Protocols and applications in enzymology seema anil belorkar & sudisha jogaiah - Own the ebook now w by Education Libraries

https://ebookmass.com/product/protocols-and-applications-in-

Instant digital products (PDF, ePub, MOBI) ready for you

Download now and discover formats that fit your needs...

Biocontrol Agents and Secondary Metabolites - Applications and Immunization for Plant Growth and Protection Sudisha Jogaiah

https://ebookmass.com/product/biocontrol-agents-and-secondarymetabolites-applications-and-immunization-for-plant-growth-andprotection-sudisha-jogaiah/ ebookmass.com

Seaweed polysaccharides : isolation, biological and biomedical applications 1st Edition Anil

https://ebookmass.com/product/seaweed-polysaccharides-isolationbiological-and-biomedical-applications-1st-edition-anil/

ebookmass.com

Bio-Based Nanomaterials : Synthesis Protocols, Mechanisms and Applications Ajay Kumar Mishra

https://ebookmass.com/product/bio-based-nanomaterials-synthesisprotocols-mechanisms-and-applications-ajay-kumar-mishra-2/ ebookmass.com

Highland Gladiator Kathryn Le Veque [Veque

https://ebookmass.com/product/highland-gladiator-kathryn-le-vequeveque-3/

ebookmass.com

Research, Ethics and Risk in the Authoritarian Field 1st Edition Marlies Glasius

https://ebookmass.com/product/research-ethics-and-risk-in-theauthoritarian-field-1st-edition-marlies-glasius/

ebookmass.com

Psychology 13th Edition David G Myers

https://ebookmass.com/product/psychology-13th-edition-david-g-myers-2/

ebookmass.com

Applied Generative AI for Beginners: Practical Knowledge on Diffusion Models, ChatGPT, and Other LLMs 1st Edition Akshay Kulkarni

https://ebookmass.com/product/applied-generative-ai-for-beginnerspractical-knowledge-on-diffusion-models-chatgpt-and-other-llms-1stedition-akshay-kulkarni/ ebookmass.com

The Dead Letter Seeley Regester

https://ebookmass.com/product/the-dead-letter-seeley-regester/

ebookmass.com

Once Upon a Mail Order Bride Linda Broday

https://ebookmass.com/product/once-upon-a-mail-order-bride-lindabroday-4/

ebookmass.com

The Vanished Days Susanna Kearsley

https://ebookmass.com/product/the-vanished-days-susanna-kearsley-3/

ebookmass.com

代找亚马逊电子书zlib没有的qq2572480550

Protocolsand Applicationsin Enzymology

Thispageintentionallyleftblank

zlib没有的qq2572480550

Protocolsand Applicationsin Enzymology

SeemaAnilBelorkar

AssistantProfessor, MicrobiologyandBioinformaticsDepartment, AtalBihariVajpayeeUniversity, Bilaspur(C.G),India

SudishaJogaiah

AssistantProfessor, LaboratoryofPlantHealthcareandDiagnostics, PGDepartmentofStudiesinBiotechnologyandMicrobiology, KarnatakUniversity,PavateNagar,Dharwad,Karnataka,India

AcademicPressisanimprintofElsevier 125LondonWall,LondonEC2Y5AS,UnitedKingdom 525BStreet,Suite1650,SanDiego,CA92101,UnitedStates 50HampshireStreet,5thFloor,Cambridge,MA02139,UnitedStates TheBoulevard,LangfordLane,Kidlington,OxfordOX51GB,UnitedKingdom

Nopartofthispublicationmaybereproducedortransmittedinanyformorbyany means,electronicormechanical,includingphotocopying,recording,oranyinformation storageandretrievalsystem,withoutpermissioninwritingfromthepublisher.Detailson howtoseekpermission,furtherinformationaboutthePublisher’spermissionspolicies andourarrangementswithorganizationssuchastheCopyrightClearanceCenterandthe CopyrightLicensingAgency,canbefoundatourwebsite: www.elsevier.com/permissions .

Thisbookandtheindividualcontributionscontainedinitareprotectedundercopyright bythePublisher(otherthanasmaybenotedherein).

Notices

Knowledgeandbestpracticeinthis ﬁeldareconstantlychanging.Asnewresearchand experiencebroadenourunderstanding,changesinresearchmethods,professional practices,ormedicaltreatmentmaybecomenecessary.

Practitionersandresearchersmustalwaysrelyontheirownexperienceandknowledgein evaluatingandusinganyinformation,methods,compounds,orexperimentsdescribed herein.Inusingsuchinformationormethodstheyshouldbemindfuloftheirownsafety andthesafetyofothers,includingpartiesforwhomtheyhaveaprofessionalresponsibility.

Tothefullestextentofthelaw,neitherthePublishernortheauthors,contributors,or editors,assumeanyliabilityforanyinjuryand/ordamagetopersonsorpropertyasa matterofproductsliability,negligenceorotherwise,orfromanyuseoroperationofany methods,products,instructions,orideascontainedinthematerialherein.

LibraryofCongressCataloging-in-PublicationData

AcatalogrecordforthisbookisavailablefromtheLibraryofCongress

BritishLibraryCataloguing-in-PublicationData

AcataloguerecordforthisbookisavailablefromtheBritishLibrary

ISBN:978-0-323-91268-6

ForinformationonallAcademicPresspublicationsvisitourwebsiteat https://www.elsevier.com/books-and-journals

Publisher: AndreGerhardWolff

AcquisitionsEditor: MichelleFisher

EditorialProjectManager: SamW.Young

ProductionProjectManager: NiranjanBhaskaran

CoverDesigner: AlanStudholme

TypesetbyTNQTechnologies

Preface ............................................................................................... xvii

CHAPTER1Enzymes past,present,andfuture .........................1

1.1 Generalintroduction. ..........................................................1

1.2 Characteristicsofenzymes... ................................................1

1.2.1Enzymeasamolecule................................................4

1.2.2Catalyticalproperty... ................................................5

1.2.3Speciﬁcity ................................................................5

1.2.4Holoenzyme... ..........................................................6

1.2.5Turnovernumber.. .....................................................7

1.2.6Reversibility... ..........................................................7

1.2.7Weightofenzyme .....................................................7

1.2.8Sensitivity ................................................................7

1.2.9Activesite. ...............................................................8

1.3 Theconceptofcell-freefermentation ....................................8

1.4 Nomenclatureofenzymes ....................................................8

1.5 Enzymes thepresentscenario.. ...........................................9

1.5.1Substratebinding. ....................................................10

1.5.2Mechanismofenzymecatalyzedreaction .....................10

1.5.3Catalysis.. ...............................................................12

1.6 Recentdevelopments ........................................................12

1.6.1Immobilization ........................................................12

1.6.2Biosensors...............................................................12

1.6.3Diagnostictools... ....................................................13

1.6.4Metagenomics .........................................................13

1.7 Thefuture .......................................................................13 References. ............................................................................14

CHAPTER2Distributionanddiversityinmicrobial enzymes ................................................................17

2.1 Originofenzymediversity... ..............................................17

2.2 Naturalnicheasadiversitysource... ...................................17

2.3 Evolutionandfunctionofenzymes... ...................................20

2.4 Acidophilicenzymes. ........................................................20

2.4.1Adaptationatcellularlevel... .....................................29

2.5 Alkaliphiles... ..................................................................34

2.6 Halophilicorganisms ........................................................36 v 代找亚马逊电子书

CHAPTER3Screeningofpotentialmicrobesforenzymes ofindustrialsigniﬁcance

3.1 History..

3.2 Highthroughputscreeningmethods needofpresentday

3.2.1Enrichmentculture..

3.2.2Uncultivablesasenzymesource a metagenomicapproach..

3.2.3Directedenzymeevolution..

3.2.4Computationalbiology(insilicostudies)....

3.3 Throughputmethodsforscreeningofenzymevariants

3.3.1Selectionscreening

3.3.2Agarplatemethod.

3.3.3Microtiterplatescreeningmethod.

3.3.4Fluorescentactivatedcellsorting..

3.3.5Invitrocompartmentalization.

3.3.6Droplets ................................................................58

3.3.7Plasmiddisplay

3.3.8Phagedisplay...

3.3.9Ribosomedisplaym-RNA.

3.3.10m-RNAdisplay

3.3.11Thec-DNAdisplay .................................................62

3.3.12Reporter-basedscreening.. .......................................63

3.3.13Fluorescence-activateddropletsorting. .......................64

3.3.14Digitalimaging

3.4 Conclusionandfutureperspective..

4.1 Historyoffermentationforsocialbeneﬁts.

4.2 LouisPasteurandfermentation ...........................................72

4.3 Fermentation ...................................................................73

4.3.1SMFfermentation... .................................................74

4.3.2Solid-statefermentation. ............................................79

4.4 Conclusionandfuturedirections

Subchapter5.4Assayofenzyme

Safetyconsiderationsandstandards.. .......................................161 Alternativemethods/procedures... ............................................161 References. ..........................................................................161

CHAPTER8Strategiestoimproveenzymeactivityfor industrialprocesses.............................................163

8.1 Introduction... ................................................................163

8.1.1Improvementofenzyme-basedprocess.. ....................163

8.1.2Strategiestoachieveefﬁcientbiocatalysis ..................166

8.1.3Heterogeneousbiocatalysis... ...................................170

8.1.4Substrateengineering. .............................................171

8.1.5Advancedstrategiesforenzymeimprovement .............171

8.1.6Alteringthesignalpeptide .......................................172

8.1.7Metagenomics .......................................................172

8.1.8Geneticengineering... .............................................173

8.2 Conclusion ....................................................................174 References. ..........................................................................174

CHAPTER9Scopeandrelevanceofindustrialapplications .....179

9.1 Introduction... ................................................................179

9.2 Enzymesinindustries .....................................................180

9.3 Commonstrategiesforenzymeimmobilization. ..................181

9.3.1Useofcarriers .......................................................181

9.3.2Covalentlinkages. ..................................................181

9.3.3Entrapment............................................................182

9.4 Someimportantindustrialenzymes.. .................................182

9.4.1Fructosyltransferase... .............................................182

9.4.2Lipases .................................................................184

9.4.3Proteases.. .............................................................187

9.5 Biocatalyticmembranes.. .................................................189

9.6 Conclusion ....................................................................190 References. ..........................................................................190

CHAPTER10Agroindustrialwastesforenzymeproduction ......197

10.1 Introduction. ................................................................197

10.2 Agroindustrialwastes:excellentrawmaterialfor production... ................................................................197

10.3 Impactofagroindustrialwastesonenvironment: aglobalconcern ...........................................................198

10.4 Wastetovalue.. ...........................................................200

CHAPTER12Biotechnologicalapplicationsofenzymesand futureprospective ..............................................225

12.1 Introduction. ................................................................225

12.2 Enzymesinmedicalﬁeld... ............................................225

12.2.1Enzymesanddiagnosis... .....................................225

12.2.2Desirablefeaturesofdiagnosticenzyme. ................227

12.2.3Indiseasediagnosis.. ..........................................227

12.2.4Coupledenzymeassay.........................................229

12.2.5Immunologicalreactions. .....................................229

12.2.6DNA-baseddiagnostic .........................................229

12.2.7Therapeuticenzymes. ..........................................230

12.3 Thebiologicaldetergents:arevolutioninlaundry industry. .....................................................................232

12.3.1Proteases. ..........................................................234

12.3.2Lipase...............................................................235

12.3.3Amylases ..........................................................235

12.3.4Cellulases ..........................................................235

12.4 Conclusion.. ................................................................236 References. ..........................................................................237

Thispageintentionallyleftblank

没有的qq2572480550

Preface

Protocolsandapplicationsinenzymology isabookpresentingintensive,coordinated,andsynchronizedinformationwiththeperfectblendofhistoricalinformation,thrustareasinresearch,andtheresultantprotocolsmuchdesiredinthe academicsandresearchlabs.

Thisbookisaone-stopsolutionforstudents,academicians,researchbeginners, andentrepreneurs.Theinitialtwochaptersrevealtheglorioushistoryofenzymologywiththepioneeringresearchanditsdeep-rootedconnectionwiththediscoveriesbyeminentchemists.Itexplainsthediversityanenzymemoleculeexhibitsand theirimplicationsonindustrialprocesses.

Thebookdescribesvariousstagesofscreeningtechnologies,conventionalVs modern,fermentations,andtheirtypesandprovidesexclusiveresearch-basedprotocolsforindustriallyimportantenzymeswhicharedesperatelyrequiredinresearch labsandindustrialresearchunits.Thus,thisbookisalinktoprotocolsdesiredbythe industriesastheintensiveoutcomeoftheresearchcommunity.Italsoelucidatesthe “Wastetovalue”termbydiscussingconversionofthetrappedenergyinwastesinto bioactivemolecules.

Thebookwillhopefullyupdatethereadersfromagriculturaltoindustrialsector withrecentadvancesinusageofenzymesasfrontiertoolsandﬁnallyfocusingon applicationsofenzymesindifferentwalksoflifeandfutureapplications.

(Dr.SeemaAnilBelorkarandDr.SudishaJogaiah)

Authors

Thispageintentionallyleftblank

zlib没有的qq2572480550

Enzymes—past,present, andfuture 1

1.1 Generalintroduction

Enzymestodayneednointroduction.Theyarewellrecognizedandacknowledged ineverywalkoflifefromalaymantoaresearchpersonentangledinmolecular biologyprotocols.Thesemagicalmoleculeshaveprovedtobeanefﬁcienttoolin almostallindustriesdirectlyorindirectly.

Theterm“Enzyme”wasproposedbyWilhelmKuhne,whichinGreekmeans “leavened”or“inyeast”in1877.EnzymeshavegainedrecognitionasaBiological catalyst,whichisgenerallyproteinswiththeexceptionsofribozymes.Thenature andactivityofanyenzymedependuponthesequenceofaminoacidsofthepolymer. Theenzymescanbeactiveasasinglechain(monomericprotein)orcanhaveanumberofpolymericchainsinformofafunctionalcomplex(oligomericprotein).The functionofanenzymeisdependentonitsstructureandconformationthatinturn dependsontheaminoacidsequence(Bergetal.,2002).Thestructureofanyenzyme isaverysensitiveandadaptivefeatureincontexttoitschangingenvironment.It cannotbethesolefeaturegoverningitsfunction(Somero,1978).

Thebasicdevelopmentofenzymologyisaccreditedtopioneeringobservations intheﬁeldthatareenlistedin Table1.1.

1.2 Characteristicsofenzymes

Enzymesarebiologicalcatalysts.Theycatalyzebiologicalreactionsinasimilar mannerasChemicalcatalystsdo.Theenzymestransformtheirsubstrates(reactants) toproductsundernormalconditionsoftemperatureandpressureincontrastto chemicalcatalyst. Fig.1.1 elucidatesthegeneralpathofenzymeaction.Itisa two-stepreaction.ThefirststepisreversibleandformsanunstableEScomplex. Ifthesubstrateisspecific,thereactionprogressesforthesecondstep;otherwise, thebreakdowncausesthereleaseofthesubstrateandabackwardreactiontakes place.Thesecondstepisirreversibleandprogressesonlyintheforwarddirection whentheenzymefindsitsspecificsubstrates. CHAPTER 1

Table1.1 Pioneeringcontributionsinenzymology.

YearNoblelaureatesContributions

2018FrancesH.ArnoldThedirectedevolutionofenzymes

GeorgeP.Smith SirGregoryP.Winter

2017JacquesDubochet JoachimFrank RichardHenderson

2016Jean-PierreSauvage

SirJ.FraserStoddart BernardL.Feringa

2015TomasLindahl PaulModrich AzizSancar

2012RobertJ.Lefkowitz BrianK.Kobilka

2009Venkatraman Ramakrishnan

ThomasA.Steitz AdaE.Yonath

2008OsamuShimomura

MartinChalﬁe RogerY.Tsien

2002JohnB.Fenn

KoichiTanaka

KurtWuthrich

1997PaulD.Boyer

JohnE.Walker

JensC.Skou

1993KaryB.Mullis

MichaelSmith

Thephagedisplayofpeptidesandantibodies

Developingcryo-electronmicroscopyforthehighresolutionstructuredeterminationofbiomolecules insolution

Thedesignandsynthesisofmolecular machines

MechanisticstudiesofDNArepair

StudiesofG-protein-coupledreceptors

Studiesofthestructureandfunctionofthe ribosome

Thediscoveryanddevelopmentofthegreen ﬂuorescentprotein,GFP

2006RogerD.KornbergThemolecularbasisofeukaryotictranscription

Developmentofsoftdesorptionionizationmethods formassspectrometricanalysesofbiological macromolecules

Developmentofnuclearmagneticresonance spectroscopyfordeterminingthethree-dimensional structureofbiologicalmacromoleculesinsolution

Elucidationoftheenzymaticmechanism underlyingthesynthesisofadenosinetriphosphate (ATP)

Fortheﬁrstdiscoveryofanion-transporting enzyme,Naþ,Kþ-ATPase

Inventionofthepolymerasechainreaction(PCR) method

Oligonucleotide-based,site-directedmutagenesis anditsdevelopmentforproteinstudies

1990EliasJamesCoreyThetheoryandmethodologyoforganicsynthesis

1989SidneyAltman

ThomasR.Cech

1975JohnWarcupCornforth VladimirPrelog

DiscoveryofcatalyticpropertiesofRNA

Thestereochemistryofenzyme-catalyzedreactions

Thestereochemistryoforganicmoleculesand reactions

1974PaulJ.FloryThephysicalchemistryofthemacromolecules 2 CHAPTER1 Enzymes—past,present,andfuture

Table1.1 Pioneeringcontributionsinenzymology. cont’d

YearNoblelaureatesContributions

1972ChristianB.Anﬁnsen

StanfordMoore

WilliamH.Stein

Ribonuclease,especiallyconcerningthe connectionbetweentheaminoacidsequenceand thebiologicallyactive conformation

Theconnectionbetweenchemicalstructureand catalyticactivityoftheactivecenterofthe ribonucleasemolecule

1970LuisF.LeloirDiscoveryofsugarnucleotidesandtheirroleinthe biosynthesisofcarbohydrates

1969DerekH.R.Barton OddHassel

Thedevelopmentoftheconceptofconformation anditsapplicationinchemistry

1965RobertBurnsWoodwardAchievementsintheartoforganicsynthesis

1964DorothyCrowfoot Hodgkin

1963KarlZiegler GiulioNatta

1962MaxFerdinandPerutz JohnCowderyKendrew

X-raytechniquesofthestructuresofimportant biochemicalsubstances

Thechemistryandtechnologyofhigh polymers

Thestructuresofglobularproteins

1961MelvinCalvinThecarbondioxideassimilationinplants

1958FrederickSangerStructureofproteins,especiallythatofinsulin

1957Lord(AlexanderR.)ToddNucleotidesandnucleotidecoenzymes

1956SirCyrilNorman Hinshelwood NikolayNikolaevich Semenov

Mechanismofchemicalreactions

1955VincentduVigneaudTheﬁrstsynthesisofapolypeptidehormone

1953HermannStaudingerMacromolecularchemistry

1950OttoPaulHermannDiels KurtAlder

1946JamesBatchellerSumner JohnHowardNorthrop WendellMeredithStanley

1929ArthurHarden

HansKarlAugustSimon vonEuler-Chelpin

Discoveryanddevelopmentofthediene synthesis

Enzymescanbecrystallized Preparationofenzymesandvirusproteinsinapure form

Thefermentationofsugarandfermentative enzymes

1926The(Theodor)SvedbergDispersesystems

1909WilhelmOstwaldCatalysisandforhisinvestigationsintothe fundamentalprinciplesgoverningchemical equilibriaandratesofreaction

1907EduardBuchnerBiochemicalresearchesandhisdiscoveryofcellfreefermentation

1902HermannEmilFischerRecognitionoftheextraordinaryserviceshehas renderedbyhisworkonsugarandpurinesyntheses

FIGURE1.1

Generalmechanismofenzyme-catalyzedreaction.

1.2.1 Enzymeasamolecule

Enzymesaremacromoleculeshavingahighmolecularweight(MW)andaredisproportionatelybiggerwhencomparedtotheirsubstrate.Generally,theMWofenzymescoversabroadrange.Thegeneralpropertiesinrelationtoitsfunctionsare explainedin Fig.1.2.

DixonandWeb(1958) tabulatedenzymesinthreeseriesorclasses(2n 12,000, 2n 16,000,and2n 19,000),where n isintegral0 4.Ahypothesiswasproposed by Wright(1962) usingexperimentaldatabutasthegroupsofenzymeswere formed,thereremainedaquestionofBiasedness. Johnstonetal.(1945) proposed abetterapproachofthecorrelationfunctionforvalidationoftheSvedberghypothesis(SvedbergandPedersen,1940).TheMWofproteinsiscalculatedbySvedberg’s equationdependingontheforceappliedandthemassofthesedimentingmolecule.

FIGURE1.2

Propertiesandfunctionsofenzymes.

Thesedimentationvelocitywasfundamentallyusedforthedeterminationofmolecularweightsofproteins.Presently,anapproachusingGelﬁltrationChromatography andSDS-PAGEaretheadvancedmethodsfortheMWdetermination.

Asweallknowthatmostenzymesareproteins,theirgeneralbehavioris colloidalasanattributeoftheirhighmolecularweight.Enzymesthatareproteinaceousarethermolabileinnature.Generally,enzymesareactiveatroomtemperature, atlowtemperaturestheyarereversiblyinhibited,andathighertemperaturestheyare irreversiblyinactivated.

Although,thistemperaturesensitivitycanbehandled,ifenzymesarestoredin theformofdriedextracts.Thestabilityoftheenzymesincreasesalongwith increasedshelflife.EveryenzymerequiresamicablepHandtemperatureconditions toexplicititshighestactivityreferredasoptimumpHandtemperature.Inzones beforeandaftertheoptimumzone,theenzymeactivitydecreases. Fig.1.3 explains themaximumenzymeactivityinresponsetooptimumpHandtemperature conditions.

1.2.2 Catalyticalproperty

Amoststrikingfeatureoftheenzymeisitscatalyticpowerthatmakesitarobust synthetictool.Incomparisontochemicalcatalysts,enzymesarewonderfulbecause theycatalyzethetransformationofsubstratesatnormaltemperatureandpressure availableinsidethebiologicalsystem.Enzymecatershighpotentialofsubstrate conversionascomparedtoreactionsthatrunbythemselves.Itremainsunalteredafterthereactioniscomplete,providingthealternativeofitsrecyclingforbetteryield.

1.2.3

Speciﬁcity

Theuniquenessoftheenzymeisitsstructuralconformationthatrendersthequality ofbeinganeffectivecatalyst.Thecatalysisconferredbytheenzymeisveryfocused

FIGURE1.3 SensitivityoftheenzymestowardpHandtemperature.

Table1.2 Differenttypesofspeciﬁcitiesexhibitedbyenzymes.

Typesofenzymespeciﬁcities

Bondspeciﬁcity (relativespeciﬁcity)

Groupspeciﬁcity (structural speciﬁcity)

Substrate speciﬁcity (absolute speciﬁcity)

Opticalspeciﬁcity (stereo-speciﬁcity)

(Cofactor speciﬁcity)

(Geometric speciﬁcity)

EnzymeActionon

Peptidase Lipase Peptidebond Esterbond

EnzymeActionon

PepsinPeptidebondlinkedtoaromaticamino acidslikephenylalanine,tyrosine,and tryptophan

EnzymeActionon

Sucrase Arginase Carbonicanhydraseo Sucrose Arginine carbonicacid.

EnzymeActionon L-aminoacidoxidase a-amylase L-aminoacids a-1-4glycosidiclinkageofstarchand glycogen

EnzymeCofactor

Glucose-6-phosphate dehydrogenase NADH

EnzymeSubstrates Alcoholdehydrogenase canoxidize Chymotrypsin

Methanoland n-propanoltoaldehydes.

Peptidebondandesterbond

andconfinedtoeitheroneoragroupofstructurallyrelatedmolecules.Thespecificitycanbeclassifiedintoabsolutespecificityandrelativespecificity.Nowadays, enzymesarefindingimmenseapplicationsongroundsofRegioselectiveandStereoselective(Muetal.,2020). Table1.2 explainsthespecificitiesofthedifferent enzymesalongwithexamplesfallingineachcategory.

1.2.4 Holoenzyme

Manyenzymeshaveanonproteinpartalongwiththem.Suchconjugatedenzymes foundassociatedwithanotherkindofmoleculesarecalledtheholoenzyme.The proteincomponentiscalledastheapoenzyme,andthenonproteincomponentis calledthecofactor.Ifthecofactorisinorganicinnature,itisreferredasprosthetic groupandifitisorganicinnature,itiscalledascoenzyme.Theprostheticgroups areintenselyassociatedwiththeapoenzymeandarenoteasilyavailable.Contrarily, thecoenzymesareverylooselyboundresultingineasyseparationfromtheprotein component.

Thecofactorisahelpermicromoleculethatconfersenzyme,itsfunctionality. Thenonproteinnaturemakesitthermostable.Themainroleofcofactorisit

1.2 Characteristicsofenzymes

becomesasigniﬁcantpartinthereactioncenteroftheenzymeforcatalysis involvingtheremovaloffunctionalgroups.Cofactorshavetheabilitytogetassociatedwithotherenzymesandhelptheirfunctionalityafterassociation.

1.2.5 Turnovernumber

Asweallknow,enzymesarebiologicalcatalyststhatremainunalteredafterthereactioniscomplete.Therateofenzymecatalystreactionsdependsuponthenumber ofsubstratemoleculesconvertedintotheproductinagivenspecifiedtime.Turnover numberisthereforedefinedastheamountofsubstrateconvertedintotheproductin 1s,inthegivenspecifiedcondition(wheretheconcentrationofsubstrateisinsaturation).Enzymesexhibitawidevarietyofturnovernumbersrangingfrom0.5to105.

1.2.6 Reversibility

Whenwefocusoncatalyzedreactions,theenzymecatalyzedreactionsdifferfrom thechemicalcatalystinawaythatitfunctionsaccordingtotheneedofthebiologicalsystem.Thenatureoftheenzymevariesinaccordancewiththeroleitis executinginthemetabolism.Therefore,enzyme-catalyzedreactionsincludeunidirectional,bidirectionaltypes,andeveneegulatoryreactions.

1.2.7 Weightofenzyme

Mostoftheenzymesrangebetween20kDand60,000kDmolecularweight.The3Dstructureisvisualizedintermsofamacromoleculewith327nminsizeandeventuallyappearstobeverylargeincontexttothecellularenvironment.Thereviewof theenzymerevealedthelargesizeoftheenzymeoffersalargesurfaceareaofthe moleculetocircumventthebindingsitesofsubstrateswithappropriateorientation.

Therearereportsoftwoesterasesverysmallmolecularweightenzymesfrom Candidalipolytica and Bacillusstearothermophilus with5.7kDa(56aminoacid residue)andthe Bacillus enzymeis1.57kDa(17aminoacids)(Matteyetal., 1998).ThehighestMWenzymesreportedarefrom Y.lipolytica withamolecular weightof120kDa,the90kDaleucineaminopeptidaseIIfrom Aspergillusoryzae (Nicaudetal.,2002),andglucoamylasefrom Arxulaadeninivorans (Swennen etal.,2002).

1.2.8 Sensitivity

Yetanotherfeatureofenzymesisthattheyareverysensitivetotheenvironmentin whichtheyarepresent.Theenvironmentalconditionsimposetremendousinﬂuence onthethree-dimensionalstructureoftheenzymemoleculeandhenceaffectitsactivityandcatalyticpowertotheextentwhetheritwillremaininactivestateorbe inactive.

ThepH,temperature,andconcentrationofionsarefewfactorsthatimpedethe biologicalfunctioningofthemolecule.Theseconsequencesareduetothe