Newchromogenicsubstrateshavebeendevelopedforthequantitativeassayofalpha-amylaseand(1→4)-β-D-glucanase.Thesewerepreparedbychemicallymodifyingamyloseorcellulosebeforedyeing,toincreasesolubility.Afterdyeing,thesubstrateswereeithersolubleorcouldbereADIlydispersedtoformfine,gelatinoussUSPensions.Assaysbasedontheuseofthesesubstratesaresensitiveandhighlyspecificforeitheralpha-amylaseor(1→4)-β-D-glucanase.Themethodofpreparationcanalsobeappliedtoobtainsubstratesforotherendo-hydrolases.

Wastesfromoliveoilandwineindustries(asexhaustedgrapemark,vineshoottrimmings,two-phaseolivemillwaste,vinassesandolivemillwastewaterwereevaluatedforlignocellulolyticenzymesproduction(ascellulases,xylanasesandferuloylesterases)bysolidstatefermentationwithAspergillusniger,AspergillusibericusandAspergillusjaponicus.TostudytheeffectofdifferentsubstratesinenzymesproductionaPlackett-Burmanexperimentaldesignwaspresented.Thevariablesthathadahigherpositiveeffectinlignocellulolyticenzymeswereurea,timeandexhaustedgrapemark.Themixtureoftwo-phaseolivemillwastewithexhaustedgrapemarkandvineshoottrimmingshadmaximaactivityofcellulases,xylanasesandferuloylesterases.

ElucidationoffungalbiomassdegradationisimportantforunderstandingtheturnoverofBIOLOGicalmaterialsinnatureandhasimportantimplicationsforindustrialbiomassconversion.Inrecentyearstherehasbeenanincreasinginterestinelucidatingthebiologicalroleofthermophilicfungiandincharacterizationoftheirindustriallyusefulenzymes.Inthepresentstudyweinvestigatedthecellulolyticpotentialof16thermophilicfungifromthethreeascomyceteordersSordariales,EurotialesandOnygenalesandfromthezygomyceteorderMucoralesthuscoveringallfungalordersthatincludethermophiles.Thermophilicfungiaretheonlydescribedeukaryotesthatcangrowattemperaturesabove45°C.All16fungiwereabletogrowoncrystallinecellulosebuttheirsecretedenzymesshowedwidelydifferentcellulolyticactivities,pHoptimaandthermostABIlities.Interestingly,incontrasttopreviousreports,wefoundthatsomefungisuchasMelanocarpusalbomycesreadilygrewoncrystallinecelluloseandproducedcellulases.Theseresultsindicatethattherearelargedifferencesinthecellulolyticpotentialofdifferentisolatesofthesamespecies.Furthermore,alltheselectedspecieswereabletodegradecellulosebutthedifferencesincellulolyticpotentialandthermostabilityofthesecretomedidnotcorrelatetothetaxonomicposition.PCRamplificationandsequencingof22cellulasegenesfromthefungishowedthatthelevelofthermostabilityofthecellulose-degradingactivitycouldnotbeinferredfromthephylogeneticrelationshipofthecellulases.

Background:D-glucose,D-xyloseandL-arabinosearethethreemajormonosaccharidesinplantcellwalls.Completeutilizationofallthreesugarsisstillabottleneckforsecond-generationcellulolyticbioethanolproduction,especiallyforL-arabinose.However,littleisknownaboutgeneexpressionprofilesduringL-arabinoseutilizationinfungiandacomparisonofthegenome-widefungalresponsetothesethreemajormonosaccharideshasnotyetbeenreported.Results:Usingnext-generationsequencingtechnology,wehaveanalyzedthetranscriptomeofN.crassagrownonL-arabinoseversusD-xylose,withD-glucoseasthereference.WefoundthatthegeneexpressionprofilesonL-arabinoseweredramaticallydifferentfromthoseonD-xylose.ItappearsthatL-arabinosecanrewirethefungalcellmetabolicpathwaywidelyandprovoketheexpressionofmanykindsofsugartransporters,hemicellulasegenesandtranscriptionfactors.Incontrast,manyfewergenes,mainlyrelatedtothepentosemetabolicpathway,wereupregulatedonD-xylose.TherewiredmetabolicresponsetoL-arabinosewassignificantlydifferentandwiderthanthatundernocarbonconditions,althoughthecarbonstarvationresponsewasinitiatedonL-arabinose.Threenovelsugartransporterswereidentifiedandcharacterizedfortheirsubstrateshere,includingoneglucosetransporterGLT-1(NCU01633)andtwonovelpentosetransporters,XAT-1(NCU01132),XYT-1(NCU05627).Onetranscriptionfactorassociatedwiththeregulationofhemicellulasegenes,HCR-1(NCU05064)wasalsocharacterizedinthepresentstudy.Conclusions:WeconductedthefirsttranscriptomeanalysisofNeurosporacrassagrownonL-arabinoseandperformedacomparativeanalysiswithcellsgrownonD-xyloseandD-glucose,whichdeepenstheunderstandingoftheutilizationofL-arabinoseandD-xyloseinfilamentousfungi.ThedatasetgeneratedbythisresearchwillbeusefulforminingtargetgenesforD-xyloseandL-arabinoseutilizationengineeringandthenovelsugartransportesidentifiedaregoodtargetsforpentoseuntilizationandbiofuelsproduction.Moreover,hemicellulaseproductionbyfungicouldbeimprovedbymodifyingthehemicellulaseregulatordiscoveredhere.

Background:Innature,lightisoneofthemostimportantenvironmentalcuesthatfungiperceiveandinterpret.Itisknownnotonlytoinfluencegrowthandconidiation,butalsocellulasegeneexpression.WethereforestudiedtherelevanceofthemaincomponentsofthelightperceptionmachineryofTrichodermareesei(Hypocreajecorina),ENV1,BLR1andBLR2,forproductionofplantcellwalldegradingenzymesinfermentationsaimedatefficientbiosynthesisofenzymemixturesforbiofuelproduction.Findings:Ourresultsindicatethatdespitecultivationinmostlydarkconditions,allthreecomponentsshowaninfluenceoncellulaseexpression.Whilewefoundtheperformanceoftheenzymemixturesecretedbyadeletionmutantinenv1tobeenhanced,thehighercellulolyticactivityobservedforΔblr2ismainlyduetoanincreasedsecretioncapacityofthisstrain.Δblr1showedenhancedbiomassaccumulation,butduetoitsobviouslylowersecretioncapacitystillwastheleastefficientstraininthisstudy.Conclusions:Weconcludethatwithrespecttoregulationofplantcellwalldegradingenzymes,thebluelightregulatorproteinsareunlikelytoactasacomplex.Theirregulatoryinfluenceoncellulasebiosynthesisinvolvesanalterationofproteinsecretion,whichmaybeduetoadjustmentoftranscriptionorposttranscriptionalregulationofupstreamfactors.Incontrast,theregulatoryfunctionofENV1seemstoinvolveadjustmentofenzymeproportionstoenvironmentalconditions.

Trichodermareesei(Hypocreajecorina)isnowadaysthemostimportantindustrialproducerofcellulaseandhemicellulaseenzymes,whichareusedforpretreatmentofcellulosicbiomassforbiofuelproduction.Inthisstudy,weintroduceanovelcomponent,GRD1(glucose-ribitoldehydrogenase1),whichshowsenzymaticactivityoncellobioseandpositivelyinfluencescellulasegenetranscription,expression,andextracellularendo-1,4-β-D-glucanaseactivity.grd1isdifferentiallytranscribedupongrowthoncelluloseandtheinductionofcellulasegeneexpressionbysophorose.Thetranscriptionofgrd1iscoregulatedwiththatofcel7a(cbh1)underinducingconditions.GRD1isfurtherinvolvedincarbonsourceutilizationonseveralcarbonsources,suchasthoseinvolvedinlactoseandD-galactosecatabolism,inseveralcasesinalight-dependentmanner.WeconcludethatGRD1representsanovelenhancerofcellulasegeneexpression,whichbycoregulationwiththemajorcellulasemayactviaoptimizationofinducingmechanisms.

ThegrowthandenzymeproductionbyTrichodermareeseiRutC-30usingdifferentlignocellulosicmaterialsascarbonsourcewereinvestigated.Cellulose,sugarbeetpulpandalkalineextractedsugarbeetpulp(resultinginpartialremovalofhemicellulose,ligninandpectin)ormixturesthereofwereusedascarbonsources.Itwasfoundthatendoglucanaseandendoxylanseactivitieswereproducedthroughoutthecultivations,whereasα-arabinosidasewasinducedlateduringthecultivation.Thehighestamountsofendoglucanse,couldbemeasuredwhenT.reeseiRutC-30wasgrownoncelluloseorcellulosecontainingmixtures.Endoxylanasewasproducedonallsubstrates,butthepresenceofcellulosewasfavourablefortheproduction.Polygalacturonaseactivitycouldbemeasuredathighvaryinglevelsthroughoutthecultivations,exceptduringgrowthoncellulose.Thevaryinglevelsmightoriginatefromtheproductionofdifferentisoenzymesofpolygalacturonase.

AGram-stain-negative,motile,rod-shaped,cellulose-degradingbacterialstrain,BIO-TAS4-2^T,whichbelongstotheBetaproteobacteria,wasisolatedfromforestsoilfromNaejangMountain,Korea,anditstaxonomicpositionwasinvestigatedbyusingapolyphasicstudy.StrainBIO-TAS4-2TgrewoptimallyatpH7.0–8.0,at30°Candinthepresenceof0–1.0 %(w/v)NaCl.Phylogenetictreesbasedon16SrRNAgenesequencesshowedthatstrainBIO-TAS4-2^TclusteredwithmembersofthegeneraAndreprevotia,SilvimonasandDeefgeaofthefamilyNeisseriaceae,withwhichitexhibited16SrRNAgenesequencesimilaritiesof93.5–94.2 %.StrainBIO-TAS4-2^TcontainedQ-8asthepredominantubiquinoneandsummedfeature3(C_16:1ϖ7cand/oriso-C_15:02-OH)andC_16:0asthemajorfattyacids.TheDNAG+Ccontentwas63.8mol%.StrainBIO-TAS4-2^Tcouldbedifferentiatedfrommembersofphylogeneticallyrelatedgenerabydifferencesinfattyacidcomposition,DNAG+Ccontentandsomephenotypicproperties.Onthebasisofphenotypic,chemotaxonomicandphylogeneticdata,strainBIO-TAS4-2^Tisconsideredtorepresentanovelspeciesinanewgenus,forwhichthenameJeongeupianaejangsanensisgen.nov.,sp.nov.isproposed,withBIO-TAS4-2^T(=KCTC22633^T=CCUG57610^T)asthetypestrain.

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.

Asequenceencodingaputativeextracellularendoglucanase(sso1354)wasidentifiedinthecompletegenomesequenceofSulfolobussolfataricus.Theencodedproteinsharessignaturemotifswithmembersofglycosidehydrolasesfamily12.Afteranunsuccessfulfirstattemptatcloningthefull-lengthcodingsequencesinEscherichiacoli,anactivebutunstablerecombinantenzymelackinga27-residueN-terminalsequencewasgenerated.This27-amino-acidsequenceshowssignificantsimilaritywithcorrespondingregionsinthesugarbindingproteinsAraS,GlcS,andTreSofS.solfataricusthatareresponsIBLeforanchoringthemtotheplasmamembrane.Astrategybasedonaneffectivevector/hostgeneticsystemforSulfolobusandonexpressioncontrolbythepromoteroftheS.solfataricusgenewhichencodestheglucosebindingproteinallowedproductionoftheenzymeinsufficientquantitiesforstudy.Infact,theenzymeexpressedinS.solfataricuswasstableandhighlythermoresistantandshowedoptimalactivityatlowpHandhightemperature.Theproteinwasdetectedmainlyintheplasmamembranefraction,confirmingthestructuralsimilaritytothesugarbindingproteins.TheresultsoftheproteinexpressioninthetwodifferenthostsshowedthattheSSO₁₃₅₄enzymeisendowedwithanendo-β-1-4-glucanaseactivityandspecificallyhydrolyzescellulose.Moreover,italsoshowssignificantbutdistinguishablespecificitytowardseveralothersugarpolymers,suchaslichenan,xylan,debranchedarabinan,pachyman,andcurdlan.

Background:Lightrepresentsanimportantenvironmentalcue,whichexertsconsiderableinfluenceonthemetabolismoffungi.StudieswiththebiotechnologicalfungalworkhorseTrichodermareesei(Hypocreajecorina)haverevealedaninterconnectionbetweentranscriptionalregulationofcellulolyticenzymesandthelightresponse.Neurosporacrassahasbeenusedasamodelorganismtostudylightandcircadianrhythmbiology.WethereforeinvestigatedwhetherlightalsoregulatestranscriptionalregulationofcellulolyticenzymesinN.crassa.Results:WeshowthattheN.crassaphotoreceptorgeneswc-1,wc-2andvvdareinvolvedinregulationofcellulasegeneexpression,indicatingthatthisphenomenonisconservedamongfilamentousfungi.ThenegativeeffectofVVDonproductionofcellulolyticenzymesistherebyaccomplishedbyitsroleinphotoadaptationandhenceitsfunctioninWhitecollarcomplex(WCC)formation.Incontrast,theinductionofvvdexpressionbytheWCCdoesnotseemtobecrucialinthisprocess.Additionally,wefoundthatWC-1andWC-2notonlyactasacomplex,butalsohaveindividualfunctionsupongrowthoncellulose.Conclusions:Genomewidetranscriptomeanalysisofphotoreceptormutantsandevaluationofresultsbyanalysisofmutantstrainsidentifiedseveralcandidategeneslikelytoplayaroleinlightmodulatedcellulasegeneexpression.Geneswithfunctionsinaminoacidmetabolism,glycogenmetabolism,energysupplyandproteinfoldingareenrichedamonggeneswithdecreasedexpressionlevelsinthewc-1andwc-2mutants.Theabilitytoproperlyrespondtoaminoacidstarvation,i.e.up-regulationofthecrosspathwaycontrolproteincpc-1,wasfoundtobebeneficialforcellulasegeneexpression.OurresultsfurthersuggestacontributionofoxidativedepolymerizationofcellulosetoplantcellwalldegradationinN.crassa.

Endogenouscellulaseactivitywasidentifiedinthegastricfluidanddigestiveglandoftheredclawcrayfish.CellulaseshowedmaximalactivityfrompH4to5andwasstableforupto2hat40°C.Cellulaseactivityinthedigestiveglandwasunaffectedbyantibiotictreatment.Takentogetherthesefindingssuggestasignificantendogenouscomponentforredclawcellulaseactivity.Partialpurificationofcellulaseactivitywasperformedusinganionexchangeandgelfiltrationchromatography.OnemajorandoneminorbandofactivitywereidentifiedsubsequentlybySDS-PAGEandzymography.Themolecularweightofthemajorbandwasestimatedat40kDawhiletheminorbandwasestimatedat30kDa.Redclawcellulaseenzymesdemonstratedbroadsubstratespecificity,hydrolysingpolysaccharidescontainingβ-1,4andmixedβ-1,4andβ-1,3glycosidicbondsbutshowedapreferenceforsolublesubstrates.Hydrolysisproductsofcellodextrinsofvariouslengthsalsoshowedthattheenzymesliberatedfreeglucose.Exposureofredclawtoantibioticsresultedinadramaticdeclineinbacterialpopulationsinthegastriccontents(>90%)butonlya40%declineincellulaseactivity.

TwentyAspergillusstrainswereevaluatedforproductionofextracellularcellulolyticandxylanolyticactivities.Aspergillusbrasiliensis,A.nigerandA.japonicasproducedthehighestxylanaseactivitieswiththeA.brasiliensisandA.nigerstrainsproducingthermostableβ-xylosidases.Theβ-xylosidaseactivitiesoftheA.brasiliensisandA.nigerstrainshadsimilartemperatureandpHoptimaat75°CandpH5andretained62%and99%,respectively,oftheseactivitiesover1hat60°C.At75°C,thesevalueswere38and44%,respectively.WhereasA.nigerisawellknownenzymeproducer,thisisthefirstreportofxylanaseandthermostableβ-xylosidaseproductionfromthenewlyidentified,non-ochratoxin-producingspeciesA.brasiliensis.

ThefungalarabinofuranosidasefromPleurotusostreatusPoAbfrecombinantlyexpressedinPichiapastorisrPoAbfanditsevolvedvariantrPoAbfF435Y/Y446Fweretestedfortheireffectivenesstoenhancetheenzymaticsaccharificationofthreelignocellulosicbiomasses,namelyArundodonax,corncobsandbrewer’sspentgrains(BSG),afterchemicalorchemical–physicalpretreatment.AlltherawmaterialsweresubjectedtoanalkalinepretreatmentbysoakinginaqueousammoniasolutionwhilstthebiomassfromA.donaxwasalsopretreatedbysteamexplosion.Thecapabilityofthewild-typeandmutantrPoAbftoincreasethefermentablesugarsrecoverywasassessedbyusingtheseenzymesincombinationwithdifferent(hemi)cellulolyticactivities.TheseenzymaticmixtureswereeitherentirelyofcommercialoriginorcontainedthecellulasefromStreptomycessp.G12CelStreprecombinantlyexpressedinEscherichiacoliinsubstitutiontothecommercialcounterparts.TheadditionofthearabinofuranosidasesfromP.ostreatusimprovedthehydrolyticefficiencyofthecommercialenzymaticcocktailsonallthepretreatedbiomasses.ThebestresultswereobtainedusingtherPoAbfevolvedvariantandarerepresentedbyincreasesofthexyloserecoveryupto56.4%.Thesedataclearlyhighlighttheimportantroleoftheaccessoryhemicellulolyticactivitiestooptimizethexylanbioconversionyields.

Thebluecrab[Callinectessapidus(Rathbun,1896)]isabenthicdecapodwithavarieddiet.Thedietincludesinvertebratesanddetritalmaterialthatcanhaverelativelylargeamountsofchitinandcellulose,bothofwhichcanbedifficulttodigestformanyorganismsandoftenrequiretheaidofspecificbacteriainthegutmicrobiome.Inthisstudy,juvenilebluecrabswerefedanoptimizeddefinedpelleteddietwitha20%replacementofwheatflourfillerwitheitherchitin,cellulose,ora14%/6%mixofboth,followedbyadietswitchtotheopposingingredient.Crabshadincreasinggrowthperformancewithincreasingamountsofcelluloseinthedietversuschitinandhadanadditionalmoltinmostcases.Thisoccurredduringtheinitialphaseandfollowingtheswitch,indicatingthatperformancecanberecovered.Subsequently,celluloseandchitindigestionassayswereusedtoshowthattheforegut,midgut,andhindgutwereallabletosignificantlydigestmorecellulosethanchitinwiththemajorityofactivityintheforegutandmidgut.Implicationsforrearinganddietformulationsaswellastheroleofcelluloseandchitindigestioninthenaturaldietarediscussed.

Background:Yarrowialipolytica,oneofthemostwidelystudied“nonconventional”oleaginousyeastspecies,isunabletogrowoncellulose.Recently,weidentifiedandoverexpressedtwoendogenousβ-glucosidasesinY.lipolytica,thusenablingthisyeasttousecello-oligosaccharidesasacarbonsourceforgrowth.Usingthisengineeredyeastplatform,wehavenowgonefurthertowardbuildingafullycellulolyticY.lipolyticaforuseinconsolidatedbioprocessingofcellulose.Results:Initially,differentessentialenzymecomponentsofacellulasecocktail(i.e,.cellobiohydrolasesandendoglucanases)wereindividuallyexpressedinY.lipolyticainordertoascertaintheviabilityofthestrategy.Accordingly,theTrichodermareeseiendoglucanaseI(TrEGI)andII(TrEGII)weresecretedasactiveproteinsin Y.lipolytica,withthesecretionyieldofEGIIbeingtwicethatofEGI.CharacterizationofthepurifiedHis-taggedrecombinantEGproteins(rhTrEGs)revealedthatrhTrEGIdisplayedhigherspecificactivitythanrhTrEGIIonbothcellotrioseandinsolublecellulosicsubstrates,suchasAvicel,β-1,3glucan,β-1,4glucan,andPASC.Similarly,cellobiohydrolases,suchasT.reeseiCBHIandII(TrCBHIandII),andtheCBHIfromNeurosporacrassa(NcCBHI)weresuccessfullyexpressedinY.lipolytica. However,theyieldoftheexpressed TrCBHIwaslow,soworkonthiswasnotpursued.Contrastingly,rhNcCBHIwasnotonlywellexpressed,butalsohighlyactiveonPASCandmoreactiveonAvicel(0.11 U/mg)thanwild-type TrCBHI(0.065 U/mg).Therefore,workwaspursuedusingacombinationof NcCBHIand TrCBHII.ThequantificationofenzymelevelsinculturesupernatantsrevealedthattheuseofahybridpromoterinsteadoftheprimarilyusedTEFpromoterprocuredfourandeighttimesmore NcCBHIand TrCBHIIexpressions,respectively.Finally,thecoexpressionofthepreviouslydescribed Y.lipolyticaβ-glucosidases,theCBHII,andEGIandIIfromT.reesei,andtheN.crassaCBHIprocuredanengineered Y.lipolyticastrainthatwasabletogrowbothonmodelcellulosesubstrates,suchashighlycrystallineAvicel,andonindustrialcellulosepulp,suchasthatobtainedusinganorganosolvprocess.Conclusions:A Y.lipolyticastraincoexpressingsixcellulolyticenzymecomponentshasbeensuccessfullydeveloped.Inaddition,theresultspresentedshowhowtherecombinantstraincanbeoptimized,forexample,usingartificialpromoterstotailorexpressionlevels.Mostsignificantly,thisstudyhasprovidedademonstrationofhowthestraincangrowonasampleofindustrialcelluloseassolecarbonsource,thusrevealingthefeasibilityofYarrowia-basedconsolidatedbioprocessfortheproductionoffuelandchemicalprecursors.Further,enzymeandstrainoptimization,coupledtoappropriateprocessdesign,willundoubtedlyleadtomuchbetterperformancesinthefuture.

Thechainconformation,chemicalcharactersandimmunomodulatoryactivityofpolysaccharidefromDendrobiumdevonianum(DDP)wereinvestigated.Resultsshowedthatmolecularweights,polydispersityindex,radiusofgyrationsofDDPwere3.99×10⁵ Da,1.27,74.1nm,respectively.Byapplyingthepolymersolutiontheory,theexponent(v)valuesof<>²>_z^1/2=kM_w^vwascalculatedas0.38,whichrevealedthatDDPexistedasaglobularshapeinaqueoussolution,andfurtherconfirmedbyAFManalysis.Furthermore,themainmonosaccharidecompositionswereManandGlcwiththeratioof29.61:1.00.Indeed,themainglycosidiclinkageswereβ-1,4-Manp,andsubstitutedwithacetylgroupsatO-2andO-3position.Notably,DDPcouldpromotetheimmunefunctionsofmacrophagesincludingNOreleaseandphagocytosis.Thus,DDPcouldbeexploredasanaturalimmune-stimulatingagentinthehealthandfunctionalfoodareaaswellaspharmaceuticalindustries.