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.

Arangeofα-L-arabinofuranosyl-(1-4)-β-D-xylo-oligosaccharides(AXOS)wereproducedbyhydrolysisofwheatflourarabinoxylan(WAX)andaciddebranchedarabinoxylan(ADWAX),inthepresenceandabsenceofanAXH-d3α-L-arabinofuranosidase,byseveralGH10andGH11β-xylanases.ThestructuresoftheoligosaccharideswerecharacterisedbyGC-MSandNMRandbyhydrolysisbyarangeofα-L-arabinofuranosidasesandβ-xylosidase.TheAXOSwerepurifiedandusedtocharacterisetheactionpatternsofthespecificα-L-arabinofuranosidases.Theseenzymes,incombinationwitheitherCellvibriomixtusorNeocallimastixpatriciarumβ-xylanase,wereusedtoproduceelevatedlevelsofspecificAXOSonhydrolysisofWAX,suchas3²-α-L-Araf-(1-4)-β-D-xylobiose(A³X),2³-α-L-Araf-(1-4)-β-D-xylotriose(A²XX),3³-α-L-Araf-(1-4)-β-D-xylotriose(A³XX),2²-α-L-Araf-(1-4)-β-D-xylotriose(XA²X),3²-α-L-Araf(1-4)-β-D-xylotriose(XA³X),2³-α-L-Araf-(1-4)-β-D-xylotetraose(XA²XX),3³-α-L-Araf-(1-4)-β-D-xylotetraose(XA³XX),2³,3³-di-α-L-Araf-(1-4)-β-D-xylotriose(A²⁺³XX),2³,3³-di-α-L-Araf-(1-4)-β-D-xylotetraose(XA²⁺³XX),2⁴,3⁴-di-α-L-Araf-(1-4)-β-D-xylopentaose(XA²⁺³XXX)and3³,3⁴-di-α-L-Araf-(1-4)-β-D-xylopentaose(XA³A³XX),manyofwhichhavenotpreviouslybeenproducedinsufficientquantitiestoallowtheiruseassubstratesinfurtherenzymicstudies.ForA^2,3XX,yieldsofapproximately16%ofthestartingmaterial(wheatarabinoxylan)havebeenachieved.Mixturesoftheα-L-arabinofuranosidases,withspecificactiononAXOS,havebeencombinedwithβ-xylosidaseandβ-xylanasetoobtainanoptimalmixtureforhydrolysisofarabinoxylantoL-arabinoseandD-xylose.

Endo-xylanase(fromBacillussubtilis)orferulicacidesterase(fromAspergillusniger)wereexpressedinwheatunderthecontroloftheendosperm-specific1DX5gluteninpromoter.Constructsbothwithandwithouttheendoplasmicreticulumretentionsignal(Lys-Asp-Glu-Leu)KDELwereused.TransgenicplantswererecoveredinallfourcasesbutnoqualitativedifferencescouldbeobservedwhetherKDELwasaddedornot.Endo-xylanaseactivityintransgenicgrainswasincreasedbetweentwoandthreefoldrelativetowildtype.Thegrainswereshrivelledandhada25%–33%decreaseinmass.Extensiveanalysisofthecellwallsshoweda10%–15%increaseinarabinosetoxyloseratio,a50%increaseintheproportionofwater-extractablearabinoxylan,andashiftintheMWofthewater-extractablearabinoxylanfrombeingmainlylargerthan85kDtobeingbetween2and85kD.Ferulicacidesterase-expressinggrainswerealsoshrivelled,andtheseedweightwasdecreasedby20%–50%.Noferulicacidesteraseactivitycouldbedetectedinwild-typegrainswhereasferulicacidesteraseactivitywasdetectedintransgeniclines.Thegraincellwallshad15%–40%increaseinwater-unextractablearabinoxylanandadecreaseinmonomericferulicacidbetween13%and34%.Inalltheplants,theobservedchangesareconsistentwithaplantresponsethatservestominimizetheeffectoftheheterologouslyexpressedenzymesbyincreasingarabinoxylanbiosynthesisandcross-linking.

Background:Thediscoveryanddevelopmentofnovelplantcellwalldegradingenzymesisakeysteptowardsmoreefficientdepolymerizationofpolysaccharidestofermentablesugarsfortheproductionofliquidtransportationbiofuelsandotherbioproducts.TheindustrialfungusTrichodermareeseiisknowntobehighlycellulolyticandisamajorindustrialmicrobialsourceforcommercialcellulases,xylanasesandothercellwalldegradingenzymes.However,enzyme-ProspectingresearchcontinuestoidentifyopportunitiestoenhancetheactivityofT.reeseienzymepreparationsbysupplementingwithenzymaticdiversityfromothermicrobes.Thegoalofthisstudywastoevaluatetheenzymaticpotentialofabroadrangeofplantpathogenicandnon-pathogenicfungifortheirabilitytodegradeplantbiomassandisolatedpolysaccharides.Results:Large-scalescreeningidentifiedarangeofhydrolyticactivitiesamong348uniqueisolatesrepresenting156speciesofplantpathogenicandnon-pathogenicfungi.Hierarchicalclusteringwasusedtoidentifygroupsofspecieswithsimilarhydrolyticprofiles.Amongmoderatelyandhighlyactivespecies,plantpathogenicspecieswerefoundtobemoreactivethannon-pathogensonsixofeightsubstratestested,withnosignificantdifferenceseenontheothertwosubstrates.Amongthepathogenicfungi,greaterhydrolysiswasseenwhentheyweretestedonbiomassandhemicellulosederivedfromtheirhostplants(commelinoidmonocotordicot).AlthoughT.reeseihasahydrolyticprofilethatishighlyactiveoncelluloseandpretreatedbiomass,itwaslessactivethansomenaturalisolatesoffungiwhentestedonxylansanduntreatedbiomass.Conclusions:Severalhighlyactiveisolatesofplantpathogenicfungiwereidentified,particularlywhentestedonxylansanduntreatedbiomass.Therewerestatisticallysignificantpreferencesforbiomasstypereflectingthemonocotordicothostpreferenceofthepathogentested.Thesehighlyactivefungiarepromisingtargetsforidentificationandcharacterizationofnovelcellwalldegradingenzymesforindustrialapplications.

Thermophilicxylanasesareofgreatinterestfortheirwideindustrialapplicationprospects.Hereweidentifiedathermophilicxylanase(XynC01)ofglycosidehydrolase(GH)family10inathermophilicfungalstrainAchaetomiumsp.Xz-8.ThededucedaminoacidsofXynC01showedthehighestidentityof≤52%toexperimentallyverifiedxylanases.XynC01wasfunctionallyexpressedinPichiapastoris,showedoptimalactivityatpH5.5and75°CwithstabilityoverabroadpHrange(pH4.0–10.0)andattemperaturesof55°Candbelow.XynC01hadthehighestcatalyticefficiency(k_cat/K_m,3710mL/s/mg)everreportedforallGH10xylanases,andwasresistanttoalltestedmetalionsandchemicalreagents.Itshydrolysisproductsofvariousxylansweresimple,mainlyconsistingofxylobioseandxylose.Undersimulatedmashingconditions,XynC01alonehadacomparableeffectonfiltrationimprovementwithUltraflofromNovozymes(20.24%vs.20.71%),andshowedbetterperformancewhencombinedwithacommercialβ-glucanase(38.50%).Combiningallexcellentpropertiesdescribedabove,XynC01mayfinddiverseapplicationsinindustrialfields,especiallyinthebrewingindustry.

Theplantcellwallisamajorbarrierthatmanyplantpathogensmustsurmountforsuccessfulinvasionoftheirplanthosts.Fullgenomesequencingofanumberofplantpathogenshasrevealedoftenlarge,complex,andredundantenzymesystemsfordegradationofplantcellwalls.Recentsurveyshavenotedthatplantpathogenicfungiarehighlycompetentproducersoflignocellulolyticenzymes,andtheirenzymeactivitypatternsreflecthostspecificity.Weproposethatplantpathogensmaycontributetobiofuelproductionasdiversesourcesofaccessoryenzymesformoreefficientconversionoflignocelluloseintofermentablesugars.

(1,3)(1,4)-β-D-Glucan(mixed-linkageglucanorMLG),acharacteristichemicelluloseinprimarycellwallsofgrasses,wasinvestigatedtodeterminebothitsroleincellwallsanditsinteractionwithcelluloseandothercellwallpolysaccharidesinvitro.BindingisothermsshowedthatMLGadsorptionontomicrocrystallinecelluloseisslow,irreversible,andtemperature-dependent.MeasurementsusingquartzcrystalmicrobalancewithdissipationmonitoringshowedthatMLGadsorbedirreversiblyontoamorphousregeneratedcellulose,formingathickhydrogel.Oligosaccharideprofilingusingendo-(1,3)(1,4)-β-glucanaseindicatedthattherewasnodifferenceinthefrequencyanddistributionof(1,3)and(1,4)linksinboundandunboundMLG.ThebindingofMLGtocellulosewasreducedifthecellulosesampleswerefirsttreatedwithcertaincellwallpolysaccharides,suchasxyloglucanandglucuronoarabinoxylan.ThetetheringfunctionofMLGincellwallswastestedbyapplyingendo-(1,3)(1,4)-β-glucanasetowallsamplesinaconstantforceextensometer.Cellwallextensionwasnotinduced,whichindicatesthatenzyme-accessibleMLGdoesnottethercellulosefibrilsintoaload-bearingnetwork.

Background:Enzymerecyclingisamethodtoreducetheproductioncostsforadvancedbioethanolbyloweringtheoveralluseofenzymes.Commercialcellulasepreparationsconsistofmanydifferentenzymesthatareimportantforefficientandcompletecellulose(andhemicellulose)hydrolysis.Thisabundanceofdifferentactivitiescomplicatesenzymerecyclingsincetheindividualenzymesbehavedifferentlyintheprocess.Previously,thegeneralperceptionwasthatβ-glucosidasescouldeasilyberecycledviatheliquidphase,astheyhavemostlybeenobservednottoadsorbtopretreatedbiomassoronlyadsorbtoaminorextent.Results:TheresultsfromthisstudywithCellic®CTec2revealedthatthevastmajorityoftheβ-glucosidaseactivitywaslostfromtheliquidphaseandwasadsorbedtotheresidualbiomassduringhydrolysisandfermentation.Adsorptionstudieswithβ-glucosidasesintwocommercialpreparations(Novozym188andCellic®CTec2)tosubstratesmimickingthecomponentsinpretreatedwheatstrawrevealedthattheAspergillusnigerβ-glucosidaseinNovozym188didnotadsorbsignificantlytoanyofthecomponentsinpretreatedwheatstraw,whereastheβ-glucosidaseinCellic®CTec2adsorbedstronglytolignin.Theextentofadsorptionofβ-glucosidasefromCellic®CTec2wasaffectedbybothtypeofbiomassandpretreatmentmethod.Withapproximately65%oftheβ-glucosidasesfromCellic®CTec2adsorbedontoligninfrompretreatedwheatstraw,theactivityoftheβ-glucosidasesintheslurrydecreasedbyonly15%.Thisdemonstratedthatsomeenzymeremainedactivedespitebeingbound.ItwaspossibletoreducetheadsorptionofCellic®CTec2β-glucosidasetoligninfrompretreatedwheatstrawbyadditionofbovineserumalbuminorpoly(ethyleneglycol).Conclusions:Contrarytotheβ-glucosidasesinNovozym188,theβ-glucosidasesinCellic®CTec2adsorbsignificantlytolignin.TheligninadsorptionobservedforCellic®CTec2isusuallynotaproblemduringhydrolysisandfermentationsincemostofthecatalyticactivityisretained.However,adsorptionofβ-glucosidasestoligninmayprovetobeaproblemwhentryingtorecycleenzymesintheproductionofadvancedbioethanol.