Pectinase assay

Pectinaseassay

Pectinasesareactuallyamixtureofenzymes,which,alongwithotherssuchascellulase,arewidelyusedinthefruitjuiceindustrywheretheyarewidelyusedtohelpextract,clarifyandmodifyfruitjuices.(Seethefootofthispageformoreinformation)

Pectinsarelargepolysaccharidemolecules,madeup(mainly)ofchainsofseveralhundredgalacturonicacidresidues.Enzymesinthispectinasegroupincludepolygalacturonases,pectinmethylesteraseandpectinlyases.Thesepectinaseenzymesactindifferentwaysonthepectins,whicharefoundintheprimarycellwallsandinthemiddlelamella.PectinsarewellknownalsofortheirABIlitytoformgels.

Pectinasesareproducedduringthenaturalripeningprocessofsomefruits,wheretogetherwithcellulases,theyhelptosoftentheircellwalls.TheseenzymesarealsosecretedbyplantpathogenssuchasthefungusMoniliniafructigenaandthesoft-rotbacteriumErwiniacarotovora,aspartoftheirstrategyforpenetratingtheplanthostcellwalls.Infact,theproductsofsuchenzymeassaults(oligosaccharins)actasasignalwhichinducesuninfectedcellstodefendthemselves.

Theprincipleofthisassaydependsuponmeasuringtheamountofwateryjuicereleasedfromtinnedapplepuree(whichisveryrichinpectin)asaresultofpectinaseaction.

Tinnedorbottledapplepuree(sauce)canbepurchasedforthisassay.

• Tomakeanextractofthefruitorvegetable,blend2cm³ofwaterforevery1goffruit.Prepareatleast25cm³ofextract.
• Collectandlabeltwoboilingtubesandplace25cm³ofapplesauceineachofthem.
• Add25cm³ofextracttooneofthetubesand25cm³ofwatertotheother,toactasacontrol.
• Useaglassrodtomixthoroughlythecontentsofbothtubesandthenleavetheminaboilingtuberackinawaterbathat35°Cforatleast30minutes.
• Taketwosimilarsizedfunnels(funnelsizeabout100cm³issuitable)andsupportthemovertwosimilarsmall(25cm³)measuringcylinders.
• Aftertheincubationperiod,pourthecontentsofthetwoboilingtubesintothetwofunnels,andallowthejuicestodrainintothemeasuringcylinders.Allowatleastfiveminutesforthedrainingtofinishandnotethevolumesofjuiceobtained.

Thedifferencesinthevolumeofjuicebetweenthetwotubesgivesameasureofthepectinaseactivityintheextract.

Ifthisexperimentisbeingcarriedoutonalargescale,theinitialdrainagefromthefunnelscouldbecollectedintesttubes,andthenthevolumeofjuicecollectedismeasuredlater.

Manyindustrialenzymeproductsaremixturesofdifferentenzymes.Thusa"pectinase"preparationmightcontainarangeofpectinasesandcellulases.Otherpreparationsmightcontainonlyasingletypeofenzyme,especiallyiftheenzymesareproducedbygenetically-modifiedstrainsorarehighlypurified.

Whenplanningyourowninvestigations,itisthereforeveryimportanttostudythedatasheetsuppliedwitheachenzyme.Thiswillindicatewhethertheproductisamixtureorcontainsjustonetypeofenzyme.

Thedatasheetalsoprovidesaroughguidetohowtheenzymemightbehave.Inpractice,however,enzymeactivityisaffectedbymanythings(e.g.pH,temperature,thepresenceofinhibitorsorcofactors)whichwillaffecttheresultsyouobtain.Inaddition,specimenactivitygraphsareoftenpreparedusingsimplesubstancesunderidealconditionsratherthanthecomplexsubstratesandsub-optimalconditionsthatmaybeencounteredinanindustrialorschoolcontext.