HighpuritydyedandcrosslinkedinsolubleAZCL-Galactan(Potato)foridentificationofenzymeactivitiesinresearch,microBIOLOGicalenzymeassaysandinvitrodiagnosticanalysis.
Substratefortheassayofendo-1,4-β-D-galactanase.
BifidobacteriumlongumendogalactanaseliberatesgalactotriosefromtypeIgalactans.
Hinz,S.W.A.,Pastink,M.I.,vandenBroek,L.A.M.,Vincken,J.P.&Voragen,A.G.J.(2005).AppliedandEnvironmentalMicrobiology,71(9),5501-5510.
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Aputativeendogalactanasegeneclassifiedintoglycosidehydrolasefamily53wasrevealedfromthegenomesequenceof
BifidobacteriumlongumstrainNCC2705(Schelletal.,Proc.Natl.Acad.Sci.USA99:14422-14427,2002).Sinceonlyafewendo-actingenzymesfrombifidobacteriahavebeendescribed,wehaveclonedthisgeneandcharacterizedtheenzymeindetail.Thededucedaminoacidsequencesuggestedthatthisenzymewaslocatedextracellularlyandanchoredtothecellmembrane.
galAwasclonedwithoutthetransmembranedomainintothepBluescriptSK(−)vectorandexpressedin
Escherichiacoli.Theenzymewaspurifiedfromthecellextractbyanion-exchangeandsizeexclusionchromatography.Thepurifiedenzymehadanativemolecularmassof329kDa,andthesubunitshadamolecularmassof94kDa,whichindicatedthattheenzymeoccurredasatetramer.TheoptimalpHofendogalactanaseactivitywas5.0,andtheoptimaltemperaturewas37°C,usingazurine-cross-linkedgalactan(AZCL-galactan)asasubstrate.The
Kmand
VmaxforAZCL-galactanwere1.62mMand99U/mg,respectively.Theenzymewasabletoliberategalactotrisaccharidesfrom(β1→4)galactansand(β1→4)galactooligosaccharides,probablybyaprocessivemechanism,movingtowardthereducingendofthegalactanchainafteraninitialmidchaincleavage.GalA"smodeofactionwasfoundtobedifferentfromthatofanendogalactanasefrom
Aspergillusaculeatus.Theenzymeseemedtobeabletocleave(β1→3)linkages.Ar
ABInosylsidechainsin,forexample,potatogalactanhinderedGalA.
CharacterizationoftheErwiniachrysanthemiganlocus,involvedingalactancatabolism.
Delangle,A.,Prouvost,A.F.,Cogez,V.,Bohin,J.P.,Lacroix,J.M.&Cotte-Pattat,N.H.(2007).JournalofBacteriology,189(19),7053-7061.
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β-1,4-Galactanisamajorcomponentoftheramifiedregionsofpectin.AnalysisofthegenomeoftheplantpathogenicbacteriaErwiniachrysanthemirevealedthepresenceofaclusterofeightgenesencodingproteinspotentiallyinvolvedingalactanutilization.ThepredictedtransportsystemwouldcompriseaspecificporinGanLandanABCtransportermadeoffourproteins,GanFGK2.Degradationofgalactanswouldbecatalyzedbytheperiplasmic1,4-β-endogalactanaseGanA,whichreleasedoligogalactansfromtrimertohexamer.Aftertheirtransportthroughtheinnermembrane,oligogalactanswouldbedegradedintogalactosebythecytoplasmic1,4-β-exogalactanaseGanB.Mutantsaffectedfortheporinorendogalactanasewereunabletogrowongalactans,buttheygrewongalactoseandonamixtureofgalactotriose,galactotetraose,galactopentaose,andgalactohexaose.Mutantsaffectedfortheperiplasmicgalactanbindingprotein,thetransporterATPase,ortheexogalactanasewereonlyabletogrowongalactose.Thus,thephenotypesofthesemutantsconfirmedthefunctionalityoftheganlocusintransportandcatabolismofgalactans.ThesemutationsdidnotaffectthevirulenceofE.chrysanthemionchicoryleaves,potatotubers,orSaintpauliaionantha,suggestinganaccessoryroleofgalactanutilizationinthebacterialpathogeny.
Large-scaleextractionofrhamnogalacturonanIfromindustrialpotatowaste.
Byg,I.,Diaz,J.,Øgendal,L.H.,Harholt,J.,Jørgensen,B.,Rolin,C.,Rolin,C.,Svava,R.&Ulvskov,P.(2012).FoodChemistry,131(4),1207-1216.
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Potatopulpisrichindietaryfibresandisanunderutilisedmaterialproducedinlargequantitiesbythepotatostarchfactories.PotatofibresareespeciallyrichinrhamnogalacturonanI(RGI).RGIisapecticpolysaccharidewithahighdegreeofbranchinganduntilnowundegradedRGIhasonlybeenextractedinsmallamountslimitingtheapplicationpossibilitiesforRGI.Thepresentpaperdescribesalarge-scaleextractionprocessprovidinglargequantitiesofundegradedRGIre
ADIlyavailable.TheextractionprocessincludesenzymaticstarchremovalusingpurifiedTermamyl,enzymaticRGIsolubilisationusingahighlypurifiedpolygalacturonase,andfinallypurificationusingdepthfiltrationandultrafiltration.TheextractedRGIhasahighmolecularweightandamonosaccharidecompositioncomparabletoRGIextractedbyanalyticalextractionprocedures.ThelargeamountofRGIavailablebythepresentedmethodallowsforthoroughstructure–functionanalysesandtailoringofRGItospecificfunctionalities.
StructuralandbiochemicalstudieselucidatethemechanismofrhamnogalacturonanlyasefromAspergillusaculeatus.
Jensen,M.H.,Otten,H.,Christensen,U.,Borchert,T.V.,Christensen,L.L.H.,Larsen,S.&Leggio,L.L.(2010).JournalofMolecularBiology,404(1),100-111.
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Wepresentherethefirstexperimentalevidenceforboundsubstrateintheactivesiteofarhamnogalacturonanlyasebelongingtofamily4ofpolysaccharidelyases,Aspergillusaculeatusrhamnogalacturonanlyase(RGL4).RGL4isinvolvedinthedegradationofrhamnogalacturonan-I,animportantpecticplantcellwallpolysaccharide.Basedonthepreviouslydeterminedwild-typestructure,enzymevariantsRGL4_H210AandRGL4_K150Ahavebeenproducedandcharacterizedbothkineticallyandstructurally,showingthatHis210andLys150arekeyactive-siteresidues.CrystalsoftheRGL4_K150Avariantsoakedwitharhamnogalacturonandigestgaveaclearpictureofsubstrateboundinthe−3/+3subsites.ThecrystallographicandkineticstudiesonRGL4,andstructuralandsequencecomparisontootherenzymesinthesameandotherPLfamilies,enableustoproposeadetailedreactionmechanismfortheβ-eliminationon[-,2)-α-L-rhamno-(1,4)-α-D-galacturonicacid-(1,-].Themechanismdifferssignificantlyfromtheoneestablishedforpectatelyases,inwhichmostoftencalciumionsareengagedincatalysis.
Simultaneousinvivotruncationofpecticsidechains.
Øbro,J.,Borkhardt,B.,Harholt,J.,Skjøt,M.,Willats,W.G.T.&Ulvskov,P.(2009).TransgenicResearch,18(6),961-969.
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Despitethewideoccurrenceofpectininnatureonlyafewsourcematerialshavebeenusedtoproducecommercialpectins.Oneofthereasonsforthisisthatmanyplantspeciescontainpectinswithhighlevelsofneutralsugarsidechainsorthatarehighlysubstitutedwithacetylorothergroups.Thesemodificationsoftenpreventgelation,whichhasbeenamajorfunctionalrequirementofcommercialpectinsuntilrecently.Wehavepreviouslyshownthatmodificationofpectinisposs
IBLethroughheterologousexpressionofpectindegradingenzymes
inplanta.TotesttheeffectofsimultaneousmodificationofthetwomainneutralpecticsidechainsinpecticrhamnogalacturonanI(RGI),weconstitutivelyexpressedtwodifferentenzymesin
Arabidopsisthalianathatwouldeithermodifythegalactanorthearabinansidechains,orbothsidechainssimultaneously.Ouranalysisshowedthatthesimultaneoustruncationofarabinanandgalactansidechainsisachievableanddoesnotseverelyaffectthegrowthof
Arabidopsisthaliana.
Theβ-1,4‐endogalactanaseAgenefromAspergillusnigerisspecificallyinducedonarabinoseandgalacturonicacidandplaysanimportantroleinthedegradationofpectichairyregions.
deVries,R.P.,Pařenicová,L.,Hinz,S.W.A.,Kester,H.C.M.,Beldman,G.,Benen,J.A.E.&Visser,J.(2002).EuropeanJournalofBiochemistry,269(20),4985-4993.
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TheAspergillusnigerβ-1,4-endogalactanaseencodinggene(galA)wasclonedandcharacterized.TheexpressionofgalAinA.nigerwasonlydetectedinthepresenceofsugarbeetpectin,D-galacturonicacidandL-arabinose,suggestingthatgalAiscoregulatedwithboththepectinolyticgenesaswellasthearabinanolyticgenes.Thecorrespondingenzyme,endogalactanaseA(GALA),containsbothactivesiteresiduesidentifiedpreviouslyforthePseudomonasfluorescensβ-1,4-endogalactanase.ThegalAgenewasoverexpressedtofacilitatepurificationofGALA.Theenzymehasamolecularmassof48.5kDaandapHoptimumbetween4and4.5.Incubationsofarabinogalactansofpotato,onionandsoywithGALAresultedinitiallyinthereleaseofD-galactotrioseandD-galactotetraose,whereasprolongedincubationresultedinD-galactoseandD-galactobiose,predominantly.MALDI-TOFanalysisrevealedthereleaseofL-arabinosesubstitutedD-galactooligosaccharidesfromsoyarabinogalactan.Thisisthefirstreportoftheabilityofaβ-1,4-endogalactanasetoreleasesubstitutedD-galacto-oligosaccharides.GALAwasnotactivetowardsD-galacto-oligosaccharidesthatweresubstitutedwithD-glucoseatthereducingend.
Investigatingthebindingofβ-1,4‐galactantoBacilluslicheniformisβ-1,4‐galactanasebycrystallographyandcomputationalmodeling.
LeNours,J.,DeMaria,L.,Welner,D.,Jørgensen,C.T.,Christensen,L.L.H.,Borchert,T.V.,Larsen,S.&LoLeggio,L.(2009).Proteins:Structure,Function,andBioinformatics,75(4),977-989.
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Microbialβ-1,4-galactanasesareglycosidehydrolasesbelongingtofamily53,whichdegradegalactanandarabinogalactansidechainsinthehairyregionsofpectin,amajorplantcellwallcomponent.TheybelongtothelargerclanGH-Aofglycosidehydrolases,whichcovermanydifferentpoly-andoligosaccharidasespecificities.CrystallographiccomplexesofBacilluslicheniformiβ-1,4-galactanaseanditsinactivenucleophilemutanthavebeenobtainedwithmethyl-β(1→4)-galactotetraoside,providing,forthefirsttime,informationonsubstratebindingtotheaglyconesideoftheβ-1,4-galactanasesubstratebindinggroove.Usingtheexperimentallydeterminedsubsitesasastartingpoint,aβ(1→4)-galactononaosewasbuiltintothestructureandsubjectedtomoleculardynamicssimulationsgivingfurtherinsightintotheresiduesinvolvedinthebindingofthepolysaccharidefromsubsite−4to+5.Inparticular,thisanalysisnewlyidentifiedaconservedβ-turn,whichcontributestosubsites−2to+3.Thisβ-turnisuniquetofamily53β-1,4-galactanasesamongallclanGH-Afamiliesthathavebeenstructurallycharacterizedandthusmightbeastructuralsignatureforendo-β-1,4-galactanasespecificity.
TheStructureofendo-β-1,4-galactanasefromBacilluslicheniformisinComplexwithTwoOligosaccharideProducts.
Ryttersgaard,C.,LeNours,J.,LoLeggio,L.,Jørgensen,C.T.,Christensen,L.L.H.,Bjørnvad,M.&Larsen,S.(2004).JournalofMolecularBiology,341(1),107-117.
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Theβ-1,4-galactanasefromBacilluslicheniformis(BLGAL)isaplantcell-wall-degradingenzymeinvolvedinthehydrolysisofβ-1,4-galactaninthehairyregionsofpectin.ThecrystalstructureofBLGALwasdeterminedbymolecularreplacementbothaloneandincomplexwiththeproductsgalactobioseandgalactotriose,catchingafirstcrystallographicglimpseoffragmentsofβ-1,4-galactan.AsexpectedforanenzymebelongingtoGH-53,theBLGALstructurerevealsa(βα)8-barrelarchitecture.However,BLGALβα-loops2,7and8arelongincontrasttothecorrespondingloopsinstructuresoffungalgalactanasesdeterminedpreviously.ThestructureofBLGALadditionallyshowsacalciumionlinkingthelongβα-loops7and8,whichreplacesadisulphidebridgeinthefungalgalactanases.Comparedtothesubstrate-bindingsubsitespredictedforAspergillusaculeatusgalactanase(AAGAL),twoadditionalsubsitesforsubstratebindingarefoundinBLGAL,−3and−4.AcomparisonofthepatternofgalactanandgalactooligosaccharidesdegradationbyAAGALandBLGALshowsthat,althoughbotharemostactiveonsubstrateswithahighdegreeofpolymerization,AAGALcandegradegalactotrioseandgalactotetraoseefficiently,whereasBLGALpreferslongeroligosaccharidesandcannothydrolyzegalactotriosetoanyappreciableextent.Thisdifferenceinsubstratepreferencecanbeexplainedstructurallybythepresenceoftheextrasubsites−3and−4inBLGAL.
MiningDictyoglomusturgidumforenzymaticallyactivecarbohydrases.
Brumm,P.,Hermanson,S.,Hochstein,B.,Boyum,J.,Hermersmann,N.,Gowda,K.&Mead,D.(2011).AppliedBiochemistryandBiotechnology,163(2),205-214.
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ThegenomeofDictyoglomusturgidumwassequencedandanalyzedforcarbohydrases.Thebroadrangeofcarbohydratesubstrateutilizationisreflectedinthehighnumberofglycosylhydrolases,54,andthehighpercentageofCAZymespresentinthegenome,3.09%ofitstotalgenes.ScreeningarandomclonelibrarygeneratedfromD.turgidumresultedinthediscoveryoffivenovelbiomass-degradingenzymeswithlowhomologytoknownmolecules.Wholegenomesequencingoftheorganismfollowedbybioinformatics-directedamplificationofselectedgenesresultedintherecoveryofsevenadditionalnovelenzymemolecules.Basedontheanalysisofthegenome,D.turgidumdoesnotappeartodegradecelluloseusingeitherconventionalsolubleenzymesoracellulosomaldegradationsystem.Thetypesandquantitiesofglycosylhydrolasesandcarbohydrate-bindingmodulespresentinthegenomesuggestthatD.turgidumdegradescelluloseviaamechanismsimilartothatusedbyCytophagahutchinsoniiandFibrobactersuccinogenes.
ExpressioncloninginKluyveromyceslactis.
vanderVlugt-Bergmans,C.J.B.&vanOoyen,A.J.J.(1999).BiotechnologyTechniques,13(1),87-92.
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Kluyveromyceslactiswasusedashostforan
Aspergillustubingensisexpressionlibrary.Anewepisomalvectorwasconstructedtodirecttheexpressionofthe
A.tubingensisCDNAsandtoallowsubsequentanalysisin
Escherichiacoli.Usingthreedifferentplateassays,18000
K.lactisrecombinantswerescreened,yielding60galactanase-,26polygalacturonase-and16cellulase-secretingcolonies.Thegalactanase-secretingrecombinantswereanalysedindetail:theyaretranscriptsofthesamegalactanasegenewithsimilaritytoan
A.aculeatusβ-1,4-galactanasegene.Theresultsofthe
K.lactissystemcomparefavourablytothoseobtainedby
Saccharomycescerevisiae.
StructuralandfunctionalcharacterizationofanovelfamilyGH1154-O-methyl-α-glucuronidasewithspecificityfordecoratedarabinogalactans.
Aalbers,F.,Turkenburg,J.P.,Davies,G.J.,Dijkhuizen,L.&vanBueren,A.L.(2015).JournalofMolecularBiology,427(24),3935-3946.
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Glycosidehydrolasesareclusteredintofamiliesbasedonaminoacidsequencesimilarities,andbelongingtoaparticularfamilycaninferbiologicalactivityofanenzyme.FamilyGH115containsα-glucuronidaseswhereseveralmembershavebeenshowntohydrolyzeterminalα-1,2-linkedglucuronicacidand4-O-methylatedglucuronicacidfromtheplantcellwallpolysaccharideglucuronoxylan.OtherGH115enzymesshownoactivityonglucuronoxylan,andtherefore,ithasbeenproposedthatfamilyGH115maybeapoly-specificfamily.Inthisstudy,werevealthataputativeperiplasmicGH115fromthehumangutsymbiontBacteroidesthetaiotaomicron,BtGH115A,hydrolyzesterminal4-O-methyl-glucuronicacidresiduesfromdecoratedarabinogalactanisolatedfromacaciatree.Thethree-dimensionalstructureofBtGH115ArevealsthatBtGH115Ahasthesamedomainarchitectureastheotherstructurallycharacterizedmemberofthisfamily,BoAgu115A;howeverthepositionoftheC-terminalmoduleisalteredwithrespecttoeachindividualenzyme.PhylogeneticanalysisofGH115aminosequencesdividesthefamilyintodistinctcladesthatmaydistinguishdifferentsubstratespecificities.Finally,weshowthatBtGH115Aα-glucuronidaseactivityisnecessaryforthesequentialdigestionofbranchedgalactansfromacaciagumbyagalactan-β-1,3-galactosidasefromfamilyGH43;however,whileB. thetaiotaomicrongrowsonlarchwoodarabinogalactan,thebacteriumisnotabletometabolizeacaciagumarabinogalactan,suggestingthatBtGH115Aisinvolvedindegradationofarabinogalactanfragmentsliberatedbyothermicrobialspeciesinthegastrointestinaltract.
EffectofmutationsontheThermostabilityofAspergillusaculeatusβ-1,4-galactanase.
Torpenholt,S.,DeMaria,L.,Olsson,M.H.,Christensen,L.H.,Skjøt,M.,Westh,P.,Jensen,J.H.&Leggio,L.L.(2015).ComputationalandStructuralBiotechnologyJournal,13,256-264.
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Newvariantsofβ-1,4-galactanasefromthemesophilicorganismAspergillusaculeatusweredesignedusingthestructureofβ-1,4-galactanasefromthethermophileorganismMyceliophthorathermophilaasatemplate.SomeofthevariantsweregeneratedusingPROPKA3.0,avalidatedpKapredictiontool,totestitsusefulnessasanenzymedesigntool.ThePROPKAdesignedvariantswereD182NandS185D/Q188T,G104D/A156R.VariantsY295FandG306Aweredesignedbyaconsensusapproach,asacomplementaryandvalidateddesignmethod.D58Nwasastabilizingmutationpredictedbybothmethods.Thepredictionswereexperimentallyvalidatedbymeasurementsofthemeltingtemperature(Tm)bydifferentialscanningcalorimetry.WefoundthattheTmiselevatedby1.1°CforG306A,slightlyincreased(intherangeof0.34to0.65°C)forD182N,D58N,Y295FandunchangedordecreasedforS185D/Q188TandG104D/A156R.TheTmchangeswereintherangepredictedbyPROPKA.Giventheexperimentalerrors,onlytheD58NandG306Ashowsignificantincreaseinthermodynamicstability.Giventhepracticalimportanceofkineticstability,thekineticsoftheirreversibleenzymeinactivationprocesswerealsoinvestigatedforthewild-typeandthreevariantsandfoundtobebiphasic.Thehalf-livesofthermalinactivationwereapproximatelydoubledinG306A,unchangedforD182Nand,disappointingly,alotlowerforD58N.Inconclusion,thisstudytestsanewmethodforestimatingTmchangesformutants,addstotheavailabledataontheeffectofsubstitutionsonproteinthermostabilityandidentifiesaninterestingthermostabilizingmutation,whichmaybebeneficialalsoinothergalactanases.
Aspergillushancockiisp.nov.,abiosyntheticallytalentedfungusendemictosoutheasternAustraliansoils.
Pitt,J.I.,Lange,L.,Lacey,A.E.,Vuong,D.,Midgley,D.J.,Greenfield,P.,Bradbury,M.I.,Lacey,E.,Busk,P.K.,Pilgaard,B.,Chooi,Y.H.&Piggott,A.M.(2017).PloSOne,12(4),e0170254.
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Aspergillushancockiisp.nov.,classifiedinAspergillussubgenusCircumdatisectionFlavi,wasoriginallyisolatedfromsoilinpeanutfieldsnearKumbia,intheSouthBurnettregionofsoutheastQueensland,Australia,andhassincebeenfoundoccasionallyfromothersubstratesandlocationsinsoutheastAustralia.ItisphylogeneticallyandphenotypicallyrelatedmostcloselytoA. leporisStatesandM.Chr.,butdiffersinconidialcolour,otherminorfeaturesandparticularlyinmetaboliteprofile.Whencultivatedonriceasanoptimalsubstrate,A. hancockiiproducedanextensivearrayof69secondarymetabolites.Elevenofthe15mostabundantsecondarymetabolites,constituting90%ofthetotalareaunderthecurveoftheHPLCtraceofthecrudeextract,werenovel.ThegenomeofA. hancockii,approximately40Mbp,wassequencedandminedforgenesencodingcarbohydratedegradingenzymesidentifiedthepresenceofmorethan370genesin114geneclusters,demonstratingthatA. hancockiihasthecapacitytodegradecellulose,hemicellulose,lignin,pectin,starch,chitin,cutinandfructanasnutrientsources.LikemostAspergillusspecies,A. hancockiiexhibitedadiversesecondarymetabolitegeneprofile,encoding26polyketidesynthase,16nonribosomalpeptidesynthaseand15nonribosomalpeptidesynthase-likeenzymes.
MetatranscriptomicsRevealstheFunctionsandEnzymeProfilesoftheMicrobialCommunityinChineseNong-FlavorLiquorStarter.
Huang,Y.,Yi,Z.,Jin,Y.,Huang,M.,He,K.,Liu,D.,Luo,H.,Zhao,D.,He,H.,Fang,Y.&Zhao,H.(2017).FrontiersinMicrobiology,8,1747.
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Chineseliquorisoneoftheworld"sbest-knowndistilledspiritsandisthelargestspiritcategorybysales.Theuniqueandtraditionalsolid-statefermentationtechnologyusedtoproduceChineseliquorhasbeenincontinuoususeforseveralthousandyears.Thediverseanddynamicmicrobialcommunityinaliquorstarteristhemaincontributortoliquorbrewing.However,littleisknownabouttheecologicaldistributionandfunctionalimportanceofthesecommunitymembers.Inthisstudy,metatranscriptomicswasusedtocomprehensivelyexploretheactivemicrobialcommunitymembersandkeytranscriptswithsignificantfunctionsintheliquorstarterproductionprocess.Fungiwerefoundtobethemostabundantandactivecommunitymembers.Atotalof932carbohydrate-activeenzymes,includinghighlyexpressedauxiliaryactivityfamily9and10proteins,wereidentifiedat62°Cunderaerobicconditions.Somepotentialthermostableenzymeswereidentifiedat50,62,and25°C(maturestage).Increasedcontentandoverexpressedkeyenzymesinvolvedinglycolysisandstarch,pyruvateandethanolmetabolismweredetectedat50and62°C.Thekeyenzymesofthecitratecyclewereup-regulatedat62°C,andtheirabundantderivativesarecrucialforflavorgeneration.Here,themetabolismandfunctionalenzymesoftheactivemicrobialcommunitiesinNFliquorstarterwerestudied,whichcouldpavethewaytoinitiateimprovementsinliquorqualityandtodiscovermicrobesthatproducenovelenzymesorhigh-valueaddedproducts.
