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.

D-Galacto-D-mannansoccurintheendospermsofawiderangeofleguminousseedsinamountsvaryingfrom0.1%(soybean)to45%(Cassiabrewsterii)ofseedweight(1).ThepolysaccharidesfromdifferentspecieshavedifferentproportionsofD-galactoseandD-mannose,butessentiallyalwaysconsistofaβ-1,4-linkedmannanbackbonewithsingleD-galactosebrancheslinedα-1,6(2).

α-Galactosidasehasbeenshowntooccurinawiderangeofplantsandanimalsandtobesynthesizedbymicroorganisms.Thisenzymehasbeenpurifiedfromseveralsourcesusingconventionalchromatographicproceduresandarangeofaffinitysupports.Oftheaffinityprocedures,thatemployingN-ɛ-aminocaproyl-α-D-galactopyranosylaminecoupledtoSepharose4BasdescribedbyHarpazetal.iseffectiveandreliableandcanbeusedtopurifyα-galactosidasefromawiderangeofBIOLOGicalmaterials.TheaffinitytechniquedescribedbyHarpazetal.wasemployedtopurifyα-galactosidasefromgreencoffeebeansandfromsoybeanseed.However,neitherofthesematerialsisagoodsourceofthisactivity.Thischapterdescribesthelarge-scalepurificationofα-galactosidaseswithhighactivityongalactomannanfromgerminatedseedsoflucerneandguaremployingtheaffinitymatrix.

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.

Enzymesmetabolizingpolysaccharideswereusedinsuchprocessesasbakingandbrewingforcountlesscenturiesbeforetherelationshipbetweenthechemicalstructureofthepolysaccharidesandtheirmodificationbyenzymeswasknown.Inthepasttwocenturies,studiesofthestructuresandfunctionsofpolysaccharidesinvolvedinthestorageofchemicalenergy,inthestructuralpartsoftissues,andasinformationcarriers,aswellastheenzymesthatmetabolizethem,havebeencarriedout.

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.

Guargalactomannanhasbeenmodifiedbytreatmentwithanα-D-galactosidaseApreparationfromlucerneseeds.ThisenzymewaspurifiedbyaffinitychromatographyonN-ϵ-aminocaproyl-α-D-galactopyranosylaminelinkedtoSepharose4B,hadahighactivitytowardsgalactomannans,andwascompletelydevoidofβ-D-mannanase.Onincubationfor2h,thisenzymeremoved>75%ofthegalactosefromguargalactomannanwithnoconcurrentdecreaseinviscosity.Eventualdecreaseinviscositywasassociatedwiththeformationofinsoluble,mannan-typeprecipitates.Thisphenomenon,althoughdirectlyrelatedtothegalactosecontentofthegalactomannan,wasalsotime-dependent.Thelimitingviscositynumberscalculatedforthe“mannanbackbones”ofα-D-galactosidase-treated,guargalactomannanhavinggalactose-mannoseratiosof38:62to15:85werethesame.Modified,guargalactomannan(at0.4%w/v)havingagalactose-mannoseratioof20:80,orless,formsagelonstorageat4°overseveralweeks.Also,gelparticlesformwhensolutionsofthesegalactomannansarepassedthroughafreeze-thawcycle.Samplescontaining<10%=""of=""galactose=""rapidly=""precipitate=""from=""solution=""even=""at=""30°.=""the=""interaction=""of=""guar=""galactomannan=""with=""xanthan=""is=""greatly=""increased=""by=""removal=""of=""galactose=""residues.=""samples=""having=""galactose-mannose=""ratios=""of=""~19:81=""interact=""with=""xanthan=""to=""essentially=""the=""same=""degree=""as=""carob=""galactomannan=""(gal/man="23:77).">

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.

Anumberofmethodshavebeendescribedfortheanalysisofthefinestructureofgalactomannans,i.e.,thedistributionofD-galactosylunitsalongtheD-mannanbackbone(1).Suchstudiesincludetheanalysisofx-raydiffractiondataofstretchedfibersofgalactomannans(2,3),¹H-and¹³C-nmr(nuclearmagneticresonance)ofnativeandpartiallydepolymerizedgalacto¬mannans(4)andarangeofchemicalprocedures(5-7),includingthoseemployingadetailedtheoreticalanalysisofthekineticsofreaction(8).Analternativeapproachinvolvesthecharacterizationandquantificationoftheoligosaccharidesproducedonhydrolysisofgalactomannansbyhighlypurifiedandwell-characterizedβ-mannanases(EC3.2.1.78)(9,10).Theβ-mannanasesemployedwerepurifiedtohomogeneitybyaffinitychromatographyongIucornannan-AH-Sepharose4B.Theywerecharacterizedbyarangeofphysicochemicaiproceduresbydeterminingthekineticsoftheiractiononβ-mannooligosaccharides,andbycharacterizingthestructuresofoligosaccharidesproducedonhydrolysisofgalactomannansandglucomannans(11).Fromthesestudies,abasicmodeldescribingthesubsitebindingrequirementsofalltheβ-mannanasesexaminedwasproposed(Fig.1).Thismodelwasthenmodifiedtoaccountfortheslightdifferencesnotedinthetypesofoligosaccharidesproducedbyβ-mannanasesfromdifferentsources.Theβ-mannanaseswhichdiffermostsignificantlyintheiractionpatternsongalactomannansarethosefromAspergillusnigerculturefiltratesandfromgerminatedguarseed.

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.

Glycosidaseswereusedtoprepareoligosaccharidestructuresofphysiologicalandmedicinalrelevance.Thestudyincludedanextensivescreeningofcrudeenzymaticpreparationsforα-andβ-galactosidase,α-andβ-mannosidase,β-N-acetylglucosaminidase,β-N-acetylgalactosaminidaseandα-L-fucosidaseactivities.Theenzymeswereassessedwithrespecttoregioselectivityofglycosyltransferontocarbohydrateacceptors.Thepurificationproceduresforindividualbiocatalystsaredescribedindetail.

Laserscanningconfocalmicroscopycombinedwithfluorescencerecoveryafterphotobleachingisaneffectivetooltomeasurethediffusioncoefficientsofmacromoleculesincross-linkedhydrogelsandpolymersolutions.Inthisstudy,theeffectsofenzymetreatmentonthediffusionofmacromolecules(FITC-dextran)inguarsolutionsandtitanium-guarhydrogelsareexamined.Enzymetreatmentwithβ-mannanase,apolymerbackbonecleavingenzyme,quicklyincreasesthediffusioncoefficientoftheprobemoleculesinbothsolutionsandhydrogelstothatinwater.Enzymetreatmentofguarsolutionsandhydrogelswithα-galactosidase,asidechaincleavingenzyme,displaysauniquebehaviorduetochangesinthefinestructureofguar.Theremovalofgalactosebranchesfromthemannanbackboneofguarcreatesadditionalhyperentanglements(i.e.,cross-links),whichreducethewaterholdingcapacityofguarandinducesyneresis.Ifthedepthatwhichthediffusioncoefficientismeasuredremainsconstant,aminimumisobservedinthediffusioncoefficientasα-galactosidaseenzymetreatmenttimeincreases.Atthesiteofmeasurement,thesamplechangesfromahomogeneousguarsystemtoaphase-separatedpolymer-richhydrogelandfinallytoadilutepolymerphaseasthepolymer-richhydrogelphaseprecipitatesbelowthesiteofmeasurement.Thediffusioncoefficientinthedilutepolymerphaseincreasestothatinwater,whilethediffusioncoefficientinthehydrogelphasecontinuestodecreasetoavalueofapproximately6×10^-8cm²/s.

Apolymerasechainreaction(PCR)wasdevelopedtodifferentiatethethickeningagentslocustbeangum(LBG)andthecheaperguarguminfinishedfoodproducts.UniversalprimersforamplificationoftheintergenicspacerregionbetweentrnL3’(UAA)exonandtrnF(GAA)geneinthechloroplast(cp)genomeandsubsequentrestrictionanalysiswereappliedtodifferentiateguargumandLBG.Thepresenceof<5% (w/w)="" guar="" gum="" powder="" added="" to="" lbg="" powder="" was="" detectable.="" based="" on="" data="" obtained="" from="" sequencing="" this="" intergenic="" spacer="" region,="" a="" second="" pcr="" method="" for="" the="" specific="" detection="" of="" guar="" gum="" dna="" was="" also="" developed.="" this="" assay="" detected="" guar="" gum="" powder="" in="" lbg="" in="" amounts="" as="" low="" as="" 1%="" (w/w).="" both="" methods="" successfully="" detected="" guar="" gum="" and/or="" lbg="" in="" ice="" cream="" stabilizers="" and="" in="" foodstuffs,="" such="" as="" dairy="" products,="" ice="" cream,="" dry="" seasoning="" mixes,="" a="" finished="" roasting="" sauce,="" and="" a="" fruit="" jelly="" product,="" but="" not="" in="" products="" with="" highly="" degraded="" dna,="" such="" as="" tomato="" ketchup="" and="" sterilized="" chocolate="" cream.="" both="" methods="" detected="" guar="" gum="" and="" lbg="" in="" ice="" cream="" and="" fresh="" cheese="" at="" levels=""><0.1%.>

Therheologicalbehaviorandsynergisticcharacterofmixedpolysaccharidesystemsareexaminedforblendsofxanthanwithenzymatically-modifiedguar.Inparticular,theenzymeα-galactosidaseisusedtoselectivelycleaveoffthegalactosesidechainsofguarinordertoobtaingalactomannanswithtailoredmoleculararchitecture:EMG1witharelativelyhighgalactosecontentof33.6%andamannose(M)togalactose(G)ratioof1.85,andEMG2withalowergalactosecontent(25.2%)andanM/Gratioof2.86.Blendsofxanthanwithenzymatically-modifiedguargumsamplesareexaminedintermsoftheirdynamicrheologicalpropertiesandcomparedtothoseofxanthan—locustbeangumblends.Theextentofsynergism,illustratedbythegelelasticmodulusG′andyieldstressτ_c,isfoundtoincreasewithincreasingextentofenzymaticmodification.Atconstantionicstrength,theEMG2andlocustbeanblendsbehavesimilarly,withincreasingextentofsynergyasthetemperatureofmixingisincreased.Additionally,atafixedmixingtemperature,theblendsmadeinwaterhaveahigherelasticmodulusthanthosemadeinsalt.Incontrast,theEMG1blendsareweakerandthedynamicmoduliareunaffectedbychangesinthemixingtemperatureorionicstrength.Theseresultsareconsistentwiththoseofotherresearchersandaredirectlyrelatedtoboththelevelofdisorderinthexanthanmoleculeaswellasthegalactosecontentandfinestructureofthegalactomannan.

Anoralcolonspecificdrugdeliveryplatformhasbeendevelopedtofacilitatetargettedreleaseoftherapeuticproteinsaswellassmallmoleculedrugs.Asimpleenzymaticprocedureisusedtomodifythemoleculararchitectureofalightlychemicallycrosslinkedgalactomannanhydrogelaswellasamodeldrug–galactomannanoligomerconjugate,fluoroisocynate(FITC)taggedguaroligomer,toentrapthemodeldrug.Theenzyme-modifiedhydrogelretainsthedruguntilitreachesthecolonicenvironmentwherebacteriasecreteenzymes(namelyβ-mannanase)todegradethegelandreleasethedrugmolecule.Laserscanningconfocalmicroscopycombinedwithfluorescencerecoveryafterphotobleachingisusedtoquantifythediffusionofthedrugconjugate.Thediffusioncoefficientofsolutesinthelightlycrosslinkedgalactomannanhydrogelisapproximatelyequaltothediffusioncoefficientintheguarsolutionforsimplediffusionaldrugloading.Afterdrugloading,α-galactosidasetreatmentgeneratesadditionalphysicalcrosslinksinthehydrogelmatrixaswellasbetweenthedrug–oligomerconjugateandthehydrogel,whichreducesdiffusionofthedrug–oligomerconjugatesignificantly.Degradationofthehydrogelbyβ-mannanaseresultsinaslowandcontrolledrateofFITC–guaroligomerdiffusion,whichgeneratesanextendedreleaseprofileforthemodeldrug.

Synergisticbiopolymerblendscomposedofxanthanandenzymaticallymodifiedguargalactomannanareinvestigatedintermsoftheirtime-dependentproperties.Inparticular,aside-chaincleavingenzyme,α-galactosidase,isusedtocleaveoffgalactosesugarunitsfromguartoproducemodifiedgalactomannanswithvaryinggalactosecontentsof25.2and16.2%.Laserscanningconfocalmicroscopyanddynamicrheologyareusedtomonitorthepropertiesofeachofthesetwomodifiedguarguminsolutionaswellasinblendswithxanthanastheyareallowedtoageoveraperiodof3weeks.Ourresultsindicatethatsolutionsofguarwithahighergalactose(25.2%)contentundergonorheologicalchangeovertheperiodofobservationandshowaconstantgelelasticmodulus(G‘)inblendswithxanthan.Confocalimagesofthesolutionsandtheblendsalsoindicatethatthesystemsarestableoveraperiodof3weeks.Incontrast,guargumwithalowergalactosecontent(16.2%)formsinterchainassociationsinsolution,developingaggregatesthatconvertitfromamacromolecularsolutiontoagel.Thisisreflectedinitsdynamicmoduliwhichincreasesignificantlywithtimeandshowatransitionfromfrequency-dependentbehaviorwithG‘ ‘(viscousmodulus)>G‘(elasticmodulus)toafrequency-independentcharacterwithG‘>G‘ ‘.Thisprocessofassociationandphaseseparationisdirectlyobservedinconfocalimagesofthemodifiedguaraswellasinitsblend,thoughnottothesameextentinthelatter.Thepresenceofasecondcomponentthusseemstoretardtheassociationprocess.Interestingly,theblendmoduliremainunchangedinmagnitudeandshowgellikefeatureseventhoughthemodeofassociationandconcomitantmicrostructurechanges.

Theinfluenceofthedegreeofbranchingofgalactomannansontheheat-inducedgelationofwheyproteinswasinvestigated,usingoscillatoryrheologicalmeasurementsatlowstrainamplitudeandmicrostructuralanalysisbyconfocallaserscanningmicroscopy.Galactomannanswerefromdifferentoriginsand/orenzymaticallymodified,withmannose-to-galactoseratiosrangingfrom1.5to3.7.Wheyproteingelswereformedat13%protein,pH7andlowionicstrength.Galactomannanconcentrationrangedfrom0to0.6%.Withintherangeofconcentrationsused,thepresenceofthegalactomannandecreasedthegellingtemperatureandhadapositiveeffectonthegelstrengthofthewheyproteingel.Theseeffectsaremorepronouncedasthedegreeofbranchingdecreases.Theeffectoftheoriginalguarsamplewasquitedifferentfromalltheothersamples,eitherintermsofrheologyormicrostructure,particularlyforthehighergalactomannanconcentration.Themixedgelsappearedasbiphasicsystems,withthepolysaccharideenrichedphasedispersedontheproteinmatrixatlowpolysaccharideconcentrations,butprogressingtoaphaseinversionathigherpolysaccharideconcentrations,especiallyforthelowerbranchedsamples.Thelinearviscoelasticityseemstobeinsensitivetosomeofthemicrostructuralchangesobservedwithinthemixedgels.ThebranchingdegreeofthegalactomannandoeshaveaneffectonmicrostructureandviscoelasticityoftheWPIgels,butthiseffectislimitedtoashortrangeofmannose-to-galactoseratios,abovewhichthiseffectisinsignificant.

Structurallymodifiedguargalactomannansfindapplicationinfoodandpetroleumindustriesasrheologymodifiers.Enzymesprovideapowerfulandconvenientmethodtomodifyguarstructure.Inthisstudy,thekineticsofenzymaticdegradationofguarsolutionswereinvestigatedusingSECandrheology.MolecularinformationfromSECrevealsthedegradationreactiontobezerothorderinguarconcentration.Further,therateconstantwasproportionaltoenzymeconcentration,demonstratingthattheenzymeactsasatruecatalyst.Thezeroshearviscositywasverysensitivetodegradation,withseveralordersofmagnitudechangebeingobservedoverthecourseofpolymerchainscission.Auniquecorrelationwasdevelopedbetweendegradationtime,guarmolecularweightandviscosity.Thisenablessuperpositionoftheviscosity-timeprofilesfordifferentenzymeconcentrationstoamastercurve;providingforaprioripredictionofguarsolutionviscosityasafunctionofdegradationtimeandenzymeconcentration.

Hydrogelscomposedofboraxcross-linkedguargalactomannansareenzymaticallydegradedusingendo-β-mannanase,anenzymewhichcleavesthepolymerchainbackbone.Dynamicrheologicalmeasurementsshowtheelastic(G‘)andviscous(G‘ ‘)modulitobesensitivetogelstructureandtoreducesignificantlyduringtheenzymatichydrolysisprocess.Thereductioninrheologicalpropertiesshowsthreedistinctregimes: aninitiallargedecrease,aslowerreductionrateatintermediatetimes,andanacceleratedreductionatlongerdegradationtimes.Incontrast,thepolymerchainmolecularweight,obtainedfromgelpermeationchromatography,reducesrapidlyatshorttimesandataslowerratesubsequently.Wethereforefindthekineticsofmodulireductiontobedictatedbytherelationshipbetweengelstructureandrheologicalproperties,ratherthanpurelytheratesofchainscission.Atshorttimes,thelargedecreaseinmoduliisanalogoustochangesinmolecularweightandcandirectlybeattributedtochainscission.Atlongtimes,correspondingtowhentheproductofpolymerconcentrationandintrinsicviscosity,c[η],reachesacriticalvalue(≤2.5),thechainsaretooshorttooverlapandthelongrangenetworkbreaksdownrapidly,leadingtoacceleratedmodulireduction.Additionally,asynergisticincreaseinthedegradationrateisobservedonusingacombinationofbackbone-cleavingβ-mannanaseenzymeandaside-chain-cleavingα-galactosidaseenzyme,ascomparedtousingonlyβ-mannanase.Thiscanbeattributedtoanenhancementofmannanaseactivityduetoremovalofthestericallyhinderinggalactosesidechains.Finally,acomparisonofgelandsolutiondegradationrevealsverysimilarbehaviorinmolecularweightchangesforbothbutcontrastingtrendsinrheology.

Thegelpropertiesofamixtureofenzymatically-modifiedguargum(EMG)andxanthan(XG)wereinvestigated.Theguargumsampletreatedwithα-galactosidasetoremovegalactoseresidueshadamannose/galactoseratioof3.02,andwascapableofformingasynergisticgelwithxanthan.Theconcentration-dependentandtemperature-dependentmodulusdatafortheEMG/XGgelwereanalyzedbythetwo-componentcascademodelwhichassumedaheterotypicassociationbetweensegmentsofEMGandXG.Theoptimalfunctionalities(numberofcross-linkingsitesperchain),f_EMGandf_XG,werefoundtobe300and30,respectively.Theformerisequaltothenumberofsegmentsizewithmorethansixunsubstitutedmannoseresiduesinagalactomannanbackbone,whilethelattercorrespondstoacross-linkdensityofoneper37repeatingunitsinaxanthanmolecule.ThecascadeanalysisofthecompositiondependenceofcriticalgellingconcentrationwasperformedbyintroducingtheeffectofthehomotypicassociationofXGorEMG,whichbecamesignificantinthegelpointmeasurement.Theenthalpychangepercross-linkfortheheterotypicassociationwasfoundtobetwotimeshigherthanthatforthehomotypicassociation.