Megazyme/Xylazyme AX Tablets/T-XAX-1000T/1,000 Tablets

endo-1,4-β-Xylanase(EC3.2.1.8)isemployedacrossabroadrangeofindustriesincludinganimalfeed,brewing,baking,biofuels,detergentsandpulp(paper).Despiteitsimportance,arapid,reliable,reproducIBLe,automatableassayforthisenzymethatisbasedontheuseofachemicallydefinedsubstratehasnotbeendescribedtodate.Reportedhereinisanewenzymecoupledassayprocedure,termedtheXylX6assay,thatemploysanovelsubstrate,namely4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-4⁵-O-glucosyl-xylopentaoside.ThedevelopmentofthesubstrateandassociatedassayisdiscussedhereandtherelationshipbetweentheactivityvaluesobtainedwiththeXylX6assayversustrADItionalreducingsugarassaysanditsspecificityandreproducibilitywerethoroughlyinvestigated.

Hydrolysisofmannan-typepolysaccharidesbyβ-mannanaseisdependentonsubstitutiononandwithinthemain-chainaswellasthesourceoftheβ-mannanaseemployed.Characterisationofreactionproductscanbeusedtodefinethesub-sitebindingrequirementsoftheenzymesaswellasthefine-structuresofthepolysaccharides.Actionofendo-arabinanaseandendo-galactanaseonarabinansandarabinogalactansisdescribed.Specificassaysforendo-arabinanaseandarabinan(infruit-juiceconcentrates)arereported.

Variousproceduresforthemeasurementofxylanaseinfermentationbroths,commercialenzymemixtures,breadimprovermixturesandfeedsamplesaredescribed.Problemsassociatedwiththeroutineuseofreducing-sugarbasedmethodsaxehighlightedandtheadvantagesandlimitationsofviscometricanddye-labelledsubstrateproceduresformeasurementoftracelevelsofactivityinfeedsamplesarediscussed.

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.

Afinebalanceexistsbetweenenzymeactivityandtheadverseeffectsassociatedwithfeedprocessing.Accurateestimationofenzymeactivityinthefeedisapre-requisitetooptimisingtheresponse.

Arabinoxylanarabinofuranohydrolase-D3(AXHd3)fromBifidobacteriumadolescentisreleasesonlyC3-linkedarabinoseresiduesfromdouble-substitutedxyloseresidues.AgenomiclibraryofB.adolescentisDSM20083wasscreenedforthepresenceoftheaxhD3gene.TwoplasmidswereidentifiedcontainingpartoftheaxhD3gene.Thenucleotidesequenceswerecombinedandthreeopenreadingframes(ORFs)werefound.ThefirstORFshowedhighhomologywithxylanasesbelongingtofamily8oftheglycosidehydrolasesandthisgenewasdesignatedxylA.ThesecondORFwastheaxhD3genebelongingtoglycosidehydrolasefamily43.Thethird(partial)ORFcodedforaputativecarboxylesterase.TheaxhD3genewasclonedandexpressedinEscherichiacoli.SeveralsubstrateswereemployedinthebiochemicalcharacterizationofrecombinantAXHd3.Theenzymeshowedthehighestactivitytowardwheatarabinoxylanoligosaccharides.Inaddition,β-xylanasefromTrichodermasp.wasabletodegradesolublewheatarabinoxylanpolymertoahigherextent,afterpretreatmentwithrecombinantAXHd3.ArabinoxylanoligosaccharidesincubatedwithacombinationofrecombinantAXHd3andanα-L-arabinofuranosidasefromAspergillusnigerdidnotresultinahighermaximalreleaseofarabinosethanincubationwiththeseenzymesseparately.

ThecompletegenomesequenceofBacillussubtilisrevealsthatsequencesencodingseveralhemicellulasesareco-localisedwithagene(xynD)encodingaputativefamily43glycosidehydrolasethathasnotyetbeencharacterised.Inthiswork,xynDhasbeenisolatedfromgenomicDNAofB.subtilissubsp.subtilisATCC6051andclonedforcytoplasmaticexpressioninEscherichiacoli.RecombinantXynD(rXynD)waspurifiedusingion-exchangechromatographyandgelpermeationchromatography.Theenzymehadamolecularmassofapproximately52kDa,ap/above9.0andreleasesα-L-arabinosefromarabinoxylo-oligosaccharidesaswellasarabinoxylanpolymerswithvaryingdegreeofsubstitution.Usingpara-nitrophenyl-α-L-arabinofuranosideassubstrate,maximumactivitywasobservedatpH5.6and45°C.TheenzymeretaineditsactivityoveralargepHrange,whileactivitywaslostafterpre-incubationabove50°C.Gas–liquidchromatographyandprotonnuclearmagneticresonancespectrometryanalysisindicatedthatrXynDspecificallyreleasesarabinofuranosylgroupsfrommono-substitutedC-(O)-2andC-(O)-3xylopyranosylresiduesonthexylanbackbone.AsrXynDdidnotdisplayendoxylanase,xylosidaseorarabinanaseactivityandwasinactiveonarabinan,weconcludethatthisenzymeisbestdescribedasanarabinoxylanarabinofuranohydrolase.

Volatilephenolshavelongbeenrecognizedasimportantflavorcontributorstothearomaofvariousalcoholicbeverages.Thetwomainflavor-activevolatilephenolsinbeerare4-vinylguaiacoland4-vinylphenol.Theyarethedecarboxylationproductsoftheprecursorsferulicacidandp-coumaricacid,respectively,whicharereleasedduringthebrewingprocess,mainlyfrommalt.Inthisstudy,thevariabilityinthereleaseoffreeandester-boundhydroxycinnamicacidsfromninemaltedbarley(HordeumvulgareL.)varietiesduringwortproductionwasinvestigated.Alargevariabilitybetweendifferentbarleymaltsandtheircorrespondingwortswasobserved.Differenceswerealsofoundbetweenfreeferulicacidlevelsfromidenticalmaltvarietiesoriginatingfromdifferentmalthouses.Duringmashing,freehydroxycinnamicacidsinwortarebothwater-extractedandenzymaticallyreleasedbycinnamoylesteraseactivity.Esteraseactivitiesclearlydifferbetweendifferentbarleymaltvarieties.Multiplelinearregressionanalysisshowedthatthereleaseofferulicacidduringmashingdidnotdependonlyonthebarleymaltesteraseactivitybutalsoontheamountofester-boundferulicacidinitiallypresentinthewortandonitsendoxylanaseactivity.Thestudydemonstratestheimportanceofselectingasuitablemaltvarietyasthefirstmeansofcontrollingthefinalvolatilephenollevelsinbeer.

Thevariabilityinryeflouralkali-extractablearabinoxylan(AE-AX)structureswasexaminedbyextensivefractionationandenzymicdegradationstudies.AXwereisolatedfromdestarchedryewater-unextractablesbysequentialextractionwithsaturatedbariumhydroxidesolution,water,1.0Msodiumhydroxide,andwater.TheisolatedAE-AXcontainedca.51%AXwithanarabinosetoxylose(A/X)ratioof0.71.FractionationoftheisolatedAE-AXbyethanolprecipitationyieldedarangeofAE-AXfractionscontainingAXmoleculeswithdifferentA/Xratiosandsubstitutionpatterns.DegradationofthesestructurallydifferentAE-AXfractionsbyanAspergillusaculeatusendoxylanase(XAA)andaBacillussubtilisendoxylanase(XBS)resultedinAXfragmentswithvariousstructuralfeatures.FurtherfractionationofthedegradedAE-AXfractionsbyethanolprecipitationshowedthatastrongcorrelationexistsbetweenthestructuralfeaturesoftheAXfragments,thatis,averagedegreeofpolymerization(DP)ofthexylanbackbone,A/Xratio,andsubstitutionpattern.ResultsindicatedthattheryeflourAE-AXconsistofacontinuumofstructuresratherthanoftwotypesofAXortwotypesofregionsintheAXmolecule.

Theimpactofvaryinglevelsofendoxylanaseactivityinwheatflouronarabinoxylan(AX)inmixedandresteddoughwasstudiedusingeightindustriallymilledwheatflourfractionswithvaryingendoxylanaseactivitylevels.Analysisofthelevelsofreducingendxylose(RX)andsolubilizedAX(S-AX)formedduringmixingandrestingandtheircorrelationwiththeendoxylanaseactivityintheflourmillingfractionsshowedthatsolubilizationofAXduringthemixingphaseismainlyduetomechanicalforces,whilesolubilizationofAXduringrestingiscausedbyendoxylanaseactivity.Moreover,solubilizationofAXduringthedoughrestingphaseismoreoutspokenthanthatduringthemixingphase.Besidesendoxylanaseactivity,thereweresignificantxylosidaseandarabinofuranosidaseactivitiesduringthedoughrestingphase.Theresultsindicatethatwheatflour-associatedendoxylanasescanalterpartoftheAXindough,therebychangingtheirfunctionalityinbreadmakingandpotentiallyaffectingdoughandendproductproperties.

Hypocreajecorinaisanimportant,filamentousfungusduetoitseffectiveproductionofhydrolyticenzymes.Geneexpressionstudiesprovidedeeperinsightintoenvironmentsensingandcellularresponsemechanisms.Reversetranscription-quantitativePCRisagene-specificandpowerfultooltomeasureevenminorchangesinmRNAcomposition.Anaccuratenormalizationstrategyisabsolutelynecessaryforappropriateinterpretationofreversetranscription-quantitativePCRresults.Onefrequentlyappliedstrategyistheusageofareferencegene.AdequatereferencegenesforHypocreahavenotbeenpublishedsofar.ByusingtheNormFinderandgeNormsoftwares,weevaluatedthemoststablegenesamongstsixpotentialreferencegenesin34samplesfromdiversecultivationconditions.Underthoseexperimentalconditions,sar1encodingforasmallGTPasewasfoundtobethemoststablegene,whereasactencodingforactinwasnotamongstthebestvalidatedones.Theinfluenceofthereferencesystemontheexpressiondataisdemonstratedbyanalysisoftwotargetgenes,encodingfortheXylanaseregulator1andforXylanaseII.Wefurthervalidatedobtainedxylanase2transcriptionrateswiththecorrespondingenzymeactivity.

Syrupformationinrefrigerateddoughsisaproblemsinceitreducesthedoughs’shelflife.Microbialexogenousxylanasesassociatedwithwheatkernelswerefoundtoplayaroleinthissyrupingphenomenon.Usingxylanase-producingmicroorganismsisolatedfromwheatkernels,weinvestigatedtheirpotencytoinducesyrupingindough.GrowthofthefungalxylanaseproducerFusariumsp.(10²colonyformingunits(CFU)/gdough)andthebacterialxylanaseproducerPaenibacillussp.(10⁴CFU/gdough)insyntheticmediaandtheirrespectiveadditiontowheatdoughcouldnotbringaboutasignificantamountofsyruping.However,whenthesespeciesweregrownonmoistwheatkernelsandanextractofthesekernelscontainingboththeorganismsanditsxylanaseswasmadeandaddedtodough,intensivesyrupingwasnoted.Thiseffectwasprimarilyattributedtothexylanasespresentintheextract.Thesefindingssuggestthattheinvolvementofxylanase-producingmicroorganismsinthesyrupingphenomenonissituatedpriortoharvest.Additionalquantitativeanalysesofmicrobialbiomasspresentonwheatkernelsrevealedthatthefungiinparticularcouldbecorrelatedtohighermicrobialexogenousxylanaseactivitiesonwheat.Ourresultsindicatethatthesyrupingislinkedtofungalxylanaseproductiononthewheatkernelsinthefield.

InHypocreajecorina,Xyr1(xylanaseregulator1)isthemaintranscriptionactivatorofhydrolase-encodinggenes,suchasxyn1,xyn2,bxl1,cbh1,cbh2,egl1,andbgl1.EventhoughXyr1mediatestheinductionsignalforallthesegenesderivedfromvariousinducingcarbonsourcesandcompounds,xyr1transcriptionitselfisnotinduciblebyanyofthesesubstances.However,cultivationonglucoseasthecarbonsourceprovokescarboncataboliterepressionofxyr1transcriptionmediatedbyCre1.Inaddition,xyr1transcriptionisrepressedbythespecifictranscriptionfactorAce1.Moreover,Xyr1ispermanentlyavailableinthecell,andnodenovosynthesisofthisfactorisneededforafirstinductionofxyn1transcription.Theconstitutiveexpressionofxyr1leadstoasignificantelevation/deregulationofthexyn1,xyn2,andbxl1transcriptioncomparedtowhatisseenfortheparentalstrain.Overall,thecorrespondingxylanolyticenzymeactivitiesareclearlyelevatedinaconstitutivelyxyr1-expressingstrain,emphasizingthisfactorasanaUSPicioustargetforgeneticallyengineeredstrainimprovement.

Endoxylanase(EC3.2.1.8)substrateselectivity,i.e.,itsrelativeactivitytowardwater-unextractablearabinoxylan(WU-AX)andwater-extractablearabinoxylan(WE-AX)substrates,isimportantforitsfunctionalityinbiotechnologicalprocessessuchasbread-makingandglutenstarchseparation.Ascreeningmethodforrapidlydeterminingsaidsubstrateselectivitywasdeveloped.EndoxylanaseactivitytowardWU-AXwasestimatedbyincubationofinsolublechromogenicsubstratewitharangeofenzymeconcentrationsinmicrotiterplates,followedbycolorimetricmeasurementofthedyereleasedinthesupernatant.AsimilarapproachusingsolublesubstrateandethanolprecipitationofunhydrolysedAXfragmentswasusedtoestimateenzymeactivitytowardWE-AX.Asubstrateselectivityfactorwasdefinedastheratioofenzymeactivitytowardinsolublesubstrateoverenzymeactivitytowardsolublesubstrate.ABacillussubtilisandanAspergillusaculeatusendoxylanase,knowntohavewidelyvaryingrelativeratesofhydrolysisofWU-AXandWE-AX,variedmostintheirsubstrateselectivity,whiletheendoxylanasesofAspergillusniger,Trichodermalongibrachiatum,andTrichodermaviridedisplayedintermediatesuchrelativeactivities.

ThisstudyreportsthevitalregulatoryinfluenceofXyr1(xylanaseregulator1)onthetranscriptionofhydrolyticenzyme-encodinggenesandhydrolaseformationonlactoseinHypocreajecorina.Whilethetranscriptionofthexyr1geneitselfisachievedbyreleaseofcarboncataboliterepression,thetranscriptformationofxyr1(xylanase1)isregulatedbyanadditionalinductionmechanismmediatedbylactose.Xyr1hasanimportantimpactonlactosemetabolismbydirectlyactivatingxyr1(xylosereductase1)transcriptionandindirectlyinfluencingtranscriptionofbga1(β-galactosidase1).ThelatterisachievedbyregulatingtheconversionofD-galactosetotheinducingcarbonsourcegalactitol.

Theeffectofacombinationofcarbohydraseandphytaseenzymesongrowthperformance,insulin-likegrowthfactor1geneexpression,insulinstatus,andinsulinreceptorsensitivityinbroilerchickensfedwheat-soybeanmealdietscontaining6%(starter)and12%(grower-finisher)offull-fatrapeseed(canolatype;lowglucosinolate,lowerucicacid)from1to42dofagewasstudied.Atotalof510one-day-oldmalebroilerchickenswererandomlyassignedto3dietarytreatments,with17penspertreatmentand10birdsperpen.ThedietarytreatmentsconsistedofacontroldietandP-andCa-deficientdietssupplementedwitheitherphytase(500U/kg)oracombinationofphytaseandamulti-carbohydraseenzyme(SuperzymeOM).Thedietswerepelletedat78°Candwerefedadlibitumthroughoutthestarter(9d),grower(18d),andfinisher(15d)phasesoftheexperiment.Overtheentiretrial,growthperformanceofbirdsfedthephytase-supplementeddietdidnotdifferfrombirdsfedthecontroldiet.Theuseofphytaseincombinationwithamulticarbohydraseenzymeimproved(P=0.007)thefeedconversionratiofrom1.90to1.84.Insulinliverreceptorsensitivityincreasedby9.3and12.3%(P=0.004)forthephytase-andthecarbohydrase-phytase-supplementeddiets,respectively.Therewasnoeffectofphytasealoneorcarbohydraseandphytasesupplementationontotalplasmacholesterol,high-densitylipoproteincholesterol,andbloodglucoselevels.However,low-densitylipoproteincholesteroldecreased(P=0.007)forthephytase-carbohydrasetreatment.Geneexpressionofinsulin-likegrowthfactor1tendedtodecreaseby32%(P=0.083)afterphytase-carbohydrasesupplementation.Thecombinationofcarbohydraseandphytaseenzymesmayserveasanattractivemeansoffacilitatingnutrientavailabilityfordigestionandthusenhancethefeedingvalueofwheat-soybeanmeal-baseddietscontainingfull-fatrapeseed.However,theextenttowhichtheeffectsofenzymeadditiononinsulinreceptorsareassociatedwithgrowthperformanceofbroilerchickenrequiresfurtherresearch.

Unlikewheatbread,thedoughofwhichhasavisco-elasticnetworkandhighgas-holdingcapacity,oatbreadgenerallyhasalowvolumeandadensestructure.Weshowedearlierthatincludingryewater-extractablecomponentsinanoatbreadbatterrecipeincreasesloafvolumebyca.30%(PaulyandDelcour,submittedasback-to-backpublication).Weherereportoneffortstoidentifytheactivefactor(s).Anionexchangechromatographyallowedenrichingtheactivefactor(s).Thisandthefactthatonlyalimitedvolumeincreasewasobservedwhenoatbatterwassupplementedwithboiledryeextractindicatethatproteinsarelikelythemostimportantcomponentsresponsibleforthevolumeincrease.Whilethemostactivefactor(s)hadapIbelow4.5,componentswithpIvaluesbetween4.5and8.5alsocontributedtooatloafvolume.Alkalineryecomponents(pI>8.5)orryearabinoxylanhadnoimpact.Ryewater-extractablecomponentssmallerthan6–8kDaalsohadapositiveimpactonloafvolume.

Loafvolumeandcrumbstructureofoatbreadarenotcomparabletothoseofbreadfromwheatflour.Hydrocolloids,surfactantsand/orenzymesareoftenincludedinoatbatterrecipesforqualityenhancementreasons.Inthisstudy,weexaminedtheimpactofwater-extractablecomponentsfrombarley,oat,ryeandwheatflouronoatbreadquality.Wespeculatedthatsuchwaterextractscontaincomponentswhichalsowouldenhancethequalityofoatbread.Asexpected,extractprotein,non-starchpolysaccharide,lipidandenzymelevelsvariedwidelyamongstthedifferentcerealfloursused.Theextractsalsovariedinfoamingpropertiesandextractviscosities.Ryeflourcontainedthehighestlevelofwater-extractablecomponents.Inclusionofryeaqueousextractresultedinthelargestloafvolumeincreaseandinsoftercrumbthannotedforcontroloatbread.Rheofermentometeranalysesshowedthatthemomentofgascellopeningwasdelayedwhenryeextractwasadded,indicatingimprovedbattergascellstabilization,whilecollapseduringbakingwasnotaffected.Theoatbreadimprovingeffectoftheryeextractislikelyduetoacombinationoftheimpactofdifferentofitsconstituentssuchasenzymesandsurfaceactivecomponents.