Megazyme/Glucomannan (Konjac; Low Viscosity)/P-GLCML/4 grams

Thisstudyinvestigatedsoftwoodhemicellulosesdegradationpathwaysduringalkalinehydrogenperoxide(AHP)pretreatmentofDouglasfir.ItwasfoundthatglucomannanismuchmoresusceptIBLetoalkalinepretreatmentthanxylan.Organicacids,includinglactic,succinic,glycolicandformicacidarethepredominantproductsfromglucomannandegradation.Atlowtreatmenttemperature(90°C),asmallamountofformicacidisproducedfromglucomannan,whereasglucomannandegradationtolacticacidandsuccinicacidbecomesthemainreactionsat140°Cand180°C.TheadditionofH₂O₂duringalkalinepretreatmentofD.firledtoasignificantremovaloflignin,whichsubsequentlyfacilitatedglucomannansolubilization.However,H₂O₂haslittledirecteffectontheglucomannandegradationreaction.Themaindegradationpathwaysinvolvedinglucomannanconversiontoorganicsacidsareelucidated.Theresultsfromthisstudydemonstratethepotentialtooptimizepretreatmentconditionstomaximizethevalueofbiomasshemicellulose.

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.

Herewepresentacomparisonofcommonlyusedmethodologiesfortheextractionandquantificationofkonjacglucomannan(KGM).Compositionalanalysisshowedthatthepurifiedkonjacflour(PKF)producedusingamodifiedextractionprocedurecontained92%glucomannan,withaweightaveragemolecularweight(M_w),polydispersityindex(PDI)anddegreeofacetylation(DA)of9.5±0.6×10⁵gmol^-1,1.2and2.8wt.%.Thesedata,plusFourier-transforminfraredspectral(FTIR)andzeroshearviscosityanalysesoftheextract(PKF)wereallconsistentwiththeliterature.ComparisonofthreeexistingmethodologiesforthequantitativeanalysisoftheKGMcontentofthePKF,namely3,5-dinitrosalicylicacid(3,5-DNS),phenol–sulphuricacidandenzymaticcolorimetricassays;indicatedthatthe3,5-DNScolorimetricassaywasthemostreproducibleandaccuratemethod,withalinearcorrelationcoefficientof0.997forsamplesrangingfrom0.5to12.5mg/ml,andrecoveriesbetween97%and103%acrossthreespikinglevels(250,500and750μg/g)ofstarch.Thesedataprovidethebasisofimprovedgoodlaboratorypractice(GLP)forthecommercialextractionandanalysisofthismultifunctionalnaturalpolymer.

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-addedproductssuchasprebioticmanno-oligosaccharides(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^-1respectively.JudgedfromthespecificityconstantsK_cat/K_m,glucomannanisthepreferredsubstrateoftheA.nigerβ-mannanase.Analysisbythinlayerchromatographyshowedthatthemainproductfromenzymatichydrolysisoflocustbeangumismannobiose,withonlylowamountsofmannotrioseandhighermanno-oligosaccharidesformed.Conclusion:Thisstudyisthefirstreportonthecloningandexpressionofathermostablemannanendo-1,4-β-mannosidasefromA.nigerinPichiapastoris.Theefficientexpressionandeaseofpurificationwillsignificantlydecreasetheproductioncostsofthisenzyme.TakingadvantageofitsacidicpHoptimumandhighthermostability,thisrecombinantβ-mannanasewillbevaluableinvariousbiotechnologicalapplications.

Background:Mannansareoneofthekeypolymersinhemicellulose,amajorcomponentoflignocellulose.TheMannanendo-1,4-β-mannosidaseor1,4-β-D-mannanase(EC3.2.1.78),commonlynamedβ-mannanase,isanenzymethatcancatalyzerandomhydrolysisofβ-1,4-mannosidiclinkagesinthemainchainofmannans,glucomannansandgalactomannans.Theenzymehasfoundanumberofapplicationsindifferentindustries,includingfood,feed,pharmaceutical,pulp/paperindustries,aswellasgaswellstimulationandpretreatmentoflignocellulosicbiomassfortheproductionofsecondgenerationbiofuel.BacilluslicheniformisisaGram-positiveendospore-formingmicroorganismthatisgenerallynon-pathogenicandhasbeenusedextensivelyforlarge-scaleindustrialproductionofvariousenzymes;however,therehasbeennopreviousreportonthecloningandexpressionofmannanendo-1,4-β-mannosidasegene(manB)fromB.licheniformis.Results:Themannanendo-1,4-β-mannosidasegene(manB),commonlyknownasβ-mannanase,fromBacilluslicheniformisstrainDSM13wasclonedandoverexpressedinEscherichiacoli.Theenzymecanbeharvestedfromthecelllysate,periplasmicextract,orculturesupernatantwhenusingthepFLAGexpressionsystem.Atotalactivityofapproximately50,000unitscouldbeobtainedfrom1-lshakeflaskcultures.Therecombinantenzymewas6×His-taggedatitsC-terminus,andcouldbepurifiedbyone-stepimmobilizedmetalaffinitychromatography(IMAC)toapparenthomogeneity.Thespecificactivityofthepurifiedenzymewhenusinglocustbeangumassubstratewas1672±96units/mg.TheoptimalpHoftheenzymewasbetweenpH6.0-7.0;whereastheoptimaltemperaturewasat50-60°C.Therecombinantβ-mannanasewasstablewithinpH5-12afterincubationfor30minat50°C,andwithinpH6-9afterincubationat50°Cfor24h.Theenzymewasstableattemperaturesupto50°Cwithahalf-lifetimeofactivity(τ1/2)ofapproximately80hat50°CandpH6.0.Analysisofhydrolyticproductsbythinlayerchromatographyrevealedthatthemainproductsfromthebioconversionoflocusbeangumandmannanwerevariousmanno-oligosaccharideproducts(M2-M6)andmannose.Conclusion:Ourstudydemonstratesanefficientexpressionandsecretionsystemfortheproductionofarelativelythermo-andalkali-stablerecombinantβ-mannanasefromB.licheniformisstrainDSM13,suitableforvariousbiotechnologicalapplications.

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.

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.

Mannantransglycosylaseisanovelcellwallenzymeactivityactingonmannan-basedplantpolysaccharidesinprimarycellwallsofmonocotyledonsanddicotyledons.Theenzymeactivitywasdetectedbyitsabilitytotransfergalactoglucomannan(GGM)polysaccharidestotritium-labelledGGM-derivedoligosaccharidesgeneratingtritium-labelledGGMpolysaccharides.Mannantransglycosylasewasfoundinarangeofplantspeciesandtissues.Highlevelsoftheenzymeactivitywerepresentinflowersofsomekiwifruit(Actinidia)speciesandinripetomato(SolanumlycopersicumL.)fruit.Lowlevelsweredetectedinmaturegreentomatofruitandactivityincreasedduringtomatofruitripeninguptotheredripestage.Essentiallyallactivitywasfoundinthetomatoskinandoutermost2mmoftissue.Mannantransglycosylaseactivityintomatoskinandouterpericarpisspecificformannan-basedplantpolysaccharides,includingGGM,galactomannan,glucomannanandmannan.Theexactstructuralrequirementsforvalidacceptorsremaintobedefined.Nevertheless,amannoseresidueatthesecondpositionofthesugarchainandtheabsenceofagalactosesubstituentonthefourthresidue(countingfromthenon-reducingend)appeartobeminimalrequirements.Mannan-basedpolysaccharidesintheplantcellwallmayhavearoleanalogoustothatofxyloglucans,introducingflexibilityandforminggrowth-restrainingnetworkswithcellulose.Thusmannantransglycosylaseandxyloglucanendotransglycosylase,theonlyotherknowntransglycosylaseactivityinplantcellwalls,maybothbeinvolvedinremodellingandrefiningthecelluloseframeworkindevelopmentalprocessesthroughoutthelifeofaplant.

ExceptinthePoaceae,littleisknownaboutthestructuresofthexyloglucansintheprimarywallsofmonocotyledons.Xyloglucanstructuresinarangeofmonocotyledonspecieswereexamined.Wallpreparationswereisolated,extractedwith6 Msodiumhydroxide,andtheextractstreatedwithaxyloglucan-specificendo-(1→4)-β-glucanasepreparation.Theoligosaccharidesreleasedwereanalyzedbyhigh-performanceanion-exchangechromatographyandbymatrix-assistedlaser-desorptionionizationtime-of-flightmassspectrometry.Oligosaccharideprofilesofthenon-commelinidmonocotyledonsweresimilartothoseofmosteudicotyledons,indicatingthexyloglucanswerefucogalactoxyloglucans,withaXXXGacoremotifandthefucosylatedunitsXXFGandXLFG.AnexceptionwasLemnaminor(Araceae),whichyieldednofucosylatedoligosaccharidesandhadbothXXXGandXXG_ncoremotifs.ExceptfortheArecales(palms)andtheDasypogonaceae,whichhadfucogalactoxyloglucans,thexyloglucansofthecommelinidmonocotyledonswerestructurallydifferent.TheZingiberalesandCommelinaleshadxyloglucanswithbothXXG_nandXXXGcoremotifs;smallproportionsofXXFGunits,butnoXLFGunits,werepresent.InthePoales,thePoaceaehadxyloglucanswithaXXG_ncoremotifandnofucosylatedunits.IntheotherPoalesfamilies,somehadbothXXXGandXXG_ncoremotifs,othershadonlyXXXG;XXFGunitswerepresent,butXLFGunitswerenot.

FourcorewoodtypeswereexaminedfromsaplingtreesoftwoclonesofPinusradiatagrowninaglasshouse.Treesweregrowneitherstraighttoproducenormalcorewood,tiltedat45°fromtheverticaltoproduceoppositecorewoodandcompressioncorewood,orrockedtoproduceflexurecorewood.MeancellulosemicrofibrilangleoftracheidwallswasestimatedbyX-raydiffractionandlongitudinalswellingmeasuredbetweenanovendryandmoisturesaturatedstate.Ligninandacetylcontentsofthewoodsweremeasuredandthemonosaccharidecompositionsofthecell-wallpolysaccharidesdetermined.Finelymilledwoodwasanalysedusingsolution-state2DNMRspectroscopyofgelsfromfinelymilledwoodinDMSO-d₆/pyridine-d₅.Althoughtherewasnosignificantdifferenceincellulosemicrofibrilangleamongthecorewoodtypes,compressioncorewoodhadthehighestlongitudinalswelling.Alignincontent>32%andagalactosylresiduecontent>6%clearlydividedseverecompressioncorewoodfromtheothercorewoodtypes.Relationshipscouldbedrawnbetweenlignincontentandlongitudinalswelling,andbetweengalactosylresiduecontentandlongitudinalswelling.The2DNMRspectrashowedthatthepresenceofH-unitsinligninwasexclusivetocompressioncorewood,whichalsohadahigher(1→4)-β-D-galactancontent,definingauniquecompositionforthatcorewoodtype.

Thepresentstudyreportsdualtetanusanddiphtheriatoxoidsloadedstablechitosan–glucomannannanoassemblies(sCh–GM-NAs)formulatedusingtandemionicgelationtechniquefororalmucosalimmunization.Thestable,lyophilizedsCh–GM-NAsexhibited~152 nmparticlesizeand~85%EEofboththetoxoids.ThelyophilizedsCh–GM-NAsdisplayedexcellentstabilityinbiomimeticmediaandpreservedchemical,conformationandbiologicalstabilityofencapsulatedtoxoids.ThehigherintracellularAPCsuptakeofsCh–GM-NAswasconcentrationandtimedependentwhichmaybeattributedtothereceptormediatedendocytosisviamannoseandglucosereceptor.ThehigherCaco-2uptakeofsCh–GM-NAswasfurtherconfirmedbyexvivointestinaluptakestudies.The invivo evaluationrevealedthatsCh–GM-NAsposedsignificantly(p < 0.001)higher=""humoral,=""mucosal=""and=""cellular=""immune=""response=""than=""other=""counterparts=""by=""eliciting=""complete=""protective=""levels=""of=""anti-tt=""and=""anti-dt=""(~0.1 iu/ml)=""antibodies.=""importantly,=""commercial=""‘dual=""antigen’=""vaccine=""administered=""through=""oral=""or=""intramuscular=""route=""was=""unable=""to=""elicit=""all=""type=""of=""immune=""response.=""conclusively,=""sch–gm-nas=""could=""be=""considered=""as=""promising=""vaccine=""adjuvant=""for=""oral=""mucosal=""immunization.="">