Cellulase assays
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Cellulaseassays
Cellulaseenzymesshowactivityduringtheripeningofsomefruits,wheretheireffectsoncellwallsresultsinsofteningofthefruit.Incasesofprogrammedcelldeath,suchastheformationofaerenchyma(largeairspacesinthecortexofplantsinfloodedsoils),andintheabscissionzonesofleavesandfruits,cellulasesareonceagainveryactive,breakingdownthecellulosewallsofthedeadcells.TwopossIBLeprotocolsaredescribedhere,ageldiffusionassaymethodandaviscosityreductionassaymethod.
Geldiffusionmethod
Afruitextractisplacedinawellinsomeagarcontainingcarboxymethylcellulose(CMC),and,ascellulasemoleculesdiffuseoutwardsfromthewell,theydestroycellulosemolecules.Themoreconcentratedtheenzymesinthewell,thelargerthezoneofcellulosedestruction. |
- 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.
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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