfromtheseextendedinvestigationsappeartoexhibitanappreciablyfavourablemortalityrateinpatientshavingbeentreatedwithalteplasecomparisontothestreptokinasetreatedsubjects. •Amixtureofstreptokinaseandstreptodornase,asproducedbyahemolyticstreptococcusgrowninaspecificenvironmentofaerated-submergedculture,isusedmeticulouslytocleanupthedebrisfromwoundsandburnseffectively.
  50. 50. 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.
  51. 51. Streptodornase •Streptodornasereferstooneoftheenzymesproducedbycertainstrainsofhaemolyticstreptococci.Itiscapableofliquefyingfibrinousandpurulentexudates. •Itisalsoemployedsometimesasawounddebridementi.e.,removalofdamagedtissue. •StreptodornaseisactuallyobtainedfromStreptococcushaemolyticusthataffordsdepolymerizationofpolymerizeddeoxyribonucleoproteins. •Itisusedextensivelyinconjunctionwithstreptokinaseasde-sloughingagenttocleanseulcersandpromotethehealingprocessprogressively.
  52. 52. 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.

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