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

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.

CommercialfarmingofthemudcrabScyllaserrataisasignificantindustrythroughoutSouthEastAsia.Thelimitedscientificknowledgeofmudcrabnutritionalrequirementsanddigestiveprocesses,however,isrecognisedasamajorconstrainttothefuturegrowthofthisindustry.Tobetterunderstandthemechanismsofdigestioninthemudcrabwehaveanalysedthediversityofdigestiveenzymesfromthemidgut(MG)gland.Significantprotease,amylase,cellulaseandxylanaseactivitiesweredetectedinsolubleextractsfromthisorgan.Temperatureprofilesforallenzymeswerebasicallysimilarwithoptimalactivitiesobservedat50°C.ExaminationofpHtolerancesrevealedoptimalactivitiesforproteaseandamylaseatpH7whilemaximumcellulaseandxylanaseactivitieswereobservedatpH5.5.Underoptimumconditions,proteaseandamylaseactivitieswereapproximatelytwoordersofmagnitudegreaterthanthoseseenforeithercellulaseorxylanase.Interestingly,MGextractswereabletoliberateglucosefromeitherstarchorcarboxymethyl(CM)-cellulosesuggestingthatarangeofcarbohydratesmaybeutilisedasenergysources.Theeffectsofdietarycarbohydratesonfeeddigestibility,digestiveenzymelevelsandgrowthwerealsostudiedbyinclusionofadditionalstarchorCM-celluloseattheexpenseofcaseininformulateddiets.Itwasshownthatamylase,cellulaseandxylanaseactivitiesinextractsfromthemidgutglandwerehighestinmudcrabsfeddietscontaining47%carbohydrate.Basedonthesefindings,wesuggestthattheABIlityofthemudcrabtomodulatedigestiveenzymeactivitiesmayrepresentamechanismtomaximiseaccesstoessentialnutrientswhenthedietaryprofilechanges.

Background:Leaf-cuttingantsliveinsymbiosiswithafungusthattheyrearforfoodbyprovidingitwithliveplantmaterial.Untilrecentlythefungus"maininferredfunctionwastomakeotherwiseinaccessIBLecellwalldegradationproductsavailabletotheants,butnewstudieshavesheddoubtonthisidea.Toprovideevidenceforthecellwalldegradingcapacityoftheattineantsymbiont,wedesignedPCRprimersfromconservedregionsofknownxylanasegenes,tobeusedinPCRwithgenomicDNAfromthesymbiontastemplate.Wealsomeasuredxylanase,cellulaseandproteinaseactivitiesinthefungusgardensinordertoinvestigatethedynamicsofdegradationactivities.Results:WeclonedaxylanasegenefromthemutualisticfungusofAcromyrmexechinatior,determineditsproteinsequence,andinserteditinayeastexpressionvectortoconfirmitssubstratespecificity.Ourresultsshowthatthefungushasafunctionalxylanasegene.Wealsoshowbylabexperimentsinvivothattheactivityoffungalxylanaseandcellulaseisnotevenlydistributed,butconcentratedinthelowerlayeroffungusgardens,withonlymodestactivityinthemiddlelayerwheregongylidiaareproducedandintermediateactivityinthenewlyestablishedtoplayer.Thisverticaldistributionappearstobenegativelycorrelatedwiththeconcentrationofglucose,whichindicatesadirectlyregulatingroleofglucose,ashasbeenfoundinotherfungiandhasbeenpreviouslysuggestedfortheantfungalsymbiont.Conclusion:ThemutualisticfungusofAcromyrmexechinatiorhasafunctionalxylanasegeneandisthuspresumablyabletoatleastpartiallydegradethecellwallsofleaves.Thisfindingsupportsasaprotrophicoriginofthefungalsymbiont.Theobserveddistributionofenzymeactivityleadsustoproposethatleaf-substratedegradationinfungusgardensisamulti-stepprocesscomparabletonormalbiodegradationoforganicmatterinsoilecosystems,butwiththecrucialdifferencethatasinglefungalsymbiontrealizesmostofthestepsthatarenormallyprovidedbyaseriesofmicroorganismsthatcolonizefallenleavesinadistinctsuccession.

Thisstudywasconductedtoevaluatetheeffectsofdietaryproteinondigestiveenzymeprofiles,growthandtailmusclecompositioninthefreshwaterredclawcrayfish,Cheraxquadricarinatus.Crayfishwerefedfivedietsthatconsistedofacommercialcrayfishpelletandexperimentaldietscontaining13%,18%,25%or32%crudeprotein(CP),foraperiodof12weeks.Analysisofdigestiveenzymeprofilesfromthemidgutgland(MG)revealedapositivecorrelationbetweenprotease,amylaseandcellulaseactivitiesanddietaryproteinlevel.Foralltreatments,carbohydraseactivitylevels(cellulaseandamylase)weresignificantlyhigherthanthosedetectedforprotease.Asdietaryproteinwaselevated,therewasageneralincreaseinspecificgrowthrate(SGR),withthehighestSGR(0.58±0.06)valuesobservedincrayfishfedthedietcontaining25%CP.Feedconversionratio(FCR)rangedbetween5.84and6.97anddidnotdiffersignificantlyamongthetreatmentgroupsincludingthereferencediet,withtheexceptionofthelow-proteindiet(13%CP)whichshowedanFCRof9.31.Finally,regressionanalysisrevealedastrongpositivecorrelationbetweenthelevelofdietaryproteinandCPcontentinthetailmuscle(P=0.004;r²)=0.99).

ArhizobacteriumwithhighantifungalactivitywasisolatedfromapotatofieldatInneruulalik,SouthGreenland.PhylogeneticanalysisbasedonmultilocussequencetypingshowedthatthebacteriumwasaffiliatedwithstrainsofPseudomonasfluorescens.Thebacterium,denotedasPseudomonasfluorescensIn5,inhibitedinvitroabroadrangeofphytopathogenicfungi,andtheantifungalactivityincreasedwithdecreasingtemperature.MicrocosmexperimentsdemonstratedthatP.fluorescensIn5protectedtomatoseedlingsfromRhizoctoniasolani.TransposonmutagenesisshowedthatthemajorcausefortheantifungalactivityofP.fluorescensIn5wasanovelnon-ribosomalpeptidesynthase(NRPS)gene.Inaddition,transposonmutagenesisshowedthatP.fluorescensIn5alsocontainedaputativequinoproteinglucosedehydrogenasegene,whichwasinvolvedingrowthinhibitionofphytopathogenicfungi.AlthoughP.fluorescensIn5containedthecapacitytosynthesizehydrogencyanide,β-1,3-glucanase,protease,andchitinase,thesedidnotseemtoplayaroleintheinvitroandmicrocosmantifungalassays.

Anovelserineproteasegene,SBcas3.3,wasidentifiedbyfunctionalscreeningofaforest-soilmetagenomiclibraryonagarplatessupplementedwithAZCL-casein.OverproductioninEscherichiacolirevealedthattheenzymeisproducedasa770-amino-acidprecursorwhichisprocessedtoamatureproteaseof~55kDa.Thelatterwaspurifiedbyaffinitychromatographyforcharacterizationwiththeazocaseinsubstrate.TheenzymeprovedtobeanalkalineproteaseshowingmaximalactivitybetweenpH9and10andat50°C.Treatmentwiththechelatingagentethylenediaminetetraaceticacidirreversiblydenaturedtheprotease,whosestabilitywasfoundtodependstrictlyoncalciumions.Theenzymeappearedrelativelyresistanttodenaturingandreducingagents,anditsactivitywasenhancedinthepresenceof10ml/lnonionicdetergent(Tween20,Tween80,orTritonX-100).Moreover,SBcas3.3displayedoxidantstability,afeatureparticularlysoughtinthedetergentandbleachingindustries.SBcas3.3wasactivatedbyhydrogenperoxideatconcentrationsupto10g/landitstillretained30%ofactivityin50g/lH₂O₂.

Cellulasesbelongtothelargefamilyofglycosylhydrolases(GHs)andareproducedbyavarietyofbacteriaandfungi.Theseextracellularenzymesactasendoglucanases(EGs),cellobiohydrolasesorβ-glucosidases.Inthispaper,wedescribemolecularscreeningforEGsfromtheGHfamily12.Usingthreehomologoussequenceboxesdeducedfromfivepreviouslyknownmembersofthefamily,weanalysed22cellulase-producingfungalstrainsobtainedfromadiverseareaofthefungalkingdom.Polymerasechainreactionsusingdegenerateprimersdesignedtothehomologousproteinboxeswereusedtoidentifythefamily12homologues.Severalfungishowedthepresenceofmultipleversionsofthegene,whileaminoacidsequenceanalysisshoweddiversityin15novelmembersofthefamily,rangingfrom26%to96%similarity.Oursequenceanalysisshowsthatthephylogenetictreeoffamily12EGscanbedividedintofoursubfamilies:12-1(fungalgroupI),12-2(fungalgroupII),12-3(StreptomycesgroupinwhichRhodothermusmarinusfits)and12-4(Thermophilesgroup).Erwiniacarotovoramayformanewsubgroup.

ThisworkdescribestheprofileoffiveproteasesandfourcarbohydrasesfromthecrustaceandecapodsMacrobrachiumaustraliense(Holthuis),Scyllaserrata(Forskal),Portunuspelagicus(Linnaeus),Penaeusesculentus,Penaeusplebejus(Hess)andMetapenaeusbennettae(Racck&Dall),feedinginthenaturalhabitat,inordertoprovideanindicationoftheirdigestivecapabilities.Theresultsraisedthefollowingpoints.First,speciesfromeachfamilyshowedaparticularsuiteofdigestiveenzymes.Second,theactivityofcellulasefromM.australiensisandS.serrata,usingAZCL-HEcelluloseasthesubstrate,wasaround90%higherthanthatobservedwithAZO-CMcellulose.However,forP.pelagicusandP.esculentus,theenzymeactivitywasbetterwithAZO-CMcellulose.Third,M.australiensedisplayedthehighestratioofamylasetoproteaseactivity.Incontrast,Portunidaespecies,P.pelagicusandS.serratashowedthelowestratios.Fourth,comparisonofthelaminarinaseactivityofM.bennettaeandP.esculentusinOctober(Spring)andDecember(earlySummer)showedasignificantdecreaseinDecember.Finally,thewidedistributionofdigestiveenzymesinthesecrustaceansmayreflectdifferentfeedinghabitsandhabitats.

VariantsoftheThermoascusaurantiacusEg1enzymewithhighercatalyticefficiencythanwild-typewereobtainedviasite-directedmutagenesis.Usingarationalmutagenesisapproachbasedonstructuralbioinformaticsandevolutionaryanalysis,twopositions(F16SandY95F)wereidentifiedasprioritysitesformutagenesis.ThemutantandparentenzymeswereexpressedandsecretedfromPichiapastorisandthesinglesitemutantsF16SandY95Fshowed1.7-and4.0-foldincreasesinK_catand1.5-and2.5-foldimprovementsinhydrolyticactivityoncellulosicsubstrates,respectively,whilemaintainingthermostability.Similartotheparentenzyme,thetwovariantswereactivebetweenpH4.0and8.0andshowedoptimalactivityattemperature70°CatpH5.0.Thepurifiedenzymeswereactiveat50°Cforover12 handretainedatleast80%ofinitialactivityfor2 hat70°C.Incontrasttotheimprovedhydrolysisseenwiththesinglemutationenzymes,noimprovementwasobservedwithathirdvariantcarryingacombinationofbothmutations,whichinsteadshoweda60%reductionincatalyticefficiency.Thisworkfurtherdemonstratesthatnon-catalyticaminoacidresiduescanbeengineeredtoenhancecatalyticefficiencyinpretreatmentenzymesofinterest.

Cellulaseandxylanasedigestiveenzymeactivitieswerecomparedinfourfreshwatercrayfish(GenusCherax)andthreemarineprawn(GenusPenaeus)species.TemperatureandpHprofilesforcellulase(endoglucanase)werefoundtobeverysimilarinallspecies,withmaximumactivityoccurringat60°CandpH5.0.TemperatureandpHprofilesforxylanase(endoxylanase)werealsoverysimilarinallcrayfishspecies,withmaximumactivityoccurringat50°CandpH5.0.Xylanaseactivitywasnotdetectedinthethreeprawnspeciesexamined.Inaddition,invitrostudiesshowedthatmostspecieswereabletoliberateglucosefromcarboxymethylcellulose,indicatingthatcellulosesubstratescanbeasourceofenergyforbothcrayfishandprawnspecies.

Synergybetweencellulolyticenzymesisessentialinbothnaturalandindustrialbreakdownofbiomass.Inadditiontosynergybetweenendo-andexo-lyticenzymes,alesserknownbutequallyconspicuoussynergyoccursamongexo-acting,processivecellobiohydrolases(CBHs)suchasCel7AandCel6AfromHypocreajecorina.Westudiedthissystemusingmicrocrystallinecelluloseassubstrateandfoundadegreeofsynergybetween1.3and2.2dependingontheexperimentalconditions.Synergybetweenenzymevariantswithoutthecarbohydratebindingmodule(CBM)anditslinkerwasstronglyreducedcomparedtothewildtypes.Oneplausibleinterpretationofthisisthatexo-exosynergydependsonthetargetingroleoftheCBM.Manyearlierworkshaveproposedthatexo-exosynergywascausedbyanauxiliaryendo-lyticactivityofCel6A.However,biochemicaldatafromdifferentassayssuggestedthattheendo-lyticactivityofbothCel6AandCel7Awere10³–10⁴timeslowerthanthecommonendoglucanase,Cel7B,fromthesameorganism.Moreover,theendo-lyticactivityofCel7Awas2–3-foldhigherthanforCel6A,andwesuggestthatendo-likeactivityofCel6Acannotbethemaincausefortheobservedsynergy.Rather,wesuggesttheexo-exosynergyfoundheredependsondifferentspecificitiesoftheenzymespossiblygovernedbytheirCBMs. 

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.

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.