Novelsubstratesfortheautomatedandmanualassayofendo-1,4-β-xylanase.
Mangan,D.,Cornaggia,C.,Liadova,A.,McCormack,N.,Ivory,R.,McKie,V.A.,Ormerod,A.&McCleary,D.V.(2017).CarbohydrateResearch,445,14-22.
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endo-1,4-β-Xylanase(EC3.2.1.8)isemployedacrossabroadrangeofindustriesincludinganimalfeed,brewing,baking,biofuels,detergentsandpulp(paper).Despiteitsimportance,arapid,reliable,reproduc
IBLe,automatableassayforthisenzymethatisbasedontheuseofachemicallydefinedsubstratehasnotbeendescribedtodate.Reportedhereinisanewenzymecoupledassayprocedure,termedtheXylX6assay,thatemploysanovelsubstrate,namely4,6-
O-(3-ketobutylidene)-4-nitrophenyl-β-4
5-
O-glucosyl-xylopentaoside.ThedevelopmentofthesubstrateandassociatedassayisdiscussedhereandtherelationshipbetweentheactivityvaluesobtainedwiththeXylX6assayversustr
ADItionalreducingsugarassaysanditsspecificityandreproducibilitywerethoroughlyinvestigated.
Comparisonofendolytichydrolasesthatdepolymerise1,4-β-D-mannan,1,5-α-L-arABInanand1,4-β-D-galactan.
McCleary,B.V.(1991).“EnzymesinBiomassConversion”,(M.E.HimmelandG.F.Leatham,Eds.),ACSSymposiumSeries,460,Chapter34,pp.437-449.AmericanChemicalSociety,Washington.
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Hydrolysisofmannan-typepolysaccharidesbyβ-mannanaseisdependentonsubstitutiononandwithinthemain-chainaswellasthesourceoftheβ-mannanaseemployed.Characterisationofreactionproductscanbeusedtodefinethesub-sitebindingrequirementsoftheenzymesaswellasthefine-structuresofthepolysaccharides.Actionofendo-arabinanaseandendo-galactanaseonarabinansandarabinogalactansisdescribed.Specificassaysforendo-arabinanaseandarabinan(infruit-juiceconcentrates)arereported.
Measurementofendo-1,4-β-D-xylanase.
McCleary,B.V.(1992).“XylansandXylanases”,(J.Visser,G.Beldman,M.A.Kusters-vanSomeronandA.G.J.Voragen,Eds.),ProgressinBiotechnology,Vol.7,Elsevier,SciencePublishersB.V.,pp.161-169.
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Variousproceduresforthemeasurementofxylanaseinfermentationbroths,commercialenzymemixtures,breadimprovermixturesandfeedsamplesaredescribed.Problemsassociatedwiththeroutineuseofreducing-sugarbasedmethodsaxehighlightedandtheadvantagesandlimitationsofviscometricanddye-labelledsubstrateproceduresformeasurementoftracelevelsofactivityinfeedsamplesarediscussed.
Newdevelopmentsinthemeasurementofα-amylase,endo-protease,β-glucanaseandβ-xylanase.
McCleary,B.V.&Monaghan,D.(2000).“ProceedingsoftheSecondEuropeanSymposiumonEnzymesinGrainProcessing”,(M.Tenkanen,Ed.),VTTInformationService,pp.31-38.
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Overthepast8years,wehavebeenactivelyinvolvedinthedevelopmentofsimpleandreliableassayprocedures,forthemeasurementofenzymesofinteresttothecerealsandrelatedindustries.Insomeinstances,differentprocedureshavebeendevelopedforthemeasurementofthesameenzymeactivity(e.g.α-amylase)inarangeofdifferentmaterials(e.g.malt,cerealgrainsandfungalpreparations).Thereasonsfordifferentproceduresmaydependonseveralfactors,suchastheneedforsensitivity,easeofuse,robustnessofthesubstratemixture,orthepossibilityforautomation.Inthispresentation,wewillpresentinformationonourmostup-to-dateproceduresforthemeasurementofα-amylase,endo-protease,β-glucanaseandβ-xylanase,withspecialreferencetotheuseofparticularassayformatsinparticularapplications.
Analysisoffeedenzymes.
McCleary,B.V.(2001).“EnzymesinFarmAnimalNutrition”,(M.BedfordandG.Partridge,Eds.),CABInternational,pp.85-107.
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Enzymesareaddedtoanimalfeedtoincreaseitsdigestibility,toremoveanti-nutritionalfactors,toimprovetheavailabilityofcomponents,andforenvironmentreasons(CampbellandBedford,1992;Walshetal.,1993).Awide-varietyofcarbohydrase,protease,phytaseandlipaseenzymesfinduseinanimalfeeds.Inmonogastricdiets,enzymeactivitymustbesufficientlyhightoallowfortherelativelyshorttransittime.Also,theenzymeemployedmustbeabletoresistunfavourableconditionsthatmaybeexperiencedinfeedpreparation(e.g.extrusionandpelleting)andthatexistinthegastrointestinaltract.Measurementoftracelevelsofenzymesinanimalfeedmixturesisdifficult.Independentoftheenzymestudied,manyoftheproblemsexperiencedaresimilar;namely,lowlevelsofactivity,extractionproblemsinactivationduringfeedpreparation,non-specificbindingtootherfeedcomponentsandinhibitionbyspecificfeed-derivedinhibitors,e.g.specificxylanaseinhibitorsinwheatflour(Debyseretal.,1999).
Measurementofpolysaccharidedegradingenzymesusingchromogenicandcolorimetricsubstrates.
McCleary,B.V.(1991).ChemistryinAustralia,September,398-401.
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Enzymicdegradationofcarbohydratesisofmajorsignificanceintheindustrialprocessingofcerealsandfruits.Intheproductionofbeer,barleyisgerminatedunderwelldefinedconditions(malting)toinducemaximumenzymesynthesiswithminimumrespirationofreservecarbohydrates.Thegrainsaredriedandthenextractedwithwaterundercontrolledconditions.Theamylolyticenzymessynthesizedduringmalting,aswellasthosepresentintheoriginalbarley,convertthestarchreservestofermentablesugars.Otherenzymesactonthecellwallpolysaccharides,mixed-linkageβ-glucanandarabinoxylan,reducingtheviscosityandthusaidingfiltration,andreducingthepossibilityofsubsequentprecipitationofpolymericmaterial.Inbaking,β-amylaseandα-amylasegivecontrolleddegradationofstarchtofermentablesugarssoastosustainyeastgrowthandgasproduction.Excessquantitiesofα-amylaseintheflourresultinexcessivedegradationofstarchduringbakingwhichinturngivesastickycrumbtextureandsubsequentproblemswithbreadslicing.Juiceyieldfromfruitpulpissignificantlyimprovedifcell-walldegradingenzymesareusedtodestroythethree-dimensionalstructureandwaterbindingcapacityofthepecticpolysaccharidecomponentsofthecellwalls.Problemsofroutineandreliableassayofcarbohydratedegradingenzymesinthepresenceofhighlevelsofsugarcompoundsareexperiencedwithsuchindustrialprocess.
Optimisingtheresponse.
Acamovic,T.&McCleary,B.V.(1996).FeedMix,4,14-19.
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Afinebalanceexistsbetweenenzymeactivityandtheadverseeffectsassociatedwithfeedprocessing.Accurateestimationofenzymeactivityinthefeedisapre-requisitetooptimisingtheresponse.
Comparativestudyofcolorectalhealthrelatedcompoundsindifferenttypesofbread:Analysisofbreadsamplespreandpostdigestioninabatchfermentationmodelofthehumanintestine.
Hiller,B.,Schlörmann,W.,Glei,M.&Lindhauer,M.G.(2011).Foodchemistry,125(4),1202-1212.
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Sevendifferenttypesofwheatandryebreadwereanalysedforcolorectalhealthrelatedcompounds,preandpostdigestion,inbatchfermentationmodelofthehumanintestine.Predigestion,higheramountsofcolorectalhealth-relateddietaryfibrecompounds(soluble/insoluble/totaldietaryfibre,arabinoxylans,β-glucans)andphytochemicals(mono-/di-phenolicacids,phyticacid,hydroxymethylfurfural)weredetectedinwholemealthaninrefinedflourtypesofbread,aswellasinryeflourtypesthaninwheatflourtypesofbread.Postdigestion,faecalbacterialmetabolitesofcolorectalhealthpromoting(acetate/propionate/butyrate,lactate,freemono-/di-phenolicacids)andimpairing(aminometabolites,bileacidmetabolites)activitieswerefoundinfermentationsupernatantsofbreadsamples.Alltypesofbreadpositivelyaffectedfaecalbacterialmetabolism;amongthedifferenttypesofbread,thehigheststimulationoforganicacidproduction(acetate/propionate/butyrate,lactate)andthelowestdetrimentalbacterialenzymeactivities(β-glucuronidase,urease)weredetectedforwheatflourbread,whereasthestrongestretardationofbacterialbileaciddegradationandthestrongeststimulationofphenolicacidmetaboliterelease(phenylpropionic/phenylpropenoicacidderivatives)wereinducedbywholemealryebread.Thisstudyforthefirsttimepresentsaqualitativeandquantitativeoverviewoverthebroadspectrumofcolorectalhealthrelatedcompoundsinhigh-andlow-fibretypesofbread,preandpostinvitrodigestion,andhighlightsthesignificanceofbreadforthepreventivenutritionalinterventionofcolorectalcancer.
Standardassaysdonotpredicttheefficiencyofcommercialcellulasepreparationstowardsplantmaterials.
Kabel,M.A.,VanderMaarel,M.J.E.C.,Klip,G.,Voragen,A.G.J.&Schols,H.A.(2006).BiotechnologyandBioengineering,93(1),56-63.
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Commercialcellulasepreparationsarepotentiallyeffectiveforprocessingbiomassfeedstocksinordertoobtainbioethanol.Inplantcellwalls,cellulosefibrilsoccurincloseassociationwithxylans(monocotyls)orxyloglucans(dicotyls).Theenzymaticconversionofcellulose/xylansisacomplexprocessinvolvingtheconcertedactionofexo/endocellulasesandcellobiasesyieldingglucoseandxylanasesyieldingxylooligomersandxylose.Anoverviewofcommonlymeasuredcellulase-,cellobiase-,andxylanase-activity,usingrespectivelyfilterpaper,cellobiose,andAZCL-dyedxylanasasubstrateof14commerciallyavailableenzymepreparationsfromseveralsuppliersispresented.Inadditiontothesestandardizedtests,theenzyme-efficiencyofdegradingnativesubstrateswasstudied.Grassandwheatbranwerefractionatedintoawaterunsolublefraction(WUS),whichwasfreeofoligosaccharidesandstarch.Additionally,cellulose-andxylan-richfractionswerepreparedbyalkalineextractionoftheWUSandwereenzymaticallydigested.Hereby,thecapabilityofcelluloseandxylanconversionofthecommercialenzymepreparationstestedwasmeasured.Theresultsobtainedshowedthattherewasalargedifferenceintheperformanceofthefourteenenzymesamples.Comparingallresults,itwasconcludedthatthechoiceofanenzymepreparationismoredependentonthecharacteristicsofthesubstrateratherthanonstandardenzyme-activitiesmeasured.
CharacteristicsofpregelatinizedAEmutantriceflourspreparedbyboilingafterpreroasting.
Nakamura,S.,Satoh,H.&Ohtsubo,K.I.(2011).JournalofAgriculturalandFoodChemistry,59(19),10665-10676.
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Asaemutantrice,suchasEM10,lacksthestarchbranchingenzymeIIb,itsamylopectincontainsmorelong-chainglucansthanthatofordinaryIndicaandJaponicaricegrains.Althoughboiledgrainsofaericecultivarsaretoohardandnonstickyfortablerice,theyarepromisingintermsofbiofunctionality,suchaspreventionofdiabetes.Thepresentpaperinvestigatesthecharacterizationofanovelgroupoffouraemutantricecultivars(EM72,EM145,EM174,andEM189).Theyweresubjectedtotheevaluationfortheirmainchemicalcomponents,physicalproperties,andenzymeactivitiesatdifferentgrainconditions(rawmilledrice,roastedrice,boiledrice,andriceboiledafterpreroasting).Thesemutantricegrainsarecharacterizedbyhighapparentamylose,highproteinandhighglucosecontents,highpastingtemperature,highα-amylaseactivities,highresistantstarch,andlowdegreeofgelatinization.Anovelmethodwasdevelopedtomaintainthehighresistantstarchcontentsofgelatinizedricegrains.Riceboiledafterpreroastingshowedahigherratioofresistantstarchandaloweramountofglucosethanordinaryboiledrice.Itbecamepossibletoproducehigh-qualityandbiofunctionalpregelatinizedricefloursbyboilingwithfrozenfruits,suchastomatoes,afterricegrainshadbeenpreroasted.Theseaemutantswerefoundtobesuitablematerialsforrice/fruitorrice/vegetableproductstoserveaspalatable,low-glucose,andhighresistantstarchriceproducts.
VariabilityinArabinoxylan,XylanaseActivity,andXylanaseInhibitorLevelsinHardSpringWheat.
Mendis,M.,Ohm,J.B.,Delcour,J.A.,Gebruers,K.,Meinhardt,S.&Simsek,S.(2013).CerealChemistry,90(3),240-248.
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Arabinoxylans(AX),xylanase,andxylanaseinhibitorshaveanimportantroleinmanycerealfoodprocessingapplications.Theeffectsofgenotype,growinglocation,andtheirinteraction(G×L)onAX,apparentxylanaseactivity,andapparentxylanaseinhibitionactivityofTriticumaestivumxylanaseinhibitor(TAXI)andxylanaseinhibitingprotein(XIP)wereinvestigatedforsixhardredandsixhardwhitespringwheatgenotypesgrownatthreelocations.DifferenceintotalAXlevelamonggenotypeswasnotdeterminedtoasignificantlevelbygenotype.Instead,variabilityintotalAXcontentwaslargelydependentonG×L.However,totalAXcontentwassignificantlydifferentbetweenthetwowheatclasses.Forbranxylanaseactivity,25%ofthevariabilitycouldbeattributedtoG×Linteraction.Moreover,therewassignificantdifferencebetweenthebranxylanaseactivitiesinthetwowheatclasses.BranTAXIactivityandXIPactivityweresignificantlydifferentamonggenotypes.Genotypecontributed72%tothevariabilityinTAXIactivityand39%inXIP.However,nosignificantdifferencewasobservedamongthetwowheatclassesforTAXIorXIPactivity.TheseresultsindicatethatTAXImightbeastableparameterinsegregatingwheatgenotypeswithvaryingxylanaseactivity.
Wheatdoughsyrupingincoldstorageisrelatedtostructuralchangesofstarchandnon-starchpolysaccharides.
Kim,H.J.,Song,Y.,Lee,S.,Lee,K.P.,Lee,B.H.&Yoo,S.H.(2017).FoodResearchInternational,99,596-602.
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Eventhoughtherefrigerateddoughindustryisgrowingquicklyduetotheconvenienceandfreshnessofrefrigerateddoughoveraprolongedstorageperiod,doughsyruping,whichisabrownishliquidthatleachesoutfromdoughduringthestorage,isaquality-diminishingfactorthatneedstoberesolved.Theobjectivesofthisstudyweretounderstanddoughsyrupingandhowitisrelatedtostructuralchangesinwater-solublearabinoxylan(WS-AX)andstarchinwheatfloursduringrefrigerationaswellastopreventsyrupingbyapplyingexogenouscellwallpolysaccharides.Doughsyrupingincreasedto6.5,6.9,and17.2%inweak,strong,andjopoomwheatflours,respectively,aftera35-daystorageperiod.Theendoxylanaseactivityofjopoomwheatflourwassubstantiallygreatercomparedtoothercommercialflours,buttheactivityofthisflourdidnotchangeoverthewholecoldstorageperiod.ThemolecularsizereductionofWS-AXwasinverselyrelatedtothedegreeofdoughsyruping.Theadditionofβ-glucan,carboxymethylcellulose,andxylaneffectivelyreducedsyrupformationinjopoomwheatflourdough.