Germinatingseedsoflucerne,guar,carobandsoybeaninitiallydepletedraffinoseseriesoligosaccharidesandthengalactomannan.Thisdepletionwasaccompaniedbyarapidincreaseandthenadecreaseinα-galactosidaselevels.Lucerneandguarcontainedtwoα-galactosidaseactivities,carobthreeandsoybeanfour.Oneoftheseineachplant,fromitslocationintheendosperm,timeofappearanceandkineticbehaviour,appearedtobeprimarilyinvolvedingalactomannanhydrolysis.ThisenzymeinlucernehadMWof23000andcouldnotbeseparatedfromβ-mannanaseby(NH₄)₂SO₄fractionation,DEAE,CMorSE-cellulosechromatographyorgelfiltration,butonlybypolyacrylamidegelelectrophoresis.Inguar,carobandsoybean,itcouldbeseparatedbyion-exchangechromatographyandgelfiltration.Inlucerne,carobandguarmostofthetotalincreaseinactivitywasduetothisenzyme.Theotherα-galactosidaseshadMWsofabout35000andcouldbeseparatedfromβ-mannanasebydissection,ionexchangecellulosechromatographyandgelfiltration.Theywerelocatedinthecotyledon-embryoandappearedtobeprimarilyinvolvedingalactosylsucroseoligosaccharidehydrolysis.

Structuralchangesingalactomannanongerminationoflucerne,carob,honeylocust,guarandsoybeanseeds,asmeasuredbyviscosity,elutionvolumesongelfiltrationandultra-centrifugationwereslightconsistentwitharapidandcompletehydrolysisofamoleculeoncehydrolysisofthemannanchainstarts.β-Mannanaseactivityincreasedandthendecreased,parallelinggalactomannandepletion.Multipleformsofβ-mannanasewereisolatedandthesewerelocatedintheendosperm.β-MannanasehadlimitedABIlitytohydrolysegalactomannanswithhighgalactosecontents.Seedscontainingthesegalactomannanshadveryactiveα-galactosidases.β-Mannosidaseswerepresentinbothendospermandcotyledon-embryoandcouldbeseparatedchromatographically.Thelevelofactivitywasjustsufficienttoaccountformannoseproductionfrommanno-oligosaccharides.

Aseriesofgalactomannanswithvaryingdegreesofgalactosesubstitutionhavebeenextractedfromtheendospermsoflegumeseedswithwaterandalkaliandtheamountofsubstitutionrequiredforwatersolubilityhasbeendetermined.Somewereheterogeneouswithrespecttothedegreeofgalactosesubstitution.Thestructuralrequirementsforhydrolysisbyplantβ-mannanasehavebeenstudiedusingtherelativeratesandextentsofhydrolysisofthesegalactomannans.Amoredetailedexaminationoftheproductsofhydrolysisofcarobgalactomannanhasbeenmade.Atleasttwocontiguousanhydromannoseunitsappeartobeneededforscission.Thisissimilartotherequirementforhydrolysisbymicrobialenzymes.Judastree(Cercissiliquastrum)endospermcontainedapolysaccharidewithauniquecompositionforalegumeseedreserve.Gelchromatographyandelectrophoresisoncelluloseacetateindicatedhomogeneity.Hydrolysiswithamixtureofβ-mannanaseandα-galactosidasegaveaglucose-mannosedisaccharideandacetolysisgaveagalactose-mannose.Theseresults,aswellasthepatternofhydrolysisbyβ-mannanasewereconsistentwithagalactoglucomannanstructure.

β-MannanaseactivitiesinthecommercialenzymepreparationsDriselaseandCellulase,inculturesolutionsofBacillussubtilis(TX1),incommercialsnailgut(Helixpomatia)preparationsandingerminatedseedsoflucerne,Leucaenaleucocephalaandhoneylocust,havebeenpurifiedbysubstrateaffinitychromatographyonglucomannan-AH-Sepharose.Onisoelectricfocusing,multipleproteinbandswerefound,allofwhichhadβ-mannanaseactivity.EachpreparationappearedasasinglemajorbandonSDS-polyacrylamidegelelectrophoresis.Theenzymesvariedintheirfinalspecificactivities,Kmvalues,optimalpH,isoelectricpointsandpHandtemperaturestabilitiesbuthadsimilarMWs.Theenzymeshavedifferentabilitiestohydrolysegalactomannanswhicharehighlysubstitutedwithgalactose.ThepreparationsDriselaseandCellulasecontainβ-mannanaseswhichcanattackhighlysubstitutedgalactomannansatpointsofsingleunsubstitutedD-mannosylresiduesiftheD-galactoseresiduesinthevicinityofthebondtobehydrolysedareallononlyonesideofthemainchain.

Anenzymictechniquehasbeendevelopedfortherapidandaccuratequantitationofthegalactomannancontentofguarseedsandmillingfractions.Thetechniqueinvolvesthemeasurementofthegalactosecomponentofgalactomannansusinggalactosedehydrogenase.Thegalactomannansareconvertedtogalactoseandmanno-oligosaccharidesusingpartiallypurifiedenzymesfromacommercialpreparationandfromgerminatedguarseeds.Simpleprocedureshavebeendevisedforthepreparationoftheseenzymes.Applicationofthetechniquetoanumberofguarvarietiesgavevaluesforthegalactomannancontentrangingfrom22.7to30.8%ofseedweight.

Aβ-D-mannosidemannohydrolaseenzymehasbeenpurifiedtohomogeneityfromgerminatedguar-seeds.Difficultiesassociatedwiththeextractionandpurificationappearedtobeduetoaninteractionoftheenzymewithotherproteinmaterial.Thepurifiedenzymehydrolysedvariousnaturalandsyntheticsubstrates,includingβ-D-manno-oligosaccharidesandreducedβ-D-manno-oligosaccharidesofdegreeofpolymerisation2to6,aswellasp-nitrophenyl,naphthyl,andmethylumbelliferylβ-D-mannopyranosides.Thepreferred,naturalsubstratewasβ-D-mannopentaose,whichwashydrolysedattwicetherateofβ-D-mannotetraoseandfivetimestherateofβ-D-mannotriose.Thisresult,togetherwiththeobservationthatα-D-mannoseisreleasedonhydrolysis,indicatesthattheenzymeisanexo-β-D-mannanase.

N.m.r.,enzymic,andchemicaltechniqueshavebeenusedtocharacterisetheD-galactose-containingtri-andtetra-saccharidesproducedonhydrolysisofcarobandL.leucocephalaD-galacto-D-mannansbyDriselaseβ-D-mannanase.Theseoligosaccharideswereshowntobeexclusively6¹-α-D-galactosyl-β-D-mannobioseand6¹-α-D-galactosyl-β-D-mannotriose.FurThermore,theseweretheonlyD-galactose-containingtri-andtetra-saccharidesproducedonhydrolysisofcarobD-galacto-D-mannanbyβ-D-mannanasesfromothersources,includingBacillussubtilis,Aspergillusniger,Helixpomatiagutsolution,andgerminatedlegumes.Acidhydrolysisoflucernegalactomannanyielded6¹-α-D-galactosyl-β-D-mannobioseand6²-α-D-galactosyl-β-D-mannobiose.

β-D-Mannosidase(β-D-mannosidemannohydrolaseEC3.2.1.25)waspurified160-foldfromcrudegut-solutionofHelixpomatiabythreechromatographicstepsandthengaveasingleproteinband(mol.wt.94,000)onSDS-gelelectrophoresis,andthreeproteinbands(ofalmostidenticalisoelectricpoints)onthin-layeriso-electricfocusing.Eachoftheseproteinbandshadenzymeactivity.Thespecificactivityofthepurifiedenzymeonp-nitrophenylβ-D-mannopyranosidewas1694nkat/mgat40°anditwasdevoidofα-D-mannosidase,β-D-galactosidase,2-acet-amido-2-deoxy-D-glucosidase,(1→4)-β-D-mannanase,and(1→4)-β-D-glucanaseactivities,almostdevoidofα-D-galactosidaseactivity,andcontaminatedwith<0.02% of="" β-d-glucosidase="" activity.="" the="" purified="" enzyme="" had="" the="" same="">K_mforborohydride-reducedβ-D-manno-oligosaccharidesofd.p.3-5(12.5mM).Theinitialrateofhydrolysisof(1→4)-linkedβ-D-manno-oligosaccharidesofd.p.2-5andofreducedβ-D-manno-oligosaccharidesofd.p.3-5wasthesame,ando-nitrophenyl,methylumbelliferyl,andnaphthylβ-D-mannopyranosideswerereADIlyhydrolysed.β-D-Mannobiosewashydrolysedatarate~25timesthatof6¹-α-D-galactosyl-β-D-mannobioseand6³-α-D-galactosyl-β-D-mannotetraose,andat~90timestherateforβ-D-mannobi-itol.

Hydrolysisofgalactomannaninendospermsofgerminatingguarisduetothecombinedactionofthreeenzymes,α-galactosidase,β-mannanaseandexo-β-mannanase.α-Galactosidaseandexo-β-mannanaseactivitiesoccurbothinendospermandcotyledontissuebutβ-mannanaseoccursonlyinendosperms.Onseedgermination,β-mannanaseandendospermicα-galactosidasearesynthesizedandactivitychangesparallelgalactomannandegradation.Galactomannandegradationandsynthesisofthesetwoenzymesareinhibitedbycycloheximide.Incontrast,endospermicexo-β-mannanaseisnotsynthesizedonseedgermination,butratherisalreadypresentthroughoutendospermtissue.Ithasnoactiononnativegalactomannan.α-Galactosidase,β-mannanaseandexo-β-mannanasehavebeenpurifiedtohomogeneityandtheirseparateandcombinedactioninthehydrolysisofgalactomannanandeffectontherateofuptakeofcarbohydratebycotyledons,studied.Resultsobtainedindicatedthatthesethreeactivitiesaresufficienttoaccountforgalactomannandegradationinvivoand,further,thatallthreearerequired.Cotyledonscontainanactiveexo-β-mannanaseandsugar-uptakeexperimentshaveshownthatcotyledonscanabsorbmannobioseintact,indicatingthatthisenzymeisinvolvedinthecompletedegradationofgalactomannanonseedgermination.

Treatmentofhot-water-solublecarobgalactomannanwithβ-D-mannanasesfromA.nigerorlucerneseedaffordsanarrayofD-galactose-containingβ-D-mannosaccharidesaswellasβ-D-manno-biose,-triose,and-tetraose(lucerne-seedenzymeonly).TheD-galactose-containingβ-D-mannosaccharidesofd.p.3–9producedbyA.nigerβ-D-mannanasehavebeencharacterised,usingenzymic,n.m.r.,andchemicaltechniques,as6¹-α-D-galactosyl-β-D-mannobiose,6¹-α-D-galactosyl-β-D-mannotriose,6³,6⁴-di-α-D-galactosyl-β-D-mannopentaose(theonlyheptasaccharide),and6³,6⁴-di-α-D-galactosyl-β-D-mannohexaose,6⁴,6⁵-di-α-D-galactosyl-β-D-mannohexaose,and6¹,6³,6⁴-tri-α-D-galactosyl-β-D-mannopentaose(theonlyoctasaccharides).Fournonasaccharideshavealsobeencharacterised.Penta-andhexa-saccharideswereabsent.Lucerne-seedβ-D-mannanaseproducedthesamebranchedtri-,tetra-andhepta-saccharides,andalsopenta-andhexa-saccharidesthatwerecharacterisedas6¹-α-D-galactosyl-β-D-mannotetraose,6³-α-D-galactosyl-β-D-mannotetraose,6¹,6³-di-α-D-galactosyl-β-D-mannotetraose,6³-α-D-galactosyl-β-D-mannopentaose,and6⁴-α-D-galactosyl-β-D-mannopentaose.NoneoftheoligosaccharidescontainedaD-galactosestubontheterminalD-mannosylgroupnorweretheysubstitutedonthesecondD-mannosylresiduefromthereducingterminal.

Purified(1→4)-β-D-mannanasefromAspergillusnigerandlucerneseedshasbeenincubatedwithmannosaccharidesandend-reduced(1→4)-β-D-mannosaccharidesand,fromtheproductsofhydrolysis,acyclicreaction-sequencehasbeenproposed.Fromtheheterosaccharidesreleasedbyhydrolysisofthehot-water-solublefractionofcarobgalactomannanbyA.nigerβ-D-mannanase,apatternofbindingbetweentheβ-D-mannanchainandtheenzymehasbeendeduced.Theproductsofhydrolysiswiththeβ-D-mannanasesfromIrpexlacteus,Helixpomatia,Bacillussubtilis,andlucerneandguarseedshavealsobeendetermined,andthedifferencesfromtheactionofA.nigerβ-D-mannanaserelatedtominordifferencesinsubstratebinding.Theproductsofhydrolysisofglucomannanareconsistentwiththoseexpectedfromthebindingpatternproposedfromthehydrolysisofgalactomannan.

ThedistributionofD-galactosylgroupsalongtheD-mannanbackbone(finestructure)ofcarobandguargalactomannanshasbeenstudiedbyacomputeranalysisoftheamountsandstructuresofoligosaccharidesreleasedonhydrolysisofthepolymerswithtwohighlypurifiedβ-D-mannanasesisolatedfromgerminatedguarseedandfromAspergillusnigercultures.Computerprogrammesweredevelopedwhichaccountedforthespecificsubsite-bindingrequirementsoftheβ-D-mannanasesandwhichsimulatedthesynthesisofgalactomannanbyprocessesinwhichtheD-galactosylgroupsweretransferredtothegrowingD-mannanchainineitherastatisticallyrandommannerorasinfluencedbynearest-neighbour/second-nearest-neighboursubstitution.Suchamodelwaschosenasitisconsistentwiththeknownpatternofsynthesisofsimilarpolysaccharides,forexample,xyloglucan;also,additiontoapreformedmannanchainwouldbeunlikely,duetotheinsolublenatureofsuchpolymers.TheD-galactosedistributionincarobgalactomannanandinthehot-andcold-water-solublefractionsofcarobgalactomannanhasbeenshowntobenon-regular,withahighproportionofsubstitutedcouplets,lesseramountsoftriplets,andanabsenceofblocksofsubstitution.TheprobabilityofsequencesinwhichalternateD-mannosylresiduesaresubstitutedislow.TheprobabilitydistributionofblocksizesforunsubstitutedD-mannosylresiduesindicatesthatthereisahigherproportionofblocksofintermediatesizethanwouldbepresentinagalactomannanwithastatisticallyrandomD-galactosedistribution.Basedonthealmostidenticalpatternsofamountsofoligosaccharidesproducedonhydrolysiswithβ-D-mannanase,itappearsthatgalactomannansfromseedofawiderangeofcarobvaritieshavethesamefine-structure.TheD-galactosedistributioninguar-seedgalactomannanalsoappearstobenon-regular,andgalactomannansfromdifferentguar-seedvarietiesappeartohavethesamefine-structure.

ArangeofgalactomannansvaryingwidelyinthecontentsofD-galactosehavebeencomparedforself-associationandtheirinteractionpropertieswithagaroseandxanthan.Whereas,ingeneral,themostinteractivegalactomannansarethoseinwhichthe(1→4)-β-D-mannanchainisleastsubstitutedbyα-D-galactosylstubs,evidenceispresentedwhichindicatesthatthedistributionofD-galactosylgroupsalongthebackbone(finestructure)canhaveasignificanteffectontheinteractionproperties.Forgalactomannanscontaining<30% of="" d-galactose,="" those="" which="" contain="" a="" higher="" frequency="" of="" unsubstituted="" blocks="" of="" intermediate="" length="" in="" the="" β-d-mannan="" chain="" are="" most="" interactive.="" for="" galactomannans="" containing="">40%ofD-galactose,thosewhichcontainahigherfrequencyofexactlyalternatingregionsintheβ-D-mannanchainaremostinteractive.Thisselectivity,onthebasisofgalactomannanfine-structure,inmixedpolysaccharideinteractionsinvitrocouldmimictheselectivityofbindingofbranchedplant-cell-wallpolysaccharidesinBIOLOGicalsystems.

ArangeofgalactomannansvaryingwidelyinthecontentofD-galactosehavebeencomparedforself-association,andtheirinteractionpropertieswithagaroseandxanthan.TheresultspresentedindicatethatingeneralthemostinteractivegalactomannansarethoseinwhichtheD-mannanmainchainbearsfewestD-galactosestubs,andconfirmthatthedistributionofD-galactosegroupsalongthemainchaincanhaveasignificanteffectontheinteractivepropertiesofthegalactomannans.Ithasbeenshownthatfreeze—thawprecipitationofgalactomannansrequiresregionsoftotallyunsubstitutedD-mannoseresiduesalongthemainchain,andthatathresholdforsignificantfreeze—thawprecipitationoccursataweight-averagelengthoftotallyunsubstitutedresiduesofapproximatelysix.ForgalactomannanshavingstructuresabovethisthresholdtheirinteractivepropertieswithotherpolysaccharidesarecontrolledbystructuralfeaturesassociatedwithtotallyunsubstitutedregionsoftheD-mannanbackbone.Incontrast,forgalactomannansbelowthisthreshold,theirinteractivepropertiesarecontrolledbystructuralfeaturesassociatedwithunsubstitutedsidesofD-mannanbackbone.

GalactomannanhasbeenextractedfromtheendospermofseedsofGleditsiatriacanthos(honeylocust)atdifferentstagesofdevelopment,whentheseedwasaccumulatingstoragematerial.Propertiesofthedifferentsampleshavebeenstudied.Themolecularsizedistributionbecamemoredisperseasgalactomannanaccumulatedandthegalactose:mannoseratiodecreasedslightly.SomepossIBLereasonsforthesechangesarediscussed.

Plantbiomassiscentraltothecarboncycleandtoenvironmentallysustainableindustriesexemplifiedbythebiofuelsector.Plantcellwalldegradingenzymesgenerallycontainnoncatalyticcarbohydratebindingmodules(CBMs)thatfulfilatargetingfunction,whichenhancescatalysis.CBMsthatbindβ-glucanchainsoftendisplaybroadspecificityrecognizingβ1,4-glucans(cellulose),β1,3-β1,4-mixedlinkedglucansandxyloglucan,aβ1,4-glucandecoratedwithα-1,6-xyloseresidues,bytargetingstructurescommontothethreepolysaccharides.Thus,CBMsthatrecognizexyloglucantargettheβ1,4-glucanbackboneandonlyaccommodatethexylosedecorations.HereweshowthattwocloselyrelatedCBMs,CBM65AandCBM65B,derivedfromEcCel5A,aEubacteriumcellulosolvensendoglucanase,bindtoarangeofβ-glucansbut,uniquely,displaysignificantpreferenceforxyloglucan.ThestructuresofthetwoCBMsrevealaβ-sandwichfold.Theligandbindingsitecomprisestheβ-sheetthatformstheconcavesurfaceoftheproteins.Bindingtothebackbonechainsofβ-glucansismediatedprimarilybyfivearomaticresiduesthatalsomakehydrophobicinteractionswiththexylosesidechainsofxyloglucan,conferringthedistinctivespecificityoftheCBMsforthedecoratedpolysaccharide.Significantly,andincontrasttootherCBMsthatrecognizeβ-glucans,CBM65Autilizesdifferentpolarresiduestobindcelluloseandmixedlinkedglucans.Thus,Gln¹⁰⁶iscentraltocelluloserecognition,butisnotrequiredforbindingtomixedlinkedglucans.Thisreportrevealsthemechanismbywhichβ-glucan-specificCBMscandistinguishbetweenlinearandmixedlinkedglucans,andshowhowtheseCBMscanexploitanextensivehydrophobicplatformtotargetthesidechainsofdecoratedβ-glucans.

TheC-terminal176aminoacidsofaThermotogamaritimamannanase(Man5)constituteacarbohydratebindingmodule(CBM)thathasbeenclassifiedintoCBMfamily27.TheisolatedCBM27domain,namedTmCBM27,bindstightly(K_as10⁵–10⁶,M^-1)toβ-1,4-mannooligosaccharides,carobgalactomannan,andkonjacglucomannan,butnottocellulose(insolubleandsoluble)orsolublebirchwoodxylan.TheX-raycrystalstructuresofnativeTmCBM27,aTmCBM27-mannohexaosecomplex,andaTmCBM27-6³,6⁴,-α-D-galactosyl-mannopentaosecomplexat2.0Å,1.6Å,and1.35Å,respectively,revealthebasisofTmCBM27"sspecificityformannans.Inparticular,thelattercomplex,whichisthefirststructureofaCBMincomplexwithabranchedplantcellwallpolysaccharide,illustrateshowthearchitectureofthebindingsitecaninfluencetherecognitionofnaturallysubstitutedpolysaccharides.

Acriticalstructuralfeatureofmanymicrobialendo-β-1,4-glucanases(EGases,orcellulases)isacarbohydratebindingmodule(CBM),whichisrequiredforeffectivecrystallinecellulosedegradation.However,CBMsareabsentfromplantEGasesthathavebeenbiochemicallycharacterizedtodate,andaccordingly,plantEGasesarenotgenerallythoughttohavethecapacitytodegradecrystallinecellulose.WereportthebiochemicalcharacterizationofatomatoEGase,SolanumlycopersicumCel8(SlCel9C1),withadistinctC-terminalnoncatalyticmodulethatrepresentsapreviouslyuncharacterizedfamilyofCBMs.InvitrobindingstudiesdemonstratedthatthismoduleindeedbindstocrystallinecelluloseandcansimilarlybindaspartofarecombinantchimericfusionproteincontaininganEGasecatalyticdomainfromthebacteriumThermobifidafusca.Site-directedmutagenesisstudiesshowthattryptophans559and573playaroleincrystallinecellulosebinding.TheSlCel9C1CBM,whichrepresentsanewCBMfamily(CBM49),isadefiningfeatureofanewstructuralsubclass(ClassC)ofplantEGases,withmemberspresentthroughouttheplantkingdom.Inaddition,theSlCel9C1catalyticdomainwasshowntohydrolyzeartificialcellulosicpolymers,celluloseoligosaccharides,andavarietyofplantcellwallpolysaccharides.

Background:Mannansarekeycomponentsoflignocellulosepresentinthehemicellulosicfractionofplantprimarycellwalls.Mannanendo-1,4-β-mannosidases(1,4-β-D-mannanases)catalyzetherandomhydrolysisofβ-1,4-mannosidiclinkagesinthemainchainofβ-mannans.Biodegradationofβ-mannansbytheactionofthermostablemannanendo-1,4-β-mannosidaseofferssignificanttechnicaladvantagesinbiotechnologicalindustrialapplications,i.e.delignificationofkraftpulpsorthepretreatmentoflignocellulosicbiomassrichinmannanfortheproductionofsecondgenerationbiofuels,aswellasforapplicationsinoilandgaswellstimulation,extractionofvegetableoilsandcoffeebeans,andtheproductionofvalue-addedproductssuchasprebioticmannooligosaccharides(MOS).Results:Ageneencodingmannanendo-1,4-β-mannosidaseor1,4-β-D-mannanmannanohydrolase(E.C.3.2.1.78),commonlytermedβ-mannanase,fromAspergillusnigerBK01,whichbelongstoglycosylhydrolasefamily5(GH5),wasclonedandsuccessfullyexpressedheterologously(upto243μgofactiverecombinantproteinpermL)inPichiapastoris.TheenzymewassecretedbyP.pastorisandcouldbecollectedfromtheculturesupernatant.ThepurifiedenzymeappearedglycosylatedasasinglebandonSDS-PAGEwithamolecularmassofapproximately53kDa.Therecombinantβ-mannanaseishighlythermostablewithahalf-lifetimeofapproximately56hat70°CandpH4.0.Theoptimaltemperature(10-minassay)andpHvalueforactivityare80°CandpH4.5,respectively.Theenzymeisnotonlyactivetowardsstructurallydifferentmannansbutalsoexhibitslowactivitytowardsbirchwoodxylan.ApparentK_mvaluesoftheenzymeforkonjacglucomannan(lowviscosity),locustbeangumgalactomannan,carobgalactomannan(lowviscosity),and1,4-β-D-mannan(fromcarob)are0.6mgmL^-1,2.0mgmL^-1,2.2mgmL^-1and1.5mgmL^-1,respectively,whiletheK_catvaluesforthesesubstratesare215s^-1,330s^-1,292s^-1and148s^-1,respectively.JudgedfromthespecificityconstantsK_cat/K_m,glucomannanisthepreferredsubstrateoftheA.nigerβ-mannanase.Analysisbythinlayerchromatographyshowedthatthemainproductfromenzymatichydrolysisoflocustbeangumismannobiose,withonlylowamountsofmannotrioseandhighermanno-oligosaccharidesformed.Conclusion:Thisstudyisthefirstreportonthecloningandexpressionofathermostablemannanendo-1,4-β-mannosidasefromA.nigerinPichiapastoris.Theefficientexpressionandeaseofpurificationwillsignificantlydecreasetheproductioncostsofthisenzyme.TakingadvantageofitsacidicpHoptimumandhighthermostability,thisrecombinantβ-mannanasewillbevaluableinvariousbiotechnologicalapplications.

Carbohydrate–proteinrecognitioniscentraltomanybiologicalprocesses.Enzymesthatactonpolysaccharidesubstratesfrequentlycontainnoncatalyticdomains,“carbohydrate-bindingmodules”(CBMs),thattargettheenzymetotheappropriatesubstrate.CBMsthatrecognizespecificplantstructuralpolysaccharidesareoftenabletoaccommodateboththevariablebackboneandtheside-chaindecorationsofheterogeneousligands.“CBM29”modules,derivedfromanoncatalyticcomponentofthePiromycesequicellulase/hemicellulasecomplex,provideanexampleofthisselectiveyetflexiblerecognition.Theydiscriminatestronglyagainstsomepolysaccharideswhileremainingrelativelypromiscuoustowardbothβ-1,4-linkedmanno-andcello-oligosaccharides.Thisfeaturemayreflectpreferential,butflexible,targetingtowardglucomannansintheplantcellwall.Thethree-dimensionalstructureofCBM29-2anditscomplexeswithcello-andmannohexaoserevealaβ-jelly-rolltopology,withanextendedbindinggrooveontheconcavesurface.Theorientationofthearomaticresiduescomplementstheconformationofthetargetsugarpolymerwhileaccommodationofbothmanno-andgluco-configuredoligo-andpolysaccharidesisconferredbyvirtueoftheplasticityofthedirectinteractionsfromtheiraxialandequatorial2-hydroxyls,respectively.Suchflexibleligandrecognitiontargetstheanaerobicfungalcomplextoarangeofdifferentcomponentsintheplantcellwallandthusplaysapivotalroleinthehighlyefficientdegradationofthiscompositestructurebythemicrobialeukaryote.

Enzymesthatdegradeplantcellwallpolysaccharidesdisplayamodulararchitecturecomprisingacatalyticdomainboundtooneormorenon-catalyticcarbohydrate-bindingmodules(CBMs).CBMsdisplayconsiderablevariationinprimarystructureandaregroupedinto59sequence-basedfamiliesorganizedintheCarbohydrate-ActiveenZYme(CAZy)database.HerewereportthecrystalstructureofCtCBM42Atogetherwiththebiochemicalcharacterizationoftwoothermembersoffamily42CBMsfromClostridiumthermocellum.CtCBM42A,CtCBM42BandCtCBM42Cbindspecificallytothearabinoseside-chainsofarabinoxylansandarabinan,suggestingthatvariouscellulosomalcomponentsaretargetedtotheseregionsoftheplantcellwall.ThestructureofCtCBM42Adisplaysabeta-trefoilfold,whichcomprises3sub-domainsdesignatedasα,βandγ.Eachoneofthethreesub-domainspresentsaputativecarbohydrate-bindingpocketwhereanaspartateresiduelocatedinacentralpositiondominatesligandrecognition.Intriguingly,theγsub-domainofCtCBM42Aispivotalforarabinoxylanbinding,whiletheconcertedactionofβandγsub-domainsofCtCBM42BandCtCBM42Cisapparentlyrequiredforligandsequestration.Thus,thisworkrevealsthatthebindingmechanismofCBM42membersisincontrastwiththatofhomologousCBM13swhererecognitionofcomplexpolysaccharidesresultsfromthecooperativeactionofthreeproteinsub-domainspresentingsimilaraffinities.

Mannanpolysaccharidesarewidespreadamongplants,wheretheyserveasstructuralelementsincellwalls,ascarbohydratereserves,andpotentiallyperformotherimportantfunctions.Previousworkhasdemonstratedthatmembersofthecellulosesynthase-likeA(CslA)familyofglycosyltransferasesfromArabidopsis(Arabidopsisthaliana),guar(Cyamopsistetragonolobus),andPopulustrichocarpacatalyseβ-1,4-mannanandglucomannansynthasereactionsinvitro.MannanpolysaccharidesandhomologsofCslAgenesappeartobepresentinalllineagesoflandplantsanalyzedtodate.Inmanyplants,theCslAgenesaremembersofextendedmultigenefamilies;however,itisnotknownwhetherallCslAproteinsareglucomannansynthases.CslAproteinsfromdiverselandplantspecies,includingrepresentativesofthemono-anddicotyledonousangiosperms,gymnosperms,andbryophytes,wereproducedininsectcells,andeachCslAproteincatalyzedmannanandglucomannansynthasereactionsinvitro.Microarrayminingandquantitativereal-timereversetranscription-polymerasechainreactionanalysisdemonstratedthattranscriptsofArabidopsisandloblollypine(Pinustaeda)CslAgenesdisplaytissue-specificexpressionpatternsinvegetativeandfloraltissues.GlycanmicroarrayanalysisofArabidopsisindicatedthatmannansarepresentthroughouttheplantandareespeciallyabundantinflowers,siliques,andstems.MannansarealsopresentinchloronemalandcaulonemalfilamentsofPhyscomitrellapatens,wheretheyareprevalentatcelljunctionsandinbuds.Takentogether,theseresultsdemonstratethatmembersoftheCslAgenefamilyfromdiverseplantspeciesencodeglucomannansynthasesandsupportthehypothesisthatmannansfunctioninmetabolicnetworksdevotedtoothercellularprocessesinadditiontocellwallstructureandcarbohydratestorage.

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.

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.