HighpuritydyedandcrosslinkedinsolubleAZCL-Barleyβ-Glucanforidentificationofenzymeactivitiesinresearch,microbiologicalenzymeassaysandinvitrodiagnosticanalysis.
Substratefortheassayofmaltβ-glucanase,lichenaseandcellulases.
Newchromogenicsubstratesfortheassayofalpha-amylaseand(1→4)-β-D-glucanase.
McCleary,B.V.(1980).CarbohydrateResearch,86(1),97-104.
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Newchromogenicsubstrateshavebeendevelopedforthequantitativeassayofalpha-amylaseand(1→4)-β-D-glucanase.Thesewerepreparedbychemicallymodifyingamyloseorcellulosebeforedyeing,toincreasesolubility.Afterdyeing,thesubstrateswereeithersolubleorcouldbereadilydispersedtoformfine,gelatinoussuspensions.Assaysbasedontheuseofthesesubstratesaresensitiveandhighlyspecificforeitheralpha-amylaseor(1→4)-β-D-glucanase.Themethodofpreparationcanalsobeappliedtoobtainsubstratesforotherendo-hydrolases.
Measurementofmaltbeta-glucanase.
McCleary,B.V.(1986).Proceedingsofthe19thConventionoftheInstituteofBrewing(Aust.andN.Z.section),181-187.
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AProcedurehasbeendevelopedfortheassayofmaltβ-glucanase[a(1→3)(1→4)-β-D-glucanase]whichemploysassubstrate,barleyβ-glucandyedwithRemazolbrilliantBlueandchemicallymodifiedwithcarboxymethylgroupstoincreasesolubility.Thedescribedassayproceduretogetherwithamodifiedextractionformatallowsanalysisofuptotenmaltsamplesinlessthan80min.Also,theprocedureisspecificforenzymesactiveonbarleyβ-glucan,isaccurateandreliable,andcanbereadilyappliedtotheanalysisofβ-glucanaseinmalt,greenmaltandwort.
Asolublechromogenicsubstratefortheassayof(1→3)(1→4)-β-D-glucanase(lichenase).
McCleary,B.V.(1986).CarbohydratePolymers,6(4),307-318.
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Asimpleprocedurefortheassayof(1→3)(1→4)-β-D-glucanase(lichenase)hasbeendeveloped.Thisassayemploysassubstratebarley(1→3)(1→4)-β-D-glucandyedwithRemazolbrilliantBlueRandchemicallymodifiedwithcarboxymethylgroupstoincreasesolubility.Preparationofthissubstraterequiredthedevelopmentofanimprovedprocedurefortheextractionandpurificationofbarleyβ-glucan.AssaysbasedontheuseofthedescribedchromogenicsubstrateatpH6•5aresensitiveandspecificforenzymesactiveonbarleyβ-glucan.
Problemscausedbybarleybeta-glucansinthebrewingindustry.
McCleary,B.V.(1986).ChemistryinAustralia,53,306-308.
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Brewing,theoldestapplicationofbio-technologyisnowamixoftradeartandmodernscience.Thisarticledescribesnewapplicationsofenzymechemistrytotrouble-shootinginbeerproduction.
Assayofmaltβ-glucanaseusingazo-barleyglucan:animprovedprecipitant.
McCleary,B.V.&Shameer,I.(1987).JournaloftheInstituteofBrewing,93(2),87-90.
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Aprocedurerecentlydescribedfortheassayofmaltβ-glucanase,whichemploysadye-labelledandchemically-modifiedbarleyβ-glucansubstrate,hasbeenimprovedbychangingtheprecipitantsolutionusedtoterminatethereaction.Thenewprecipitantsolutioncontains0•4%(w/v)zincacetateand4%(w/v)sodiumacetatedissolvedin80%(v/v)aqueousmethylcellosolve.Withthisprecipitanttheprocedurecanbedirectlyappliedtotheassayofcellulaseactivity,andwithminormodification,totheassayoflichenaseactivity.
Activityofarabinoxylanhydrolyzingenzymesduringmashingwithbarleymaltorbarleymaltandunmaltedwheat.
Debyser,W.,Delvaux,F.&Delcour,J.A.(1998).JournalofAgriculturalandFoodChemistry,46(12),4836-4841.
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Pilotscalebrewswerepreparedeitherwith100%barleymalt(BM100)or60%barleymaltand40%unmaltedwheat(BM60W40).Arabinoxylanandβ-glucanhydrolyzingenzymeactivitiesweredeterminedduringmashingusingtwotemperatureprofiles.ThemeasuredenzymicactivitiesincreasedfortheBM100andBM60W40mashesintheearlystagesofmashing.Theendoxylanaseandα-L-arabinofuranosidaseactivitiesremainedconstantat50°Cbutrapidlydecreasedabove50°C.At72°C,theendoxylanaseandα-L-arabinofuranosidaseactivitieswerealmostcompletelylost.Theβ-D-xylosidaseactivityonlydecreasedslowlyat63°C.Theβ-glucanaseactivitydecreasedrapidlyat50°Candwascompletelylostafter15minat50°C.Fromthexylose(Xyl)levels(ameasureforarabinoxylancontent)intheBM100worts(1.28−1.33g/L),asolubilizationof0.23−0.26%Xyl(%ofcerealdrymatter)duringmashingwascalculated.TheXyllevelsintheBM60W40worts(0.92−1.11g/L)correspondedwithasolubilizationof0.12to0.15%Xylduringmashing.
Purification,characterizationandstructuralanalysisofanabundantβ-1,3‐glucanasefrombananafruit.
Peumans,W.J.,Barre,A.,Derycke,V.,Rougé,P.,Zhang,W.,May,G.D.,Delcour,J.A.,VanLeuven,F.&VanDamme,E.J.(2000).EuropeanJournalofBiochemistry,267(4),1188-1195.
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Anabundant,catalyticallyactiveβ-1,3-endoglucanase(EC3.2.1.39)hasbeenisolatedfromthepulpripeofbananas.Biochemicalanalysisofthepurifiedprotein,molecularmodelling,andmolecularcloningofthecorrespondinggeneindicatethatthisbananaenzymecloselyresemblespreviouslycharacterizedplantβ-glucanaseswithrespecttoitsamino-acidsequence,structureandbiologicalactivity.Theresultsdescribedinthispaperdemonstrateboththeoccurrenceofanabundantactiveβ-1,3-endoglucanasesinfruitsandalsoreaddressthequestionofthepossibleinvolvementoftheseenzymesintheripeningand/orsofteningprocess.
Tenacibaculumskagerrakensesp.nov.,amarinebacteriumisolatedfromthepelagiczoneinSkagerrak,Denmark.
Frette,L.,Jørgensen,N.O.G.,Irming,H.&Kroer,N.(2004).InternationalJournalofSystematicandEvolutionaryMicrobiology,54(2),519-524.
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AnumberofbacteriawereisolatedfromseawaterinSkagerrak,Denmark,at30mdepth.Twooftheisolates,strainsD28andD30T,belongedtotheFlavobacteriaceaewithintheCytophaga–Flavobacterium–Bacteroidesgroup.Sequencingof16SrRNAgenesofthetwostrainsindicatedstronglythattheybelongedtothegenusTenacibaculumandthattheyshowedgreatestsimilaritytothespeciesTenacibaculumamylolyticumandTenacibaculummesophilum.DNA–DNAhybridizationvalues,DNAbasecompositionandphenotypiccharacteristicsseparatedtheSkagerrakstrainsfromtheotherspecieswithinTenacibaculum.Thus,itisconcludedthatthestrainsbelongtoanovelspecieswithinthegenusTenacibaculum,forwhichthenameTenacibaculumskagerrakensesp.nov.isproposed,withstrainD30T(=ATCCBAA-458T=DSM14836T)asthetypestrain.
Evaluationofcellulolyticandhemicellulolyticabilitiesoffungiisolatedfromcoffeeresidueandsawdustcomposts.
Eida,M.F.,Nagaoka,T.,Wasaki,J.&Kouno,K.(2011).MicrobesEnviron,26(3),220-227.
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Thisstudyfocusedontheevaluationofcellulolyticandhemicellulolyticfungiisolatedfromsawdustcompost(SDC)andcoffeeresiduecompost(CRC).Toidentifyfungalisolates,theITSregionoffungalrRNAwasamplifiedandsequenced.Toevaluateenzymeproduction,isolateswereinoculatedontowheatbranagarplates,andenzymeswereextractedandtestedforcellulase,xylanase,β-glucanase,mannanase,andproteaseactivitiesusingdifferentazurinecross-linked(AZCL)substrates.Intotal,18isolatesfromSDCand29isolatesfromCRCwereidentifiedandevaluated.Fourgenera(Aspergillus,Galactomyces,Mucor,andPenicillium)andfivegenera(Aspergillus,Coniochaeta,Fusarium,Penicillium,andTrichoderma/Hypocrea)weredominantinSDCandCRC,respectively.Penicilliumsp.,Trichodermasp.,andAspergillussp.displayedhighcellulolyticandhemicellulolyticactivities,whileMucorisolatesexhibitedthehighestβ-glucanaseandmannanaseactivities.TheenzymeanalysesrevealedthatPenicillium,Aspergillus,andMucorisolatessignificantlycontributedtothedegradationofSDC,whereasPenicillium,Aspergillus,andTrichodermaisolateshadadominantroleinthedegradationofCRC.Notably,isolatesSDCF5(P.crustosum),CRCF6(P.verruculosum),andCRCF2andCRCF16(T.harzianum/H.lixii)displayedhighactivityregardingcelluloseandhemicellulosedegradation,whichindicatesthatthesespeciescouldbebeneficialfortheimprovementofbiodegradationprocessesinvolvinglignocellulosicmaterials.
Patternsoffunctionalenzymeactivityinfungusfarmingambrosiabeetles.
Licht,H.H.D.F.&Biedermann,P.H.W.(2012).FrontiersinZoology,9(1),13.
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Introduction:Inwood-dwellingfungus-farmingweevils,theso-calledambrosiabeetles(Curculionidae:ScolytinaeandPlatypodinae),woodintheexcavatedtunnelsisusedasamediumforcultivatingfungibythecombinedactionofdigginglarvae(whichcreatemorespaceforthefungitogrow)andofadultssowingandpruningthefungus.Thebeetlesareobligatelydependentonthefungusthatprovidesessentialvitamins,aminoacidsandsterols.However,towhatextentmicrobialenzymessupportfungusfarminginambrosiabeetlesisunknown.Herewemeasure(i)13plantcell-walldegradingenzymesinthefungusgardenmicrobialconsortiumoftheambrosiabeetleXyleborinussaxesenii,includingitsprimaryfungalsymbionts,inthreecompartmentsoflaboratorymaintainednests,atdifferenttimepointsaftergalleryfoundationand(ii)fourspecificenzymesthatmaybeeitherinsectormicrobiallyderivedinX.saxeseniiadultandlarvalindividuals.Results:Wediscoveredthattheactivityofcellulasesinambrosiafungusgardensisrelativelysmallcomparedtotheactivitiesofothercellulolyticenzymes.Enzymeactivityinallcompartmentsofthegardenwasmainlydirectedtowardshemicellulosecarbohydratessuchasxylan,glucomannanandcallose.Hemicellulolyticenzymeactivitywithinthebroodchamberincreasedwithgalleryage,whereasirrespectiveoftheageofthegallery,thehighestoverallenzymeactivityweredetectedinthegallerydumpmaterialexpelledbythebeetles.Interestinglyendo-β-1,3(4)-glucanaseactivitycapableofcallosedegradationwasidentifiedinwhole-bodyextractsofbothlarvaeandadultX.saxesenii,whereasendo-β-1,4-xylanaseactivitywasexclusivelydetectedinlarvae.Conclusion:Similartocloselyrelatedfungiassociatedwithbarkbeetlesinphloem,themicrobialsymbiontsofambrosiabeetleshardlydegradecellulose.Instead,theirenzymeactivityisdirectedmainlytowardscomparativelymoreeasilyaccessiblehemicellulosecomponentsoftheray-parenchymacellsinthewoodxylem.Furthermore,thedetectionofxylanolyticenzymesexclusivelyinlarvae(whichfeedonfunguscolonizedwood)andnotinadults(whichfeedonlyinfungi)indicatesthatonlylarvae(pre-)digestplantcellwallstructures.ThisimpliesthatinX.saxeseniiandlikelyalsoinmanyotherambrosiabeetles,adultsandlarvaedonotcompeteforthesamefoodwithintheirnests-incontrast,larvaeincreasecolonyfitnessbyfacilitatingenzymaticwooddegradationandfunguscultivation.
Evolutionarytransitionsinenzymeactivityofantfungusgardens.
DeFineLicht,H.H.,Schiøtt,M.,Mueller,U.G.&Boomsma,J.J.(2010).Evolution,64(7),2055-2069.
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Fungus-growing(attine)antsandtheirfungalsymbiontspassedthroughseveralevolutionarytransitionsduringtheir50millionyearoldevolutionaryhistory.Thebasalattinelineagesoftenshiftedbetweentwomaincultivarclades,whereasthederivedhigher-attinelineagesmaintainedanassociationwithamonophyleticcladeofspecializedsymbionts.Inconjunctionwiththetransitiontospecializedsymbionts,theantsadvancedincolonysizeandsocialcomplexity.Hereweprovideacomparativestudyofthefunctionalspecializationinextracellularenzymeactivitiesinfungusgardensacrosstheattinephylogeny.Weshowthat,relativetosisterclades,gardensofhigher-attineantshaveenhancedactivityofprotein-digestingenzymes,whereasgardensofleaf-cuttingantsalsohaveincreasedactivityofstarch-digestingenzymes.However,theenzymeactivitiesoflower-attinefungusgardensaretargetedprimarilytowardpartialdegradationofplantcellwalls,reflectingaplesiomorphicstateofnondomesticatedfungi.Theenzymeprofilesofthehigher-attineandleaf-cuttinggardensappearparticularlysuitedtodigestfreshplantmaterialsandtoaccessnutrientsfromlivecellswithoutmajorbreakdownofcellwalls.Theadaptivesignificanceofthelower-attinesymbiontshiftsremainsunclear.Oneoftheseshiftswasobligate,butdigestiveadvantagesremainedambiguous,whereastheotherremainedfacultativedespiteprovidinggreaterdigestiveefficiency.
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.
Diversityofmicrobialcarbohydrate-activeenzymesinDanishanaerobicdigestersfedwithwastewatertreatmentsludge.
Wilkens,C.,Busk,P.K.,Pilgaard,B.,Zhang,W.J.,Nielsen,K.L.,Nielsen,P.H.&Lange,L.(2017).BiotechnologyforBiofuels,10(1),158.
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Background:Improvedcarbohydrate-activeenzymes(CAZymes)areneededtofulfillthegoalofproducingfood,feed,fuel,chemicals,andmaterialsfrombiomass.Littleisknownabouthowthediversemicrobialcommunitiesinanaerobicdigesters(ADs)metabolizecarbohydratesorwhichCAZymesthatarepresent,makingtheADsauniquenichetolookforCAZymesthatcanpotentiatetheenzymeblendscurrentlyusedinindustry.Results:EnzymaticassaysshowedthatfunctionalCAZymesweresecretedintotheADenvironmentsinfourfull-scalemesophilicDanishADsfedwithprimaryandsurplussludgefrommunicipalwastewatertreatmentplants.MetagenomesfromtheADswereminedforCAZymeswithHomologytoPeptidePatterns(HotPep).19,335CAZymeswereidentifiedofwhich30%showed50%orloweridentitytoknownproteinsdemonstratingthatADsmakeupapromisingpoolfordiscoveryofnovelCAZymes.Afunctionwasassignedto54%ofallCAZymesidentifiedbyHotPep.Manydifferentα-glucan-actingCAZymeswereidentifiedinthefourmetagenomes,andthemostabundantfamilywasglycosidehydrolasefamily13,whichcontainsα-glucan-actingCAZymes.CellulyticandxylanolyticCAZymeswerealsoabundantinthefourmetagenomes.Thecellulyticenzymeswerelimitedalmosttoendoglucanasesandβ-glucosidases,whichreflectthelargeamountofpartlydegradedcelluloseinthesludge.NodockerindomainswereidentifiedsuggestingthatthecellulyticenzymesintheADsstudiedoperateindependently.OfxylanolyticCAZymes,especiallyxylanasesandβ-xylosidase,butalsoabatteryofaccessoryenzymes,werepresentinthefourADs.Conclusions:OurfindingssuggestthattheADsareagoodplacetolookfornovelplantbiomassdegradingandmodifyingenzymesthatcanpotentiatebiologicalprocessesandprovidebasisforproductionofarangeofadded-valueproductsfrombiorefineries.
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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Chineseliquorisoneoftheworldsbest-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.
