(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.

Twovariantsofanendo-β-1,4-mannanasefromthedigestivetractofbluemussel,Mytilusedulis,werepurifiedbyacombinationofimmobilizedmetalionaffinitychromatography,sizeexclusionchromatographyintheabsenceandpresenceofguanidinehydrochlorideandionexchangechromatography.Thepurifiedenzymeswerecharacterizedwithregardtoenzymaticproperties,molecularweight,isoelectricpoint,aminoacidcompositionandN-terminalsequence.Theyaremonomericproteinswithmolecularmassesof39 216and39 265Da,respectively,asmeasuredbyMALDI-TOFmassspectrometry.Theisoelectricpointsofbothenzymeswereestimatedtobearound7.8,howeverslightlydifferent,byisoelectricfocusinginpolyacrylamidegel.TheenzymesarestablefrompH4.0to9.0andhavetheirmaximumactivitiesatapHabout5.2.Theoptimumtemperatureofbothenzymesisaround50–55°C.Theirstabilitydecreasesrapidlywhengoingfrom40to50°C.TheN-terminalsequences(12residues)wereidenticalforthetwovariants.Theycanbecompletelyrenaturedafterdenaturationin6Mguanidinehydrochloride.TheenzymesreADIlydegradethegalactomannansfromlocustbeangumandivorynutmannanbutshownocross-specificityforxylanandcarboxymethylcellulose.Thereisnobindingabilityobservedtowardscelluloseandmannan.

Separationandcharacterizationofcomplexmixturesofoligosaccharidesisquitedifficultand,dependingonelutionconditions,structuralinformationisoftenlost.Therefore,theuseofaporous-graphitized-carbon(PGC)-HPLC-ELSD-MSⁿ-methodasanalyticaltoolfortheanalysisofoligosaccharidesderivedfromplantcellwallpolysaccharideshasbeeninvestigated.ItisdemonstratedthatPGC-HPLCcanbewidelyusedforneutralandacidicoligosaccharidesderivedfromcellwallpolysaccharides.FurThermore,itisanon-modifyingtechniquethatenablesthecharacterizationofcellwalloligosaccharidescarrying,e.g.acetylgroupsandmethylesters.Neutraloligosaccharidesareseparatedbasedontheirsizeaswellasontheirtypeoflinkageandresulting3D-structure.Seriesoftheplanarβ-(1,4)-xylo-andβ-(1,4)-gluco-oligosaccharidesareretainedmuchmorebythePGCmaterialthantheseriesofβ-(1,4)-galacto-,β-(1,4)-manno-andα-(1,4)-gluco-oligosaccharides.Chargedoligomerssuchasα-(1,4)-galacturonicacidoligosaccharidesarestronglyretainedandareelutedonlyafteradditionoftrifluoroaceticaciddependingontheirnetcharge.Online-MS-couplingusinga1:1splitterenablesquantitativedetectionofELSDaswellassimpleidentificationofmanyoligosaccharides,evenwhenseparationofoligosaccharideswithinacomplexmixtureisnotcomplete.Consequently,PGC-HPLC-separationincombinationwithMS-detectiongivesapowerfultooltoidentifyawiderangeofneutralandacidicoligosaccharidesderivedfromvariouscellwallpolysaccharides.

Ingeneral,cellulasesandhemicellulasesaremodularenzymesinwhichthecatalyticdomainisappendedtooneormorenoncatalyticcarbohydratebindingmodules(CBMs).CBMs,byconcentratingtheparentalenzymeattheirtargetpolysaccharide,increasethecapacityofthecatalyticmoduletobindthesubstrate,leadingtoapotentiationincatalysis.ClostridiumthermocellumhypotheticalproteinCthe_0821,definedhereasC.thermocellumMan5A,isamodularproteincomprisinganN-terminalsignalpeptide,afamily5glycosidehydrolase(GH5)catalyticmodule,afamily32CBM(CBM32),andaC-terminaltypeIdockerinmodule.RecentproteomicstudiesrevealedthatCthe_0821isoneofthemajorcellulosomalenzymeswhenC.thermocellumisculturedoncellulose.HereweshowthattheGH5catalyticmoduleofCthe_0821displaysendomannanaseactivity.C.thermocellumMan5Ahydrolyzessolublekonjacglucomannan,solublecarobgalactomannan,andinsolubleivorynutmannanbutdoesnotattackthehighlygalactosylatedmannanfromguargum,suggestingthattheenzymeprefersunsubstitutedβ-1,4-mannosidelinkages.TheCBM32ofC.thermocellumMan5Adisplaysapreferenceforthenonreducingendsofmannooligosaccharides,althoughtheproteinmoduleexhibitsmeasurableaffinityfortheterminiofβ-1,4-linkedglucooligosaccharidessuchascellobiose.CBM32potentiatestheactivityofC.thermocellumMan5Aagainstinsolublemannansbuthasnosignificanteffectonthecapacityoftheenzymetohydrolyzesolublegalactomannansandglucomannans.TheproductprofileofC.thermocellumMan5AisaffectedbythepresenceofCBM32.

Pseudomonasfluorescenssubsp.cellulosawhenculturedinthepresenceofcarobgalactomannandegradedthepolysaccharide.Toisolategene(s)fromP.fluorescenssubsp.cellulosaencodingendo-β-1,4-mannanase(mannanase)activity,agenomiclibraryofPseudomonasDNA,constructedinlamBDaZAPII,wasscreenedformannanase-expressingclonesusingthedye-labelledsubstrate,azo-carobgalactomannan.Thenucleotidesequenceofthepseudomonadinsertfromamannanase-positiveclonerevealedasingleopenreadingframeof1257bpencodingaproteinofM_r46,938.ThededucedN-terminalsequenceoftheputativepolypeptideconformedtoatypicalprokaryoticsignalpeptide.Truncatedderivativesofthemannanase,lacking54and16residuesfromtheN-andC-terminusrespectivelyofthematureformoftheenzyme,didnotexhibitcatalyticactivity.Inspectionoftheprimarystructureofthemannanasedidnotrevealanyobviouslinkersequencesorproteinmotifscharacteristicofthenon-catalyticdomainslocatedinotherPseudomonasplantcellwallhydrolases.Thesedataindicatethatthemannanaseisnon-modulator,comprisingasinglecatalyticdomain.ComparisonofthemannanasesequencewiththoseintheSWISSPROTdatabaserevealedgreatestsequencehomologywiththemannanasefromBacillussp.ThusthePseudomonasenzymebelongstoglycosylhydrolaseFamily26,afamilycontainingmannanasesandendoglucanases.Analysisofthesubstratespecificityofthemannanaseshowedthattheenzymehydrolysedmannanandgalactomannan,butdisplayedlittleactivitytowardsotherpolysaccharideslocatedintheplantcellwall.TheenzymehadapHoptimumofapprox.7.0,wasresistanttoproteolysisandhadanM_rof46,000whenexpressedbyEscherichiacoli.

Howthediversepolysaccharidespresentinplantcellwallsareassembledandinterlinkedintofunctionalcompositesisnotknownindetail.Here,usingtwonovelmonoclonalantibodiesandacarbohydrate-bindingmoduledirectedagainstthemannangroupofhemicellulosecellwallpolysaccharides,weshowthatmolecularrecognitionofmannanpolysaccharidespresentinintactcellwallsisseverelyrestricted.Insecondarycellwalls,mannanesterificationcanpreventproberecognitionofepitopes/ligands,anddetectionofmannansinprimarycellwallscanbeeffectivelyblockedbythepresenceofpectichomogalacturonan.Maskingbypectichomogalacturonanisshowntobeawidespreadphenomenoninparenchymasystems,andmaskedmannanwasfoundtobeafeatureofcellwallregionsatpitfields.Directfluorescenceimagingusingamannan-specificcarbohydrate-bindingmoduleandsequentialenzymetreatmentswithanendo-β-mannanaseconfirmedthepresenceofcrypticepitopesandthatthemaskingofprimarycellwallmannanbypectinisapotentialmechanismforcontrollingcellwallmicro-environments.

Thegrowingfieldofbiotechnologyisinconstantneedofbindingproteinswithnovelproperties.Notjustbindingspecificitiesandaffinitiesbutalsostructuralstabilityandproductivityareimportantcharacteristicsforthepurposeoflarge‐scaleapplications.Inordertofindsuchmolecules,librariesarecreatedbydiversifyingnaturallyoccurringbindingproteins,whichinthosecasesserveasscaffolds.Inthisstudy,weinvestigatedtheuseofathermostablecarbohydratebindingmodule,CBM4‐2,fromaxylanasefoundinRhodothermusmarinus,asadiversity‐carryingscaffold.Acombinatoriallibrarywascreatedbyintroducingrestrictedvariationat12positionsinthecarbohydratebindingsiteoftheCBM4‐2.Despitethesmallsizeofthelibrary(1.6×10⁶clones),variantsspecifictowardsdifferentcarbohydratepolymers(birchwoodxylan,Avicelandivorynutmannan)aswellasaglycoprotein(humanIgG4)weresuccessfullyselectedfor,usingthephagedisplaymethod.Investigatedclonesshowedahighproductivity(onaverage69mgofpurifiedprotein/lshakeflaskculture)whenproducedinEscherichiacoliandtheywereallstablemoleculesdisplayingahighmeltingtransitiontemperature(75.7±5.3°C).AllourresultsdemonstratethattheCBM4‐2moleculeisasuitablescaffoldforcreatingvariantsusefulindifferentbiotechnologicalapplications.

ThephytopathogenicfungusSclerotium(Athelia)rolfsiiformsonemajorendo-β-1,4-D-mannanase(EC3.2.1.78)undernon-inducedandderepressedconditions,i.e.afterdepletionofglucosewhichwasusedastheonlycarbohydratesubstrateforitscultivation.Thismannanasewaspurifiedtoelectrophoretichomogeneitybyammoniumsulfateprecipitation,hydrophobicinteractionchromatography,anionexchangechromatographyandgelfiltration.Theenzymeisaglycoproteinwithamolecularmassof46.5±2kDa(SDS-PAGE),anisoelectricpointof2.75,andapHoptimumof3.0–3.5.Theenzymeisespeciallystableintheacidicregionwithanexceptionalhalf-lifeofactivityof41daysatpH4.5and50°C.Itexertsactivityonβ-1,4-mannanfromivorynut,whichishydrolyzedmainlytomannobioseandmannotriose,aswellasonglucomannan,galactomannan,galactoglucomannan,andmannooligosaccharidesnotsmallerthanmannotetraose.Themainend-productsmannotrioseandtoalesserextentmannobioseinhibititsactivitymoderately.

Thehydrolysisoftheplantcellwallbymicrobialglycosidehydrolasesandesterasesistheprimarymechanismbywhichstoredorganiccarbonisutilizedinthebiosphere,andthustheseenzymesareofconsiderablebiologicalandindustrialimportance.Plantcellwall-degradingenzymesingeneraldisplayamodulararchitecturecomprisingcatalyticandnon-catalyticmodules.TheX4modulesinglycosidehydrolasesrepresentalargefamilyofnon-catalyticmoduleswhosefunctionisunknown.HereweshowthattheX4modulesfromaCellvibriojaponicusmannanase(Man5C)andarabinofuranosidase(Abf62A)bindtopolysaccharides,andthustheseproteinscompriseanewfamilyofcarbohydrate-bindingmodules(CBMs),designatedCBM35.TheMan5C-CBM35bindstogalactomannan,insolubleamorphousmannan,glucomannan,andmanno-oligosaccharidesbutdoesnotinteractwithcrystallinemannan,cellulose,cello-oligosaccharides,orotherpolysaccharidesderivedfromtheplantcellwall.Man5C-CBM35alsopotentiatesmannanaseactivityagainstinsolubleamorphousmannan.Abf62A-CBM35interactswithunsubstitutedoat-speltxylanbutnotsubstitutedformsofthehemicelluloseorxylo-oligosaccharides,andrequirescalciumforbinding.Thisisinsharpcontrasttootherxylan-bindingCBMs,whichinteractinacalcium-independentmannerwithbothxylo-oligosaccharidesanddecoratedxylans.

TheenzymaticdegradationofsinglecrystalsofmannanIwiththecatalyticcoredomainofaβ-mannanase(EC3.2.1.78orMan5A)fromTrichodermareeseiwasinvestigatedbytransmissionelectronmicroscopyandelectrondiffraction.Theenzymeattacktookplaceattheedgeofthecrystalsandprogressedtowardstheircentres.Quiteremarkablythecrystallineintegrityofthecrystalswaspreservedalmosttotheendofthedigestionprocess.Thisbehaviourisconsistentwithanendo-mechanism,wheretheenzymeinteractswiththeaccessiblemannanchainslocatedatthecrystalperipheryandcleavesonemannanmoleculeatatime.Theendomodeofdigestionofthecrystalswasconfirmedbyananalysisofthesolubledegradationproducts.

Innature,algaeproducecelluloseIwhereallglucanchainsarealignedparallel.However,thepresenceofcelluloseIIwithanti-parallelglucanchainshasbeenreportedforcertainDerbesia(Chlorophyceaealgae)cellwalls;ifthisistrue,itwouldmeananewbiologicalprocessforsynthesizingcellulosethathasnotyetbeenrecognized.Toanswerthisquestion,weexaminedcellulosestructureinDerbesiacellwalls,intactaswellastreatedwithcelluloseisolationprocedures,usingsum-frequency-generationspectroscopy,infrared(IR)spectroscopyandX-raydiffraction(XRD).Derbesiawallscontainlargeamountsofmannanandsmallamountsofcrystallinecellulose.EvidenceforcelluloseIIintheintactcellwallswasnotfound,whereascelluloseIIinthetrifluoroaceticacid(TFA)treatedcellwallsamplesweredetectedbyIRandXRD.Acontrolexperimentconductedwithball-milledAvicelcellulosesamplesshowedthatcelluloseIIstructurecouldbeformedasaresultofTFAtreatmentanddryingofamorphouscellulose.ThesedatasuggestthatthecelluloseIIstructuredetectedintheTFA-treatedDerbesiagametophytewallsamplesismostlikelyduetoreorganizationofamorphouscelluloseduringthesamplepreparation.OurresultscontradictthepreviousreportofcelluloseIIinnativealgacellwalls.EvenifthecrystallinecelluloseIIexistsinintactDerbesiagametophytecellwalls,itsamountwouldbeverysmall(belowthedetectionlimit)andthusbiologicallyinsignificant.

β-mannanasehasshowncompellingbiologicalfunctionsbecauseofitsregulatoryrolesinmetabolism,inflammation,andoxidation.Thisstudyseparatedandpurifiedtheβ-mannanasefromBacillussubtilisBE-91,whichisapowerfulhemicellulose-degradingbacteriumusinga“two-step”methodcomprisingultrafiltrationandgelchromatography.Thepurifiedβ-mannanase(about28.2 kDa)showedhighspecificactivity(79,859.2 IU/mg).TheoptimumtemperatureandpHwere65°Cand6.0,respectively.Moreover,theenzymewashighlystableattemperaturesupto70°CandpH4.5-7.0.Theβ-mannanaseactivitywassignificantlyenhancedinthepresenceofMn⁺,Cu²⁺,Zn²⁺,Ca²⁺,Mg²⁺,andAl³⁺andstronglyinhibitedbyBa²⁺,andPb²⁺.K_mandV_maxvaluesforlocustbeangumwere7.14 mg/mLand107.5 μmol/min/mLversus1.749 mg/mLand33.45 µmol/min/mLforKonjacglucomannan,respectively.Therefore,β-mannanasepurifiedbythisworkshowsstabilityathightemperaturesandinweaklyacidicorneutralenvironments.Basedonsuchdata,theβ-mannanasewillhavepotentialapplicationsasadietarysupplementintreatmentofinflammatoryprocesses.

MethylationinwaterwithNaOH/MeIisappliedtostudytheinfluenceofthestereochemistryonrelativereactivitiesofD-mannosyl(M)comparedtoD-glucosyl(G)unitsinkonjacglucomannan(KGM).ThepHiskeptconstantat13.6overthecourseofthereactionandaliquotsareremovedaftervarioustimeintervals.MethyldistributioninGandMresiduesisdeterminedafterperethylation,hydrolysis,andconversiontoO-ethyl-O-methyl-alditolacetates.TheorderofrelativerateconstantsdeterminedfortheO-methylKonjacglucomannans(M-KGMs)indegreeofsubstitution(DS)range0.3–0.8isG-k₆>M-k₆>G-k₂≈M-k₂>M-k₃>G-k₃.OligosaccharidesobtainedbypartialhydrolysisafterfullprotectionofM-KGMwithMeI-d₃arelabeledwithm-amino-benzoicacidandmeasuredbyliquidchromatography–electrosprayionization–massspectrometry.DS/DPprofilesareinfullagreementwithrandomdistributionofmethylgroups.ThermalpropertiesofM-KGMsareanalyzedbydifferentialscanningcalorimetryandthermogravimetricanalysis.DecompositiontemperatureincreaseswithDS,whilethetemperatureofanendothermicchangedecreases.

Thegene(1488-bp)encodinganovelGH10endo-β-1,4-xylanase(XylM)consistingofanN-terminalcatalyticGH10domainandaC-terminalricin-typeβ-trefoillectindomain-like(RICIN)domainwasidentifiedfromLuteimicrobiumxylanilyticumHY-24.TheGH10domainofXylMwas72%identicaltothatofMicromonosporalupineendo-β-1,4-xylanaseandtheRICINdomainwas67%identicaltothatofActinospicarobiniaehypotheticalprotein.Therecombinantenzyme(rXylM:49kDa)exhibitedmaximumactivitytowardbeechwoodxylanat65°CandpH6.0,whiletheoptimumtemperatureandpHofitsC-terminaltruncatedmutant(rXylM△RICIN:35kDa)were45°Cand5.0,respectively.Afterpre-incubationof1hat60°C,rXylMretainedover80%ofitsinitialactivity,butthethermostabilityofrXylM△RICINwassharplydecreasedattemperaturesexceeding40°C.Thespecificactivity(254.1Umg^-1)ofrXylMtowardoatspeltsxylanwas3.4-foldhigherthanthat(74.8Umg^-1)ofrXylM△RICINwhenthesamesubstratewasused.rXylMdisplayedsuperiorbindingcapacitiestoligninandinsolublepolysaccharidescomparedtorXylM△RICIN.Enzymatichydrolysisofβ-1,4-D-xylooligosaccharides(X₃-X₆)andbirchwoodxylanyieldedX3asthemajorproduct.TheresultssuggestthattheRICINdomaininXylMmightplayanimportantroleinsubstrate-bindingandbiocatalysis.