Megazyme/AZCL-Curdlan/I-AZCUR/3 grams

Apsychrophilic,aerobicbacteriumdesignatedA2i^TwasisolatedfrommarinesedimentrecoveredfromshallowwaterssurroundingAdelaideIsland,Antarctica(67°34"S,68°07"W).Theorganismexhibitedxylanolyticandlaminarinolyticactivityandwashalotolerant.Basiccharacterizationshowedthatitwasgram-negative,non-motile,yellow-pigmented(β,β-carotene-3,3"-diol)andpositiveforoxidaseandcatalasesynthesis.Analysisofthe16SrDNAsequencesuggeststhattheorganismbelongstotheFlexibacter-Cytophaga-Bacteroidesphylum.Onthebasisofits16SrDNAsequence,thebacteriumis96.8%similartoFlavobacteriumcolumnareATCC43622-itsclosestrelation.ThegenomicDNAG+Ccontentwas35mol%.Growthonxylanoccursoptimallyat15°C,thoughgrowthalsooccursat0°C,andthedoublingtimesare9.6and34.8h,respectively.Themaximumgrowthtemperatureonxylanisat24°C.Thebacteriumisaneutrophile,growingacrossthepHrange5.6-8.4andhavinganoptimumatpH7.5.Analysisofthe16SrDNAsequence,togetherwithphenotypiccharacterization,suggeststhattheorganismisamemberofthegenusFlavobacterium.DNA-DNAhybridizationexperimentshaveshownthatitisanovelspecies;itisproposed,therefore,thattheorganismbedesignatedasthetypestrainofFlavobacteriumfrigidariumsp.nov.(=ATCC700810^T=NCIMB13737^T).

Wedescribetheidentificationandexpressioncloningoftwonovelenzymes,aβ-glucanaseandanasparticprotease,secretedfromthebasidiomycetousyeastPhaffiarhodozyma.ACDNAlibraryfromP.rhodozymaCBS6938wasconstructed,andfull-lengthcDNAencodinganendo-1,3(4)-β-glucanase(bg1)andanasparticprotease(pr1)wereclonedbyexpressioncloninginSaccharomycescerevisiaeW3124.Thebg1cDNAencodesa424-residueprecursorproteinwithaputativesignalpeptide.Thepr1cDNAencodesa405-residueprepropolypeptidewithan81-residueleaderpeptide.Theasparticproteasewaspurifiedandcharacterized.Ithasamolecularmassof36 kDa,anisoelectricpointofpH7.5,apHactivityoptimumat4.0–6.0,andatemperatureactivityoptimumaround40°C.Bothenzymesshowonlylowsequenceidentitytootherknownenzymes.

ThediversityofculturablebacteriaassociatedwithseaicefromfourpermanentlycoldfjordsofSpitzbergen,ArcticOcean,wasinvestigated.Atotalof116psychrophilicandpsychrotolerantstrainswereisolatedunderaerobicconditionsat4°C.TheisolatesweregroupedusingamplifiedrDNArestrictionanalysisfingerprintingandidentifiedbypartialsequencingof16SrRNAgene.Thebacterialisolatesfellinfivephylogeneticgroups:subclassesαandγofProteobacteria,theBacillus–Clostridiumgroup,theorderActinomycetales,andtheCytophaga–Flexibacter–Bacteroides(CFB)phylum.Over70%oftheisolateswereaffiliatedwiththeProteobacteriaγsubclass.Basedonphylogeneticanalysis(<98% sequence="" similarity),="" over="" 40%="" of="" arctic="" isolates="" represent="" potentially="" novel="" species="" or="" genera.="" most="" of="" the="" isolates="" were="" psychrotolerant="" and="" grew="" optimally="" between="" 20="" and="" 25°c.="" only="" a="" few="" strains="" were="" psychrophilic,="" with="" an="" optimal="" growth="" at="" 10–15°c.="" the="" majority="" of="" the="" bacterial="" strains="" were="" able="" to="" secrete="" a="" broad="" range="" of="" cold-active="" hydrolytic="" enzymes="" into="" the="" medium="" at="" a="" cultivation="" temperature="" of="" 4°c.="" the="" isolates="" that="" are="" able="" to="" degrade="" proteins="" (skim="" milk,="" casein),="" lipids="" (olive="" oil),="" and="" polysaccharides="" (starch,="" pectin)="" account="" for,="" respectively,="" 56,="" 31,="" and="" 21%="" of="" sea-ice="" and="" seawater="" strains.="" the="" temperature="" dependences="" for="" enzyme="" production="" during="" growth="" and="" enzymatic="" activity="" were="" determined="" for="" two="" selected="" enzymes,="" α-amylase="" and="" β-galactosidase.="" interestingly,="" high="" levels="" of="" enzyme="" productions="" were="" measured="" at="" growth="" temperatures="" between="" 4="" and="" 10°c,="" and="" almost="" no="" production="" was="" detected="" at="" higher="" temperatures="" (20–30°c).="" catalytic="" activity="" was="" detected="" even="" below="" the="" freezing="" point="" of="" water="" (at="" −5°c),="" demonstrating="" the="" unique="" properties="" of="" these="" enzymes.="">

Thediscoveryofnew,highlyactive,biomass-degrADIngenzymesisimportanttothedevelopmentofasustainablebiofuelsindustry.Dictyoglomusturgidum,aThermophilic,anaerobiceubacteriumthatfermentscelluloseandproducesethanolandhydrogen,waschosenasacandidatetoscreenfornovelenzymes.Anovelthermostableendoglucanase,CelA,wasidentifiedandpurifiedduringscreeningofashotgunlibraryofDictyoglomusturgidumandsubsequentlysubclonedandexpressedinE.coli.ThecelAgenecodingfora312aminoacidproteinshowedlowhomologytoproteinsoutsidethegenusDictoglomiandlackedanapparentsignalpeptide.CelAhadabroadsubstraterange,possessingbothendoandexoactivityonsolubleandinsolubleβ-(1,4)-linkedglucose-containingsubstratesaswellasendoactivityonsolubleandinsolubleβ-(1,4)-linkedmannosecontainingsubstrates.ThespecificactivityofCelAwas226U/mgusingβ-glucan,66U/mgusingglucomannan,and63U/mgusingCMCassubstrates.Thehightemperatureoptimumof70°Cto80°Candwidesubstraterangeoftheenzymemightmakeitanexcellenttoolforbiomassdegradationathightemperature.

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.

ThenucleotidesequenceoftheβgIII_Agene,encodingtheextracellularβ-1,3-glucanaseII_A(βgIII_A)oftheyeast-lyticactinomyceteOerskoviaxanthineolyticaLLG109,wasdetermined.SequencecomparisonshowsthattheβgIII_Aenzymehasover80%identitytotheβgIIIisoenzyme,anendo-β-1,3-glucanasehavinglowyeast-lyticactivitysecretedbythesamebacterium.TheβgIII_Aenzymelacksaglucan-ormannan-bindingdomain,suchasthoseobservedinβ-1,3-glucanasesandproteaseshavinghighyeast/fungus-lyticactivity.Itcanbeincludedintheglycosylhydrolasefamily16.GenefusionexpressioninBacillussubtilisDN1885followedbypreliminarycharacterizationoftherecombinantgeneproductindicatesthatβgIII_AhasapIof3.8to4.0andisactiveonbothlaminarinandcurdlan,havinganacidoptimumpHactivity(ca.4.0).

Anovelaerobic,psychrotolerantmarinebacteriumwasisolatedat4°CfromseawatersamplescollectedfromSpitzbergenintheArctic.Thestrainwasapolar-flagellated,Gram-negativebacteriumthatgrewoptimallyat10–15°CandpH7–8inmediacontaining2–3 %NaCl(w/v),usingvariouscarbohydratesandorganicacidsassubstrates.Themainfattyacidcomponentsincluded16 : 0(12.7 %oftotalfattyacids),straight-chainsaturatedfattyacidmethylester(FAME)and16 : 1ω7c(40.2 %)monounsaturatedFAME.Phylogeneticanalysisrevealedacloserelationship(99 %16SrRNAgenesequencesimilarity)betweenthenovelisolateandPseudoalteromonaselyakoviiKMM162^TandsomeotherspeciesofthegenusPseudoalteromonas.TheDNAG+Ccontentofthenovelstrainwas39mol%.DNA–DNAhybridizationshowedonly47.6 %DNA–DNArelatednesswithP.elyakoviiKMM162^T,44.2 %withPseudoalteromonasdistinctaKMM638^Tand22.6 %withPseudoalteromonasnigrifaciensNCIMB8614^TBasedonphylogeneticandphenotypiccharacteristics,thisisolaterepresentsanovelspeciesofthegenusPseudoalteromonasforwhichthenamePseudoalteromonasarcticaisproposed;thetypestrainisA37-1-2^T(=LMG23753^T=DSM18437^T).

ThegenomeofDictyoglomusturgidumwassequencedandanalyzedforcarbohydrases.Thebroadrangeofcarbohydratesubstrateutilizationisreflectedinthehighnumberofglycosylhydrolases,54,andthehighpercentageofCAZymespresentinthegenome,3.09%ofitstotalgenes.ScreeningarandomclonelibrarygeneratedfromD.turgidumresultedinthediscoveryoffivenovelbiomass-degradingenzymeswithlowhomologytoknownmolecules.Wholegenomesequencingoftheorganismfollowedbybioinformatics-directedamplificationofselectedgenesresultedintherecoveryofsevenadditionalnovelenzymemolecules.Basedontheanalysisofthegenome,D.turgidumdoesnotappeartodegradecelluloseusingeitherconventionalsolubleenzymesoracellulosomaldegradationsystem.Thetypesandquantitiesofglycosylhydrolasesandcarbohydrate-bindingmodulespresentinthegenomesuggestthatD.turgidumdegradescelluloseviaamechanismsimilartothatusedbyCytophagahutchinsoniiandFibrobactersuccinogenes.

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.

PenicilliumochrochloronBiourge,whichwasisolatedfromrottensweetpotato,canproduceplantcellwalldegradingenzymes(PCWDEs)withhighviscosityreducingcapABIlityforethanolproductionusingfreshsweetpotatotubersasfeedstock.Theenzymepreparationwascharacterizedbyabroadenzymespectrumincluding13kindsofenzymeswiththeactivitytohydrolyzecellulose,hemicellulose,pectin,starchandprotein.Themaximumviscosityreducingcapabilitywasobservedwhentheenzymepreparationwasobtainedafterfivedaysfermentationusing20g/Lcorncobassolecarbonsource,4.5g/LNH₄NO₃assolenitrogensource,andaninitialmediumpHof6.5.Thesweetpotatomashtreatedwiththeenzymepreparationexhibitedmuchhigherfermentationefficiency(92.58%)comparedwithcommercialcellulase(88.06%)andcontrol(83.5%).Theenzymeproductionwasthenscaleduptothe0.5,5,and100L,andtheviscosityreducingrateswerefoundtobe85%,90%,and91%,respectively.Thus,P.ochrochloronBiourgedisplayspotentialviscosityreducingcapabilityforethanolproduction.

Theeffectofinitialsolidandmoisturecontents,temperatureandtimeofincubationontheproductionofpolygalacturonase,phytase,acidphosphatase,xylanaseandβ-glucanasebyAspergillusniger377-4duringsolidstatefermentationwasstudied.Parametersofenzymesynthesiswereoptimizedusingstatisticalexperimentaldesigns.Itwasshownthatthecapacityofstraintosynthesizetheaforementionedenzymescouldbemodifiedwithinawiderangebycultureparametersselection.Theoptimalpolygalacturonaseproductionefficiencywasachievedwiththeinitialmediummassof19.9gandhumidityof59.9%,after77.7hofincubationat28.9°C.Thebestcombinationofcultureparametersforphytasesynthesiswas:initialmediummass19.9g,moistures50%,temperature33°Candincubationtime83.9h.Thehighestactivityofacidphosphatasewasobtainedafter81.3hofincubationat27°C,withinitialsubstratemassof17.8gandmoistnesscontentof60%.Theinitialsolidandmoisturecontentstosynthesizexylanasewere19.9gand50%,respectively,withincubationtimeof73hat29.6°C.Thehighestefficiencyofβ-glucanasebiosynthesiswasobtainedwhenA.niger377-4wascultivatedfor80.4hat27°Conainitialmediummassof20gandinitiallevelofmoistness59.9%.

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.

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.

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.

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.