Megazyme/Galactan (Potato)/PGALPOT/3 grams

Potatotuberpectinisrichingalactan(oligomerofβ-1,4-linkedgalactosylresidues).Wehaveexpressedafungalendo-galactanasecDNAinpotatoundercontrolofthegranuleboundstarchsynthasepromotertoobtainexpressionoftheenzymeintubersduringgrowth.Thetransgenicplantsdisplayednoalteredphenotypecomparedwiththewildtype.Fungalendo-galactanaseactivitywasquantifiedinthetransgenictubers,anditsexpressionwasverifiedbyWesternblotanalysis.Theeffectoftheendo-galactanaseactivityonpotatotuberpectinwasstudiedbyFouriertransforminfraredmicrospectroscopy,immuno-goldlabeling,andsugaranalysis.Allanalysesrevealedalterationsinpectincomposition.MonosaccharidecompositionoftotalcellwallsandisolatedrhamnogalacturonanIfragmentsshowedareductioningalactosylcontentto30%inthetransformantscomparedwiththewildtype.Increasedsolubilityofpectinfromtransgeniccellwallsbyendo-polygalacturonase/pectinmethylesterasedigestionpointstootherchangesinwallarchitecture.

Potatopulpisahigh-volumeside-streamfromindustrialpotatostarchmanufacturing.Enzymaticallysolubilizedβ-1,4-galactan-richpotatopulppolysaccharidesofmolecularweights>100kDa(SPPP)arehighlybifidogenicinhumanfecalsamplefermentationsinvitro.Theobjectiveofthepresentstudywastousepotatoβ-1,4-galactanandtheSPPPassubstratesforenzymaticproductionofpotentiallyprebioticcompoundsoflowerandnarrowermolecularweight.Anovelendo-1,4-β-galactanasefromEmericellanidulans(anamorphAspergillusnidulans),GHfamily53,wasproducedinarecombinantPichiapastorisstrain.TheenzymewaspurifiedbyCu²⁺affinitychromatographyanditsoptimalreactionconditionsweredeterminedtopH5and49°Cviaastatisticalexperimentaldesign.ThespecificactivityoftheE.nidulansenzymeexpressedinP.pastoriswassimilartothatofanendo-1,4-β-galactanasefromAspergillusnigerusedasbenchmark.TheE.nidulansenzymeexpressedinP.pastorisgeneratedaspectrumpoly-andoligo-saccharideswhichwerefractionatedbymembranefiltration.Thepotentialgrowthpromotingpropertiesofeachfractionwereevaluatedbygrowthofbeneficialgutmicrobesandpathogenicbacteria.Allthegalactan-andSPPP-derivedproductspromotedthegrowthofprobioticstrainsofBifidobacteriumlongumandLactobacillusacidophilusandgenerallydidnotsupportthepropagationofClostridiumperfringensinsingleculturefermentations.NotablythegrowthofB.longumwassignificantlyhigher(p<0.05) or="" at="" least="" as="" good="" on="" galactan-="" and="" sppp-derived="" products="" as="" fructooligosaccharides="" (fos).="" except="" in="" one="" case="" these="" products="" did="" not="" support="" the="" growth="" of="" the="" pathogen="">Cl.perfringenstoanysignificantextent.

Thestructureofarabinanandgalactandomainsinassociationwithcellulosemicrofibrilswasinvestigatedusingenzymaticandalkalidegradationprocedures.Sugarbeetandpotatocellwallresidues(called‘natural’composites),richinpecticneutralsugarsidechainsandcellulose,aswellas‘artificial’composites,createdbyinvitroadsorptionofarabinanandgalactansidechainsontoprimarycellwallcellulose,werestudied.Thesecompositesweresequentiallytreatedwithenzymesspecificforpecticsidechainsandhotalkali.Thedegradationapproachusedshowedthatmostofthearabinanandgalactansidechainsareinstronginteractionwithcelluloseandarenothydrolysedbypecticsidechain-degradingenzymes.Itseemsunlikelythatisolatedarabinanandgalactanchainsareabletotetheradjacentmicrofibrils.However,cellulosemicrofibrilsmaybetetheredbydifferentpecticsidechainsbelongingtothesamepecticmacromolecule.

β-1,4-Galactangalactosyltransferase(GT)activitywassolubilizedfrompotatomicrosomalmembranesinthepresenceof78mM3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonicacid.ThesolubilizedGTactivitytransferred¹⁴[C]galactosefromUDP-¹⁴[C]galactoseontotheacceptor-substratescomposedofrhamnogalacturonan(RG)withshortgalactanchains(RG-A,approximately1.2MDa,mol%Gal/Rha=0.7;RG-B,approximately21kDa,mol%Gal/Rha=1.2).However,shorterRGcontainingshortgalactanchains(approximately2kDaand1.2kDa),RGoligomerswithoutgalactosyl-residues,galactan,andgalactooligomersdidnotactasacceptor-substrates.OptimalpHfor¹⁴[C]incorporationontoRG-AandRG-Bwasaround5.6and7.5,respectively.The¹⁴[C]-labelledproductssynthesizeduponRG-AandRG-BcouldbedigestedwithaRGspecificlyaseintosmallerRGfragments.1,4-β-Endogalactanasecouldnotdigesttheformerproduct,whereasthelatterproductwasdigestedto¹⁴[C]galactobioseand¹⁴[C]galactose.ThisdemonstratesthatatleasttwoGTactivitiesweresolubilizedfrompotatomicrosomalmembranes.OnehadoptimalpHaround5.6totransfergalactosylresiduesontoRG-A,whereastheotherhadoptimalpHaround7.5totransfergalactosylresiduesontoRG-B.BothsynthesizedgalactanattachedtotheRGbackboneofRG-AandRG-B,andthegalactansynthesizedontotheRG-Bacceptorwas1,4-β-linked.

Thebiosynthesisofgalactanwasinvestigatedusingmicrosomalmembranesisolatedfromsuspension-culturedcellsofpotato(SolanumtuberosumL.var.AZY).IncubationofthemicrosomalmembranesinthepresenceofUDP-[¹⁴C]galactoseresultedinaradioactiveproductinsolublein70%methanol.Theproductreleasedonly[¹⁴C]galactoseuponacidhydrolysis.TreatmentoftheproductwithAspergillusnigerendo-1,4-β-galactanasereleased65–70%oftheradioactivitytoa70%-methanol-solublefraction.Toaminorextent,[¹⁴C]galactosewasalsoincorporatedintoproteins,howeverthesegalactoproteinswerenotasubstrateforAspergillusnigerendo-1,4-β-galactanase.Thus,themajorityofthe¹⁴C-labelledproductwas1,4-β-galactan.Compoundsreleasedbytheendo-1,4-β-galactanasetreatmentweremainly[¹⁴C]galactoseand[¹⁴C]galactobiose,indicatingthatthesynthesized1,4-β-galactanwaslongerthanatrimer.Invitrosynthesisof1,4-β-galactanwasmostactivewith6-d-oldcells,whichareinthemiddleofthelineargrowthphase.TheoptimalsynthesisoccurredatpH6.0inthepresenceof7.5 mMMn²⁺.AspergillusaculeatusrhamnogalacturonaseAdigestedatleast50%ofthelabelledproducttosmallerfragmentsofapprox.14 kDa,suggestingthatthesynthesized[¹⁴C]galactanwasattachedtotheendogenousrhamnogalacturonanI.WhenrhamnogalacturonaseAdigestsofthelabelledproductweresubsequentlytreatedwithendo-1,4-β-galactanase,radioactivitywasnotonlyfoundas[¹⁴C]galactoseor[¹⁴C]galactobiosebutalsoaslargerfragments.Thelargerfragmentswerelikelythe[¹⁴C]galactoseor[¹⁴C]galactobiosestillattachedtotherhamnogalacturonanbackbonesincetreatmentwithβ-galactosidasetogetherwithendo-1,4-β-galactanasedigestedallradioactivitytothefractionelutingas[¹⁴C]galactose.Thedataindicatethatthemajorityofthe[¹⁴C]galactanwasattacheddirectlytotherhamnoseresiduesinrhamnogalacturonanI.Thus,isolatedmicrosomalmembranescontainenzymeactivitiestobothinitiateandelongate1,4-β-galactansidechainsintheendogenouspecticrhamnogalacturonanI.

TheC-terminalmoduleofxylanase10AfromStreptomyceslividansisafamily13carbohydrate-bindingmodule(CBM13).CBM13bindsmono-andoligo-saccharideswithassociationconstantsof1×10²M^-1–1×10³M^-1.Itappearstobespecificonlyforpyranosesugars.CBM13bindsinsolubleandsolublexylan,holocellulose,pachyman,lichenan,arabinogalactanandlaminarin.Theassociationconstantforbindingtosolublexylanis(6.2±0.6)×10³/molofxylanpolymer.Site-directedmutationindicatestheinvolvementofthreefunctionalsitesonCBM13inbindingtosolublexylan.Thesitesaresimilarinsequence,andarepredictedtohavesimilarstructures,totheα,βandγsitesofricintoxinB-chain,whichisalsoinfamily13.TheaffinityofasinglebindingsiteonCBM13forsolublexylanisonly≈(0.5±0.1)×10³/molofxylan.ThebindingofCBM13tosolublexylaninvolvesadditiveandco-operativeinteractionsbetweenthethreebindingsites.ThismechanismofbindinghasnotpreviouslybeenreportedforCBMsbindingpolysaccharides.CBM13isthefirstbacterialmodulefromfamily13tobedescribedindetail.

Theextracellularbga1-encodedβ-galactosidaseofHypocreajecorina(Trichodermareesei)wasoverexpressedunderthepyruvatkinase(pki1)promoterregionandpurifiedtoapparenthomogeneity.Themonomericenzymeisaglycoproteinwithamolecularmassof118.8±0.5kDa(MALDI-MS)andanisoelectricpointof6.6.Bga1isactivewithseveraldisaccharides,e.g.lactose,lactuloseandgalactobiose,aswellaswitharyl-andalkyl-β-D-galactosides.Basedonthecatalyticefficiencies,lactitolandlactobionicacidarethepoorestsubstratesando-nitrophenyl-β-D-galactosideandlactulosearethebest.ThepHoptimumforthehydrolysisofgalactosidesis5.0,andtheoptimumtemperaturewasfoundtobe60°C.Bga1isalsocapableofreleasingD-galactosefromβ-galactansandisthusactuallyagalacto-β-D-galactanase.β-GalactosidaseisinhibitedbyitsreactionproductD-galactoseandtheenzymealsoshowsasignificanttransferaseactivitywhichresultsintheformationofgalacto-oligosaccharides.

β-1,4-Galactansareabundantpolysaccharidesinplantcellwalls,whicharegenerallyfoundassidechainsofrhamnogalacturonanI.RhamnogalacturonanIisamajorcomponentofpectinwithabackboneofalternatingrhamnoseandgalacturonicacidresiduesandsidechainsthatincludeα-1,5-arabinans,β-1,4-galactans,andarabinogalactans.Manyenzymesarerequiredtosynthesizepectin,butfewhavebeenidentified.Pectinismostabundantinprimarywallsofexpandingcells,butβ-1,4-galactanisrelativelyabundantinsecondarywalls,especiallyintensionwoodthatformsinresponsetomechanicalstress.WeinvestigatedenzymesinglycosyltransferasefamilyGT92,whichhasthreemembersinArabidopsisthaliana,whichwedesignatedGALACTANSYNTHASE1,(GALS1),GALS2andGALS3.Loss-of-functionmutantsinthecorrespondinggeneshadadecreasedβ-1,4-galactancontent,andoverexpressionofGALS1resultedinplantswith50%higherβ-1,4-galactancontent.Theplantsdidnothaveanobviousgrowthphenotype.Heterologouslyexpressedandaffinity-purifiedGALS1couldtransferGalresiduesfromUDP-Galontoβ-1,4-galactopentaose.GALS1specificallyformedβ-1,4-galactosyllinkagesandcouldaddsuccessiveβ-1,4-galactosylresiduestotheacceptor.TheseobservationsconfirmtheidentityoftheGT92enzymeasβ-1,4-galactansynthase.Theidentificationofthisenzymecouldprovideanimportanttoolforengineeringplantswithimprovedbioenergyproperties.

Theproductionofglycatedlysozyme(LZM),withgalactose,galactooligosaccharides(GOSs)andpotatogalactanthroughtheMaillardreaction,wasinvestigated.Thepercentblockedlysine,estimatedfromthefurosinecontent,reachedamaximumvalueof11.2%forLZM:galactanconjugatesafter1dayincubationataa_wof0.65.Amaximumpercentblockedlysineof7.0and13.5%wereobtainedforLZM:galactose/GOSconjugatesatalowera_wof0.45after3and7days,respectively.However,thelowpercentblockedlysineandthehighproteinaggregationindexofLZM:galactose/GOSconjugatesata_w0.79and0.65revealedtheprevalenceofthedegradationoftheAmadoricompoundsandtheproteincross-linking.MassspectrometryofLZMconjugatesrevealedtheformationofdifferentglycoforms.GlycatedLZMscontaininguptosevengalactosemoietieswereformed;whileonlymono-anddiglycatedLZMswithGOSsweredetected.2–3molofgalactanwereconjugatedto1molofLZM.Responsesurfacemethodology,basedona5-leveland3-factorcentralcompositedesign,revealedthatmolarratioandtemperaturewerethemostsignificantvariablesfortheglycationofLZMwithGOSs.Theoptimalconditionsleadingtoahighpercentblockedlysine(16.11%)withalowproteinaggregationindex(0.11)wereidentified:temperatureof49.5°C,LZM:GOSmolarratioof1:9anda_wof0.65.Tothebestofourknowledge,thisisthefirststudyontheoptimizationofLZMglycationwithGOSs.

ThesubcellularlocalizationandtopologyofrhamnogalacturonanI(RG-I)β(1→4)galactosyltransferase(s)(β[1→4]GalTs)frompotato(SolanumtuberosumL.)wereinvestigated.Usingtwo-stepdiscontinuoussucrosestepgradients,galactosyltransferase(GalT)activitythatsynthesized70%-methanol-insolubleproductsfromUDP-[¹⁴C]Galwasdetectedinboththe0.5Msucrosefractionandthe0.25/1.1Msucroseinterface.TheformerfractioncontainedmainlysolubleproteinsandthelatterwasenrichedinGolgivesiclesthatcontainedmostoftheUDPaseactivity,aGolgiMarker.Bygel-filtrationanalysis,productsof180–2,000Dawerefoundinthesolublefraction,whereasintheGolgi-enrichedfractiontheproductswerelargerthan80kDaandcouldbedigestedwithrhamnogalacturonanlyaseandβ(1,4)endogalactanasetoyieldsmallerrhamnogalacturonanoligomers,galactobioseandgalactose.Theendogalactanaserequiresβ(1→4)galactanswithatleastthreegalactosylresiduesforcleavage,indicatingthattheenzyme(s)presentinthe0.25/1.1MSucinterfacetransferredoneormoregalactosylresiduestopre-existingβ(1→4)galactansproducingRG-Isidechainsintotallongerthanatrimer.Thus,theβ(1→4)GalTactivitythatelongatesβ(1→4)-linkedgalactanonRG-IwaslocatedintheGolgiapparatus.Thisβ(1→4)GalTactivitywasnotreducedaftertreatmentoftheGolgivesicleswithproteinase,butapproximately75%oftheactivitywaslostaftertreatmentwithproteinaseinthepresenceofTritonX-100.Inaddition,theβ(1→4)GalTactivitywasrecoveredinthedetergentphaseaftertreatmentofGolgivesicleswithTritonX-114.Takentogether,theseobservationssupportedtheviewthattheRG-Iβ(1→4)GalTthatelongatesβ(1→4)galactanwasmainlylocatedintheGolgiapparatusandintegratedintothemembranewithitscatalyticsitefacingthelumen.

Galactan-richrhamnogalacturonanI(RGI),exhibitingpromisinghealthbenefits,isthemostabundantpolysaccharideinpotatopulpby-product.Inthepresentstudy,themicrowave-assistedalkalineextractionofgalactan-richRGIwasinvestigated.Solid/liquidratiowasidentifiedasthemostsignificantparameteraffectinglinearlyyieldandgalactose/rhamnosecontents.Microwavepowerandsolid/liquidratioexhibitedasignificantadverseinteractiveeffectontheyield.GalactosecontentofextractedpolysaccharidescanbemodulatedbycompromisingbetweenKOHconcentrationandextractiontime,whichexhibitedadverseinteraction.Optimumconditionswereidentifiedusingtheestablishedpredictedmodelsandconsistedoftreatmentofpotatocellwallatsolid/liquidratioof2.9%(w/v)with1.5 MKOHundermicrowavepowerof36.0 Wfor2.0 min.Yieldofintactgalactan-richRGIof21.6%andproductivityof192.0 g/L hwereachieved.ThefunctionalpropertiesofRGI-richpolysaccharideswerecomparableorsuperiortopotatogalactanandorangeshomogalacturonan.

Prebioticsareselectivelyfermentedbythegastrointestinalmicroflora,resultinginbenefitstohumanhealth.TheseedmucilageofHyptissuaveolenscontainsneutralandacidicpolysaccharidesinaratioof1:1.Theneutralpolysaccharidesconsistofgalactose,glucoseandmannosewhereastheacidicpolysaccharidescontainfucose,xyloseand4-O-methylglucuronicacid-residues.Thegrowthofprobioticsinthepresenceoftotal,acidicorneutralpolysaccharidesandoligosaccharideswastestedusingturbiditymeasurements.Themajority(11outof14)ofthetestedprobioticstrainssignificantlygrewintheneutralfraction.Growthoccurredwithsometimedelay,butmaybelongerlastingthanwithotherlowermolecularprebiotics.TheextentofgrowthincreasedwithneutralpolysaccharidesfromH.suaveolenscorrespondingtotheexternallyavailablegalactoseunits(20%).Inconclusion,neutralpoly-andoligosaccharidesfromH.suaveolenshaveaprebioticpotentialcharacterizedbyadelayedbutlonglastingeffect.