Megazyme/Galactomannan (Carob; High Viscosity)/PGALMH/5 grams

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.

Xyloglucaniswidelybelievedtofunctionasatetherbetweencellulosemicrofibrilsintheprimarycellwall,limitingcellenlargementbyrestrictingtheabilityofmicrofibrilstoseparatelaterally.Totestthebiomechanicalpredictionsofthis“tetherednetwork”model,weassessedtheabilityofcucumber(Cucumissativus)hypocotylwallstoundergocreep(long-term,irreversibleextension)inresponsetothreefamily-12endo-β-1,4-glucanasesthatcanspecificallyhydrolyzexyloglucan,cellulose,orboth.Xyloglucan-specificendoglucanase(XEGfromAspergillusaculeatus)failedtoinducecellwallcreep,whereasanendoglucanasethathydrolyzesbothxyloglucanandcellulose(Cel12AfromHypocreajecorina)inducedahighcreeprate.Acellulose-specificendoglucanase(CEGfromAspergillusniger)didnotcausecellwallcreep,eitherbyitselforincombinationwithXEG.Testswithadditionalenzymes,includingafamily-5endoglucanase,confirmedtheconclusionthattocausecreep,endoglucanasesmustcutbothxyloglucanandcellulose.Similarresultswereobtainedwithmeasurementsofelasticandplasticcompliance.BothXEGandCel12Ahydrolyzedxyloglucaninintactwalls,butCel12AcouldhydrolyzeaminorxyloglucancompartmentrecalcitranttoXEGdigestion.XyloglucaninvolvementintheseenzymeresponseswasconfirmedbyexperimentswithArabidopsis(Arabidopsisthaliana)hypocotyls,whereCel12Ainducedcreepinwild-typebutnotinxyloglucan-deficient(xxt1/xxt2)walls.Ourresultsareincompatiblewiththecommondepictionofxyloglucanasaload-bearingtetherspanningthe20-to40-nmspacingbetweencellulosemicrofibrils,buttheydoimplicateaminorxyloglucancomponentinwallmechanics.Thestructurallyimportantxyloglucanmaybelocatedinlimitedregionsoftightcontactbetweenmicrofibrils.

ThegenomesequenceofthehyperthermophilicbacteriumThermotogamaritimaencodesanumberofglycosylhydrolases.Manyoftheseenzymeshavebeenshowninvitrotodegradespecificglycosidesthatpresumablyserveascarbonandenergysourcesfortheorganism.However,becauseofthebroadsubstratespecificityofmanyglycosylhydrolases,itisdifficulttodeterminethephysiologicalsubstratepreferencesforspecificenzymesfrombiochemicalinformation.Inthisstudy,T.maritimawasgrownonarangeofpolysaccharides,includingbarleyβ-glucan,carboxymethylcellulose,carobgalactomannan,konjacglucomannan,andpotatostarch.Inallcases,significantgrowthwasobserved,andcelldensitiesreached10⁹cells/ml.Northernblotanalysesrevealeddifferentsubstrate-dependentexpressionpatternsforgenesencodingthevariousendo-actingβ-glycosidases;thesepatternsrangedfromstrongexpressiontonoexpressionundertheconditionstested.Forexample,cel74(TM0305),ageneencodingaputativeβ-specificendoglucananse,wasstronglyexpressedonallsubstratestested,includingstarch,whilenoevidenceofexpressionwasobservedonanysubstrateforlam16(TM0024),xyl10A(TM0061),xyl10B(TM0070),andcel12A(TM1524),whicharegenesthatencodealaminarinase,twoxylanases,andanendoglucanase,respectively.Thecel12B(TM1525)gene,whichencodesanendoglucanase,wasexpressedonlyoncarboxymethylcellulose.Anextracellularmannanaseencodedbyman5(TM1227)wasexpressedoncarobgalactomannanandkonjacglucomannanandtoalesserextentoncarboxymethylcellulose.Anunexpectedresultwasthefindingthatthecel5A(TM1751)andcel5B(TM1752)genes,whichencodeputativeintracellular,β-specificendoglucanases,wereinducedonlywhenT.maritimawasgrownonkonjacglucomannan.Toinvestigatethebiochemicalbasisofthisfinding,therecombinantformsofMan5(M_r,76,900)andCel5A(M_r,37,400)wereexpressedinEscherichiacoliandcharacterized.Man5,aT.maritimaextracellularenzyme,hadameltingtemperatureof99°Candanoptimuntemperatureof90°C,comparedto90and80°C,respectively,fortheintracellularenzymeCel5A.WhileMan5hydrolyzedbothgalactomannanandglucomannan,noactivitywasdetectedonglucansorxylans.Cel5A,however,notonlyhydrolyzedbarleyβ-glucan,carboxymethylcellulose,xyloglucan,andlicheninbutalsohadactivitycomparabletothatofMan5ongalactomannanandhigheractivitythanMan5onglucomannan.ThebiochemicalcharacteristicsofCel5A,thefactthatCel5AwasinducedonlywhenT.maritimawasgrownonglucomannan,andtheintracellularlocalizationofCel5Asuggestthatthephysiologicalroleofthisenzymeincludeshydrolysisofglucomannanoligosaccharidesthataretransportedfollowinginitialhydrolysisbyextracellularglycosidases,suchasMan5.

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.