Megazyme/Arabinogalactan (Larch Wood)/PARGAL/25 grams

Therecentlyisolatedbacterialstrain80/3representsoneofthemostabundant16SrRNAphylotypesdetectedinthehealthyhumanlargeintestineandbelongstotheRuminococcaceaefamilyofFirmicutes.Thecompletedgenomesequencereportedhereisthefirstforamemberofthisimportantfamilyofbacteriafromthehumancolon.Thegenomecomprisestwolargechromosomesof2.24and0.73Mbp,leadingustoproposethenameRuminococcusbicirculansforthisnewspecies.Analysisofthecarbohydrateactiveenzymecomplementsuggestsanabilitytoutilizecertainhemicelluloses,especiallyβ-glucansandxyloglucan,forgrowththatwasconfirmedexperimentally.Theenzymaticmachineryenablingthedegradationofcelluloseandxylanbyrelatedcellulolyticruminococciishoweverlackinginthisspecies.Whilethegenomeindicatedthecapacitytosynthesizepurines,pyrimidinesandall20aminoacids,onlygenesforthesynthesisofnicotinate,NAD⁺,NADP⁺andcoenzymeAweredetectedamongtheessentialvitaminsandco-factors,resultinginmultiplegrowthrequirements.Invivo,thesegrowthfactorsmustbesuppliedfromthediet,hostorothergutmicroorganisms.OtherfeaturesofecologicalinterestincludetwotypeIVpilins,multipleextracytoplasmicfunction-sigmafactors,aureaseandabilesalthydrolase.

Togetherwithfungi,saprophyticbacteriaarecentraltothedecompositionandrecyclingofbiomassinforestenvironments.TheBacteroidetesphylumisabundantindiversehabitats,andseveralspecieshavebeenshowntobeabletodeconstructawidevarietyofcomplexcarbohydrates.ThegenusChitinophagaisoftenenrichedinhotspotsofplantandmicrobialbiomassdegradation.WepresentaproteomicassessmentoftheabilityofChitinophagapinensistogrowonanddegrademannanpolysaccharides,usinganagaroseplate-basedmethodofproteincollectiontominimisecontaminationwithexopolysaccharidesandproteinsfromlysedcells,andtoreflecttherealisticsettingofgrowthonasolidsurface.WeshowthatselectPolysaccharideUtilisationLoci(PULs)areexpressedindifferentgrowthconditions,andidentifyenzymesthatmaybeinvolvedinmannandegradation.Bycomparingproteomicandenzymaticprofiles,weshowevidencefortheinducedexpressionofenzymesandPULsincellsgrownonmannanpolysaccharidescomparedwithcellsgrownonglucose.Inaddition,weshowthatthesecretionofputativebiomass-degradingenzymesduringgrowthonglucosecomprisesasystemfornutrientscavenging,whichemploysconstitutivelyproducedenzymes.Significanceofthisstudy:Chitinophagapinensisbelongstoabacterialgenuswhichisprominentinmicrobialcommunitiesinagriculturalandforestenvironments,whereplantandfungalbiomassisintensivelydegraded.Suchdegradationishugelysignificantintherecyclingofcarboninthenaturalenvironment,andtheenzymesresponsibleareofbiotechnologicalrelevanceinemergingtechnologiesinvolvingthedeconstructionofplantcellwallmaterial.Thebacteriumhasacomparativelylargegenome,whichincludesmanyuncharacterisedcarbohydrate-activeenzymes.Wepresentthefirstproteomicassessmentofthebiomass-degradingmachineryofthisspecies,focusingonmannan,anabundantplantcellwallhemicellulose.Ourfindingsincludetheidentificationofseveralnovelenzymes,whicharepromisingtargetsforfuturebiochemicalcharacterisation.Inaddition,thedataindicatetheexpressionofspecificPolysaccharideUtilisationLoci,inducedinthepresenceofdifferentgrowthsubstrates.Wealsohighlighthowaconstitutivesecretionofenzymeswhichdeconstructmicrobialbiomasslikelyformspartofanutrientscavengingprocess.

Background:Pectinisanabundantcomponentinmanyfruitandvegetablewastesandcouldthereforebeanexcellentresourceforbiorefinery,butiscurrentlyunderutilized.Fungalpectinasesalreadyplayacrucialroleforindustrialpurposes,suchasforfoodstuffprocessing.However,theregulationofpectinasegeneexpressionisstillpoorlyunderstood.Foranoptimalutilizationofplantbiomassforbiorefineryandbiofuelproduction,adetailedanalysisoftheunderlyingregulatorymechanismsiswarranted.Inthisstudy,weappliedthegeneticresourcesofthefilamentousascomycetespeciesNeurosporacrassatoscreenfortranscriptionfactorsthatplayamajorroleinpectinaseinduction.Results:Thepectindegradationregulator-1(PDR-1)wasidentifiedthroughatranscriptionfactormutantscreeninN.crassa.TheΔpdr-1mutantexhibitedaseveregrowthdefectonpectinandalltestedpectin-relatedpoly-andmonosaccharides.BiochemicalaswellastranscriptionalanalysesofWTandtheΔpdr-1mutantrevealedthatwhilePDR-1-mediatedgeneinductionwasdependentonthepresenceofL-rhamnose,italsostronglyaffectedthedegradationofthehomogalacturonanbackbone.Theexpressionoftheendo-polygalacturonasegh28-1wasgreatlyreducedintheΔpdr-1mutant,whiletheexpressionlevelsofallpectatelyasegenesincreased.Moreover,apdr-1overexpressionstraindisplayedsubstantiallyincreasedpectinaseproduction.PromoteranalysisofthePDR-1regulonallowedrefinementoftheputativePDR-1DNA-bindingmotif.Conclusions:PDR-1ishighlyconservedinfilamentousascomycetefungiandispresentinmanypathogenicandindustriallyimportantfungi.OurdatademonstratethatthefunctionofPDR-1inN.crassacombinesfeaturesoftworecentlydescribedtranscriptionfactorsinAspergillusniger(RhaR)andBotrytiscinerea(GaaR).Theresultspresentedinthisstudycontributetoabroaderunderstandingofhowpectindegradationisorchestratedinfilamentousfungiandhowitcouldbemanipulatedforoptimizedpectinaseproduction.

Thehumangutmicrobiotautilizescomplexcarbohydratesasmajornutrients.Therequirementforefficientglycandegradingsystemsexertsamajorselectiveselectionpressureonthismicrobialcommunity.Thus,weproposethatthismicrobialecosystemrepresentsasubstantialresourcefordiscoveringnovelcarbohydrateactiveenzymes.TotestthishypothesiswescreenedthepotentialenzymaticfunctionsofhypotheticalproteinsencodedbygenesofBacteroidesthetaiotaomicronthatwereupregulatedbyarabinogalactanarabinogalactanproteinsorAGPs.AlthoughAGPsareubiquitousinplants,thereisapaucityofinformationontheirdetailedstructure,thefunctionoftheseglycansinplantaandthemechanismsbywhichtheyaredepolymerizedinmicrobialecosystems.HerewehavediscoveredanewpolysaccharidelyasefamilythatisspecificfortheL-rhamnose-alpha1,4-D-glucuronicacidlinkagethatcapsthesidechainsofcomplexAGPs.Thereactionproductgeneratedbythelyase,delta4,5-unsaturateduronicacid,isremovedfromAGPbyaglycosidehydrolaselocatedinfamilyGH105,producingthefinalproduct4-deoxy-β-L-threo-hex-4-enepyranosyl-uronicacid.Thecrystalstructureofamemberofthenovellyasefamilyrevealedacatalyticdomainthatdisplaysan(alpha/alpha6)6barrelfold.Inthecentreofthebarrelisadeeppocket,which,basedonmutagenesisdataandaminoacidconservation,comprisestheactivesiteofthelyase.Atyrosineistheproposedcatalyticbaseinthebeta-eliminationreaction.Thisstudyillustrateshowhighlycomplexglycanscanbeusedasascaffoldtodiscovernewenzymefamilieswithinmicrobialecosystemswherecarbohydratemetabolismisamajorevolutionarydriver.

Duringtheindustrialextractionofstarchfrompotatoes(Seresta),somestarchremainswithinundisruptedpotatocellsinthefibrousside-stream.Theaimofthisstudywastoinvestigateifenzymaticdegradationofcellwallpolysaccharides(CWPs)canenhancestarchrecoveryandlowerthewaterholdingcapacity(WHC)ofthe“fibre”fraction.Theuseofapectinase-richpreparationrecovered58%ofthestarchpresentinthe“fibre”fraction.Also,the“fibre”fractionretainedonly40%ofthewaterpresentinthenon-enzymetreated“fibre”.Thiswascausedbythedegradationofpectins,inparticulararabinogalactansidechainscalculatedasthesumofgalactosylandarabinosylresidues.

InthecurrentstudyforthefirsttimewereinvestigatedthechemicalcompositionandantioxidantactivityofpolysaccharidesisolatedfromthreeindigenousforBulgariaspeciesofPlantagogenus-PlantagomajorL.,PlantagolanceolataL.andPlantagomediaL.Crudepolysaccharideswereextractedfromfreshleaveswithwateranddiluteacidandtheiryieldwasbetween0.64%and2.79%.Thechemicalcompositionofwater-extractablepolysaccharides(WEPs)andtotalacid-extractablepolysaccharides(TAEPs)ofPlantagoleaveswasevaluatedbyHPLCanalysis.Thephytochemicaldatarevealedthepresenceofbranchedheteropolysaccharideswithdifferentneutral/acidicmonosaccharideratio.ThepredominantmonosaccharideunitofWEPswasgalacturonicacid(62.64%-70.58%).Additionally,therewereregisteredsmallamountsofarabinoseandrhamnose.InTAEPsamongwithgalacturonicacid(36.93%-41.46%),significantamountsofneutralmonosaccharidesasgalactose(22.80%-46.11%)andrhamnose(16.96%-35.74%)weredetermined.TwotypesofanalyseswereusedtoevaluatetheantioxidantactivityofPlantagoisolatedpolysaccharides:DPPHandFRAPassay.BasedonDPPHmethod,WEPsexhibitedstrongerradicalscavengingability(29.39%-40.08%)comparedtoTAEPs(19.44%-24.15%).Inparallel,WEPsshowedgreaterrateofferricreducingpower(103.71-137.83µMTE/5mgPs)comparedtoTAEPs(34.63-117.66µMTE/5mgPs).AlthoughlowerthansyntheticBHT,Plantagopolysaccharidesrevealedantioxidantpotentialandcouldbefurtherexploredaspromisingnaturalantioxidantsforthenutraceuticalandpharmaceuticalindustries.