Megazyme/Lichenan (Icelandic Moss)/PLICHN/4 grams

WeimprovedtheCaCCOprocessforricestrawbyitsincorporationwithastepoflimepretreatmentatroomtemperature(RT).WefirstlyoptimizedtheRT-limepretreatmentforthelignocellulosicpart.Whentheratiooflime/dry-biomasswas0.2(w/w),theRTlime-pretreatmentfor7-dresultedinaneffectontheenzymaticsaccharificationofcelluloseandxylanequivalenttothatofthepretreatmentat120°Cfor1h.Sucrose,starchandβ-1,3-1,4-glucan,whichcouldbeoftendetectedinricestraw,weremostlystableundertheRT-limepretreatmentcondition.Then,thepretreatmentconditionintheconventionalCaCCOprocesswasmodifiedbytheadaptationoftheoptimizedRTlime-pretreatment,resultinginsignificantlybettercarbohydraterecoveriesviaenzymaticsaccharificationthanthoseoftheCaCCOprocess(120°Cfor1h).Thus,theimprovedCaCCOprocess(theRT-CaCCOprocess)couldpreserve/pretreatthefeedstockatRTinawetformwithminimumlossofcarbohydrates.

Celluloseformsthemajorload-bearingnetworkoftheplantcellwall,whichsimultaneouslyprotectsthecellanddirectsitsgrowth.Althoughtheprocessofcellulosesynthesishasbeenobserved,littleisknownaboutthebehaviorofcelluloseinthewallaftersynthesis.UsingPontamineFastScarlet4B,adyethatfluorescespreferentiallyinthepresenceofcelluloseandhasexcitationandemissionwavelengthssuitableforconfocalmicroscopy,weimagedthearchitectureanddynamicsofcelluloseinthecellwallsofexpandingrootcells.WefoundthatcelluloseexistsinArabidopsis(Arabidopsisthaliana)cellwallsinlargefibrillarbundlesthatvaryinorientation.Duringanisotropicwallexpansioninwild-typeplants,weobservedthatthesecellulosebundlesrotateinatransversetolongitudinaldirection.Wealsofoundthatcelluloseorganizationissignificantlyalteredinmutantslackingeitheracellulosesynthasesubunitortwoxyloglucanxylosyltransferaseisoforms.Ourresultssupportamodelinwhichcelluloseisdepositedtransverselytoaccommodatelongitudinalcellexpansionandreorientedduringexpansiontogenerateacellwallthatisfortifiedagainststrainfromanydirection.

Theplantcellwallisacomplexmaterialinwhichthecellulosemicrofibrilsareembeddedwithinameshofotherpolysaccharides,someofwhicharelooselytermed“hemicellulose.”Onesuchhemicelluloseisxyloglucan,whichdisplaysaβ-1,4-linkedD-glucosebackbonesubstitutedwithxylose,galactose,andoccasionallyfucosemoieties.BothxyloglucanandtheenzymesresponsIBLeforitsmodificationanddegradationarefindingincreasingprominence,reflectingboththedriveforenzymaticbiomassconversion,theirroleindetergentapplications,andtheutilityofmodifiedxyloglucansforcellulosefibermodification.Herewepresenttheenzymaticcharacterizationandthree-dimensionalstructuresinligand-freeandxyloglucan-oligosaccharidecomplexedformsoftwodistinctxyloglucanasesfromglycosidehydrolasefamiliesGH5andGH12.Theenzymes,PaenibacilluspabuliXG5andBacilluslicheniformisXG12,bothdisplayopenactivecentergroovesgraftedupontheirrespective(β/α)₈andβ-jellyrollfolds,inwhichthesidechaindecorationsofxyloglucanmaybeaccommodated.Fortheβ-jellyrollenzymetopologyofGH12,bindingofxylosylandpendantgalactosylmoietiesistolerated,buttheenzymeissimilarlycompetentinthedegradationofunbranchedglucans.Inthecaseofthe(β/α)₈GH5enzyme,kineticallyproductiveinteractionsaremadewithbothxyloseandgalactosesubstituents,asreflectedinbothahighspecificactivityonxyloglucanandthekineticsofaseriesofarylglycosides.Thedifferentialstrategiesfortheaccommodationofthesidechainsofxyloglucanpresumablyfacilitatetheactionofthesemicrobialhydrolasesinmilieuswherediverseanddifferentlysubstitutedsubstratesmaybeencountered.

Molecularengineeringofligand-bindingproteinsiscommonlyusedforidentificationofvariantsthatdisplaynovelspecificities.UsingthisapproachtointroducenovelspecificitiesintoCBMs(carbohydrate-bindingmodules)hasnotbeenextensivelyexplored.Here,wereporttheengineeringofaCBM,CBM4-2fromtheRhodothermusmarinusxylanaseXyn10A,andtheidentificationoftheX-2variant.Ascomparedwiththewild-typeprotein,thisengineeredmoduledisplayshigherspecificityforthepolysaccharidexylan,andalowerpreferenceforbindingxylo-oligomersratherthanbindingthenaturaldecoratedpolysaccharide.ThemodeofbindingofX-2differsfromotherxylan-specificCBMsinthatitonlyhasonearomaticresidueinthebindingsitethatcanmakehydrophobicinteractionswiththesugarringsoftheligand.TheevolutionofCBM4-2hasthusgeneratedaxylan-bindingmodulewithdifferentbindingpropertiestothosedisplayedbyCBMsavailableinNature.

PttCel9Aisamembrane-bound,family9glycosylhydrolasefromPopulustremulaxtremuloidesthatisupregulatedduringsecondarycellwallsynthesis.ThecatalyticdomainofPttCel9A,Δ_1-105PttCel9A,waspurified,anditsactivitywascomparedtoTfCel9AandTfCel9BfromThermobifidafusca.Sincearomaticaminoacidsinvolvedinsubstratebindingatsubsites−4,−3,and−2aremissinginPttCel9A,theactivityofTfCel9AmutantenzymesW256S,W209A,andW313Gwasalsoinvestigated.Δ_1-105PttCel9Ahydrolyzedacomparativelynarrowrangeofpolymericsubstrates,andthepreferredsubstratewas(carboxymethyl)cellulose4M.Moreover,Δ_1-105PttCel9Adidnothydrolyzeoligosaccharidesshorterthancellopentaose,whereasTfCel9AandTfCel9Bhydrolyzedcellotetraoseandcellotriose,respectively.ThesedatasuggestthatthepreferredsubstratesofPttCel9Aarelong,low-substituted,solublecellulosicpolymers.At30°CandpH6.0,thek_catforcellohexaoseofΔ_1-105PttCel9A,TfCel9A,andTfCel9Bwere0.023±0.001,16.9±2.0,and1.3±0.2,respectively.Thecatalyticefficiency(k_cat/k_m)ofTfCel9Bwas39%ofthatofTfCel9A,whereasthecatalyticefficiencyofΔ_1-105PttCel9Awas0.04%ofthatofTfCel9A.Removingtryptophanresiduesatsubsites−4,−3,and−2decreasedtheefficiencyofcellohexaosehydrolysisbyTfCel9A.MutationofW313toGhadthemostdrasticeffect,producingamutantenzymewith1%ofthecatalyticefficiencyofTfCel9A.TheapparentnarrowsubstraterangeandcatalyticefficiencyofPttCel9Aarecorrelatedwithalackofaromaticaminoacidsinthesubstratebindingcleftandmaybenecessarytopreventexcessivehydrolysisofcellwallpolysaccharidesduringcellwallformation.

Inthischapter,wedescribethekeystepsofthe“designer”oligosaccharidemicroarrayapproachwefollowedtoprovethecarbohydratebindingactivityanddefinetheoligosaccharideligandsforDectin-1,anatypicalC-typelectin-likesignalingreceptorofthemammalianinnateimmunesystemwithakeyroleinanti-fungalimmunity.Theterm“designer”microarray,whichweintroducedinthecourseoftheDectin-1studyreferstoamicroarrayofoligosaccharideprobesgeneratedfromligand-bearingglycoconjugatestorevealtheoligosaccharideligandstheyharbor,sothatthesecanbeisolatedandcharacterized.Oligosaccharideprobesweregeneratedfromtwopolysaccharides,onethatwasboundbyDectin-1andknowntoberichinβ1,3-glucosesequenceandanotherthatwasnotboundandwasrichinβ1,6-glucosesequenceandservedasanegativecontrol.Theapproachinvolved:classicELISA-typebindingassaystoselectthepolysaccharides;partialdepolymerizationofthepolysaccharidesbychemicalhydrolysis;fractionationbysizeoftheglucanoligosaccharidesobtainedanddeterminationoftheirchainlengthsbymassspectrometry;detectionofDectin-1ligand-positiveandligand-negativeoligosaccharidesusingtheneoglycolipid(NGL)technology;methylationanalysisofoligosaccharidestoderiveglucoselinkageinformation,andincorporationofthenewlygeneratedglucanoligosaccharideprobesintomicroarraysencompassingdiversemammalian-typeandexogenoussequencesformicroarrayanalysisofDectin-1.

AnendoglycanasegeneofPaenibacilluscookiiSS-24wasclonedandsequenced.ThisPgl8AgenehadanopenreADIngframeof1,230bpthatencodedaputativesignalsequence(31aminoacids)andmatureenzyme(378aminoacids:41,835Da).Theenzymewasmosthomologoustoaβ-1,3-1,4-glucanaseofBacilluscirculansWL-12with84%identity.Therecombinantenzymehydrolyzedcarboxymethylcellulose,swollencelluloses,chitosanandlichenanbutnotAvicel,chitinpowderorxylan.Withchitosanasthesubstrate,theoptimumtemperatureandhydrolysisproductsoftherecombinantenzymevariedatpH4.0and8.0.ThisisthefirstreportthatcharacterizeschitosanaseactivityunderdifferentpHconditions.

High-resolution,three-dimensionalstructuresofthearchetypalglycosidehydrolasefamily16(GH16)endo-xyloglucanasesTm-NXG1andTm-NXG2fromnasturtium(Tropaeolummajus)havebeensolvedbyx-raycrystallography.KeystructuralfeaturesthatmodulatetherelativeratesofsubstratehydrolysistotransglycosylationintheGH16xyloglucan-activeenzymeswereidentifiedbystructure–functionstudiesoftherecombinantlyexpressedenzymesincomparisonwithdataforthestrictxyloglucanendo-transglycosylasePtt-XET16-34fromhybridaspen(Populustremula×Populustremuloides).ProductionoftheloopdeletionvariantTm-NXG1-ΔYNIIGyieldedanenzymethatwasstructurallysimilartoPtt-XET16-34andhadagreatlyincreasedtransglycosylation:hydrolysisratio.ComprehensivebioinformaticanalysesofXTHgeneproducts,togetherwithdetailedkineticdata,stronglysuggestthatxyloglucanaseactivityhasevolvedasagainoffunctioninanancestralGH16XETtomeetspecificbiologicalrequirementsduringseedgermination,fruitripening,andrapidwallexpansion.

Theeffectsoffinestructureandmolecularsizeontherheologicalpropertiesofsixmixed-linkage(1→3),(1→4)-β-D-glucans(β-glucans)inthesolutionandgelstatewerestudied.Molecularsizecharacterizationwascarriedoutwithhigh-performancesizeexclusionchromatographycombinedwitharefractiveindexdetector.Samplesweredividedintotwogroupsaccordingtothevaluesofapparentmolecularweight(M_w)ofthepeakfractionofthemainelutingpeakcalculatedas200×10³foranoat,abarley,andawheatβ-glucanand∼100×10³foranoatandabarleyβ-glucan,andalichenansample.Allpolysaccharidesanalyzedby2DNMRspectroscopyandhigh-performanceanion-exchangechromatographyofthecellulosicoligomersreleasedbytheactionoflichenaseshowedthetypicalfinestructureofmixed-linkagelinear(1→3),(1→4)-β-D-glucan.Followinglichenasedigestionofβ-glucans,themolarratiosoftri-totetrasaccharides(DP3/DP4)werefoundtofollowtheorderoflichenan(24.5)>wheat(3.7)>barley(2.8–3.0)>oat(2.1).Differencesincriticalconcentration(c**),viscosity,viscoelasticandshearthinningpropertiesamongsamplesweredependentmainlyondifferencesinmolecularsizeofthepolymericchainsaswellasontheβ-glucanfinestructure.Allβ-glucanisolateswereabletoformgels,asprobedbydynamicrheometry;withdecreasingmolecularsizeandincreasingDP3/DP4ratio,thegelationtimedecreasedandthegelationrate(I_E=[dlogG/dt]_max)increased.Differentialscanningcalorimetry(DSC)showedthatcerealβ-glucangelsexhibitratherbroadendothermicgel→soltransitionsat55–80°C,whilelichenangelsgiveasharpertransition,implyingamorecooperativeprocess.TheDSCkineticdatashowedsimilarresponsestothatfromdynamicrheometry;therateofdevelopmentoftheendothermincreasedwithincreasingDP3/DP4ratioofthepolysaccharide.Furthermore,thestoragemodulus(G′)andtheapparentmeltingenthalpyvalues(plateauΔH)increasedwithdecreasingmolecularsizeandwithincreasingDP3/DP4ratio.Themeltingtemperatureofthegelnetwork,asdeterminedbyDSCanddynamicrheometry,wasfoundtoincreasewiththemolecularsizeandtheDP3/DP4ratioofβ-glucans;theT_mforlichenanwas∼89°Candforcerealβ-glucansvariedinthenarrowrangeof∼65–72°C.Largedeformationmechanicaltests(compressionmode)uptofailurerevealedanincreaseinstrengthandadecreaseinbrittlenessofmixed-linkageβ-glucangelswithincreasingDP3/DP4ratioandmolecularsizeofthepolysaccharide.

The(1→3)(1→4)-β-D-glucansfromfourcerealsources(oats,wheat,barleyandrye)andonelichensource(Icelandicmoss)wereusedtotesttwoproposedstructurallybasedhypothesesaboutthegellingmechanismofthesepolymers.Structureswereevaluatedusinghighperformanceanionexchangechromatographyoftheoligosaccharidefragmentsreleasedbya(1→3)(1→4)-β-D-glucan-4-glucanohydrolase.Thisdeterminedtherelativeamountsofcellodextrinunits,ofdifferentdegreesofpolymerisation,whicharejoinedbyβ-(1→3)linkagesintheintactpolysaccharidechain.Oatβ-glucanhadthelowestβ-(1→3)-linkedcellotriosylunitcontentandlichenanhadthehighest.Strongcorrelationswerefoundbetweenthefractionofβ-(1→3)-linkedcellotriosylunitsintheβ-glucansandtheelasticityof6%gelsinwater,asmeasuredbydynamicrheometry.Differentialscanningcalorimetryshowedthattheβ-(1→3)-linkedcellotriosylunitcontentwasalsocorrelatedwiththeonsetandpeaktemperatureswhen6%β-glucangelsweremelted.Nocorrelationwasfoundbetweenthelonger(DP6–9)β-(1→3)-linkedcellodextrinoligosaccharidecontentandeitherthegelelasticityormeltingcharacteristics.Thesefindingsareconsistentwithamodelinwhichrunsofconsecutiveβ-(1→3)-linkedcellotriosylunitsformthejunctionzonesinthegelnetwork,butnotwithamodelinwhichlongerβ-(1→3)-linkedcellodextrinsassociate,asincellulosefibres,toproducethegelnetwork.Microscopicimagesoftheβ-glucangelsfromthefivespeciesrevealedthatthemicrostructurewasnothomogeneousinanyofthesamples,whichmayberelatedtothevariabilityintheenthalpyofmeltingofgels.Therewasacoarseningofgelstructureastheβ-(1→3)-linkedcellotriosylunitcontentincreased.

Thedigestionofdietaryfibersisamajorfunctionofthehumanintestinalmicrobiota.Sofarthisfunctionhasbeenattributedtothemicroorganismsinhabitingthecolon,andmanystudieshavefocusedonthisdistalpartofthegastrointestinaltractusingeasilyaccessiblefecalmaterial.However,microbialfermentations,supportedbythepresenceofshort-chainfattyacids,aresUSPectedtooccurintheuppersmallintestine,particularlyintheileum.Usingafosmidlibraryfromthehumanilealmucosa,wescreened20,000clonesfortheiractivitiesagainstcarboxymethylcelluloseandxylanschosenasmodelsofthemajorplantcellwall(PCW)polysaccharidesfromdietaryfibres.ElevenpositiveclonesrevealedabroadrangeofCAZymeencodinggenesfromBacteroidesandClostridialesspecies,aswellasPolysaccharideUtilizationLoci(PULs).Thefunctionalglycosidehydrolasegeneswereidentified,andoligosaccharidebreak-downproductsexaminedfromdifferentpolysaccharidesincludingmixed-linkageβ-glucans.CAZymesandPULswerealsoexaminedfortheirprevalenceinhumangutmicrobiome.Severalclustersofgenesoflowprevalenceinfecalmicrobiomesuggestedtheybelongtounidentifiedstrainsratherspecificallyestablishedupstreamthecolon,intheileum.Thus,theilealmucosa-associatedmicrobiotaencompassestheenzymaticpotentialforPCWpolysaccharidedegradationinthesmallintestine.

Thepossibilityofusingadditiveenzymestoimprovethesaccharificationoflignocellulosicsubstrateswithcommercialcellulolyticenzymeswasstudied.Reed(Phragmitescommunis)andrice(Oryzasativa)strawpowderswerepretreatedwithNaOH/steamviaahigh-temperatureexplosionsystem.Thesaccharificationofuntreatedreedandricestrawpowdersbycommercialenzymes(Celluclast1.5 L + Novozym188)wasnotsignificantlyincreasedbytheadditionofxylanases(Xyn10J,XynX),acellulase(Cel6H),andaβ-1,3-1,4-glucanase(BGlc8H)withbroadsubstratespecificity.Thesaccharificationofthepretreatedreedandricestrawpowdersbythecommercialenzymeswasincreasedby10.4and4.8 %,respectively,bytheadditionofBGlc8H.InthepresenceofCa²⁺andBGlc8H,thesaccharificationofthepretreatedreedandricestrawpowdersbythecommercialenzymeswasincreasedby18.5and11.7 %,respectively.NosucheffectofCa²⁺wasobservedwithXyn10J,XynX,orCel6H.Theresultssuggestthattheenzymaticconversionoflignocellulosicbiomasstoreducingsugarscouldbeenhancedbycertainadditiveenzymessuchasβ-1,3-1,4-glucanase,andthattheenhancementcouldfurtherbeincreasedbyCa²⁺.

Suberin,amajorconstituentofthepotatoperiderm,isknowntopromotetheproductionofthaxtomins,thekeyvirulencefactorsofthecommonscab-causingagentStreptomycesscabiei.Inthepresentstudy,wespeculatedthatsuberinaffectedtheproductionofglycosylhydrolases,suchascellulases,byS.scabiei,anddemonstratedthatsuberinpromotedglycosylhydrolaseactivitywhenaddedtocellulose-,xylan-,orlichenin-containingmedia.Furthermore,secretomeanalysesrevealedthattheadditionofsuberintoacellulose-containingmediumincreasedtheproductionofglycosylhydrolases.Forexample,theproductionof13outofthe14cellulasesproducedbyS.scabieiincellulose-containingmediumwasstimulatedbythepresenceofsuberin.Inmostcases,thetranscriptionofthecorrespondingcellulase-encodinggeneswasalsomarkedlyincreasedwhenthebacteriumwasgrowninthepresenceofsuberinandcellulose.Thelevelofasubtilase-likeproteaseinhibitorwasmarkedlydecreasedbythepresenceofsuberin.WeproposedamodelfortheonsetofS.scabieivirulencemechanismsbybothcelluloseandsuberin,themaindegradationproductofcellulosethatactsasaninducerofthaxtominbiosyntheticgenes,andsuberinpromotingthebiosynthesisofsecondarymetabolitesincludingthaxtomins.

Mixed-linkageglucan:xyloglucanendotransglucosylase(MXE)isoneofthethreeactivitiesoftherecentlycharacterisedhetero-trans-β-glucanase(HTG),whichamongland-plantsisknownonlyfromEquisetumspecies.ThebiochemicaldetailsoftheMXEreactionwereincompletelyunderstood-detailsthatwouldpromoteunderstandingofMXEsroleinvivoandenableitsfulltechnologicalexploitation.WeinvestigatedHTGssiteofattackononeofitsdonorsubstrates,mixed-linkage(1→3),(1→4)-β-D-glucan(MLG),withradioactiveoligosaccharidesofxyloglucanasacceptorsubstrate.ComparingthreedifferentMLGpreparations,weshowedthattheenzymefavoursthosewithahighcontentofcellotetraoseblocks.Thereactionproductswereanalysedbyenzymicdigestion,thin-layerchromatography,HPLCandgel-permeationchromatography.EquisetumHTGconsistentlycleavedtheMLGatthethirdconsecutiveβ-(1→4)-bondfollowing(towardsthereducingterminus)aβ-(1→3)-bond.Itthenformedaβ-(1→4)-bondbetweentheMLGandthenon-reducingterminalglucoseresidueofthexyloglucanoligosaccharide,consistentwithitsXTHsubfamilymembership.Usingsize-homogeneousbarleyMLGasdonorsubstrate,weshowedthatHTGdoesnotfavouranyparticularregionoftheMLGchainrelativethepolysaccharidesreducingandnon-reducingtermini;rather,itselectsitstargetcellotetraosylunitstochasticallyalongtheMLGmolecule.Thisworkimprovesourunderstandingofhowenzymescanexhibitpromiscuoussubstratespecificitiesandprovidesthefoundationstoexplorestrategiesforengineeringnovelsubstratespecificitiesintotransglycanases.

WeestablishedtransgeniclinesofNicotianatabacumexpressinghybridgenesofSynechocystissp.PCC6803Δ12(desA)acyllipiddesaturaseandSynechococcusvulcanusδ9(desC)acyllipiddesaturasewithorwithoutsequencecodingfortransitpeptideofRubiscosmallsubunitofArabidopsisthalianaundercontrolofaconstitutivepromoter.Reliableincreaseoflinoleicacidportion(C18:2;δ9,12)accompaniedwithdecreaseofα-linolenicacid(C18:3;δ9,12,15)relativeamountwasdetectedforplantsexpressinghybriddesA::licBM3gene.Noreliablechangesweredetectedinfattyacidprofilesandunsaturationindexofplantstransformedwithδ9desaturasegenedesC::licBM3lackingsignalsofintracellulartargetingwhileexpressionofthisgenewithArabidopsisthalianaRubiscosmallsubunittransitpeptidesequencecausedgrowthofC18:3α-linolenicacidpartsimultaneouslywithreductionofC18:2linoleicacidpart,aswellasincreaseofunsat-urationindex.Nochangesinrelativeamountofδ9-monounsaturatedfattyacidswereobservedinanyofstudiedlines.Allplantsexpressingdesaturasegenesexhibitedenhancedlevelsofsuperoxidedismutase(SOD)activityaftercoldtreatmentincontrasttocontrollineswithsuppressedSODactivityaftercoldtreatment.

β-1,3-1,4-Glucanase(BGlc8H)fromPaenibacillussp.X4wasmutatedbyerror-pronePCRortruncatedusingterminationprimerstoimproveitsenzymeproperties.ThecrystalstructureofBGlc8Hwasdeterminedataresolutionof1.8Åtostudythepossiblerolesofmutatedresiduesandtruncatedregionsoftheenzyme.Inmutationexperiments,threeclonesofEP2-6,2-10,and5-28werefinallyselectedthatexhibitedhigherspecificactivitiesthanthewildtypewhenmeasuredusingtheircrudeextracts.EnzymevariantsofBG_2-6,BG_2-10,andBG_5-28weremutatedattwo,two,andsixaminoacidresidues,respectively.TheseenzymeswerepurifiedhomogeneouslybyHi-TrapQandCHT-IIchromatography.SpecificactivityofBG5-28was2.11-foldhigherthanthatofwild-typeBG_wt,whereasthoseofBG_2-6andBG_2-10were0.93-and1.19-foldthatofthewildtype,respectively.TheoptimumpHvaluesandtemperaturesofthevariantswerenearlythesameasthoseofBG_wt(pH5.0and40°C,respectively).However,thehalf-lifeoftheenzymeactivityandcatalyticefficiency(k_cat/K_m)ofBG_5-28were1.92-and2.12-foldgreaterthanthoseofBG_wtat40°C,respectively.ThecatalyticefficiencyofBG5-28increasedto3.09-foldthatofBG_wtat60°C.TheseincreasesinthethermostabilityandcatalyticefficiencyofBG_5-28mightbeusefulforthehydrolysisofβ-glucanstoproducefermentablesugars.OfthesixmutatedresiduesofBG_5-28,fiveresidueswerepresentinmatureBGlc8Hprotein,andtwoofthemwerelocatedinthecorescaffoldofBGlc8Handtheremainingthreeresidueswereinthesubstrate-bindingpocketformingloopregions.Intruncationexperiments,threeformsofC-terminaltruncatedBGlc8Hweremade,whichcomprised360,286,and215aminoacidresiduesinsteadofthe409residuesofthewildtype.Noenzymeactivitywasobservedforthesetruncatedenzymes,suggestingthecompletescaffoldoftheα₆/α₆-double-barrelstructureisessentialforenzymeactivity.

Background:Effectiveandsimplemethodsthatleadtohigherenzymaticefficienciesarehighlysough.Hereweproposedafoldon-triggeredtrimerizationofthetargetenzymeswithsignificantlyimprovedcatalyticperformancesbyfusingafoldondomainattheC-terminusoftheenzymesviaelastin-likepolypeptides(ELPs).Thefoldondomaincomprises27residuesandcanformstrimerswithhighstability.Results:Lichenaseandxylanasecanhydrolyzelichenanandxylantoproducevalueaddedproductsandbiofuels,andtheyhavegreatpotentialsasbiotechnologicaltoolsinvariousindustrialapplications.Wetookthemastheexamplesandcomparedthekineticparametersoftheengineeredtrimericenzymestothoseofthemonomericandwildtypeones.Whencomparedwiththemonomericones,thecatalyticefficiency(k_cat/K_m)ofthetrimericlichenaseandxylanaseincreased4.2-and3.9-fold.Thecatalyticconstant(k_cat)ofthetrimericlichenaseandxylanaseincreased1.8-foldand5.0-foldthantheircorrespondingwild-typecounterparts.Also,thespecificactivitiesoftrimericlichenaseandxylanaseincreasedby149%and94%thanthoseofthemonomericones.Besides,therecoveryofthelichenaseandxylanaseactivitiesincreasedby12.4%and6.1%duringthepurificationprocessusingELPsasthenon-chromatographictag.ThepossiblereasonisthefoldondomaincanreducethetransitiontemperatureoftheELPs.Conclusion:Thetrimericlichenaseandxylanaseinducedbyfoldonhaveadvantagesinthecatalyticperformances.Besides,theywereeasiertopurifywithincreasedpurificationfoldanddecreasedthelossofactivitiescomparedtotheircorrespondingmonomericones.Trimerizingofthetargetenzymestriggeredbythefoldondomaincouldimprovetheiractivitiesandfacilitatethepurification,whichrepresentsasimpleandeffectiveenzyme-engineeringtool.Itshouldhaveexcitingpotentialsbothinindustrialandlaboratoryscales.