Cellulaseenzymesshowactivityduringtheripeningofsomefruits,wheretheireffectsoncellwallsresultsinsofteningofthefruit.Incasesofprogrammedcelldeath,suchastheformationofaerenchyma(largeairspacesinthecortexofplantsinfloodedsoils),andintheabscissionzonesofleavesandfruits,cellulasesareonceagainveryactive,breakingdownthecellulosewallsofthedeadcells.TwopossIBLeprotocolsaredescribedhere,ageldiffusionassaymethodandaviscosityreductionassaymethod.

Geldiffusionmethod

• Prepareanagargelcontaining1.7%agarand0.5%CMC(carboxymethylcellulose).Pourthisgelintopetridishesandallowittoset.
• Afterthegelhasset,useanarrowcork-borertopunchsmallcylindersinthegel.Then,usingamountedneedle,removeeachofthesecylinderstocreateaseriesofsimilarsizedwellsintheagar.Fourormorewellscanbeputinasingledish,providedtheyarespacedapart.
• Placesimilarvolumesofextractsoffruitsintheeachofthewells.Inonewell,placesomedistilledwater,asacontrol.Incubatethedishesforatleast24hoursat30°C.
• Aftertheincubationperiodisfinished,usetapwatertorinseoutthecontentsofthewells,andthenfloodthedisheswithCongoredsolutionfor15minutes.Thenrinsethedisheswith1MNaClsolutionforatleast10-15minutes.

Wellscontainingcellulaseshouldhaveaclearzonearoundthem,andthediameterofthezonegivesameasureofthecellulaseactivityinthatwell.

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Viscosityreductionmethod

Thetechniqueisbasedontheactionofcellulaseenzymeswhichshortenthelengthsofcellulosemoleculesinaviscoussolutionofwallpaperpasteandcauseittobecomelessviscous(runnier).

• Makeupa2%(w/v)wallpaperpastesolution,sufficienttoprovide25cm³foreachsampletobetested.
• Place25cm³ofthepasteinaboilingtubeandadd2to5cm³offruitextract.Mixthoroughly.
• Thenpourthemixtureintothebarrelofasyringe,heldinaretortstand,pointingdownwardsintoasmallbeaker.Notethetimetakenforallthemixturetodrainthroughthesyringenozzleintothebeaker.
• Incubatethemixtureinawater-bathat30°C,checkingthechangeinviscosityaboutevery30minutes.

Themoreactivetheenzyme,thegreaterthereductioninviscosity,andsotheshorterthedrainagetimes.

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