HighpuritydyedandcrosslinkedinsolubleAZCL-Caseinforidentificationofenzymeactivitiesinresearch,microBIOLOGicalenzymeassaysandinvitrodiagnosticanalysis.
Substratefortheassayofproteases.
PurificationandcharacterizationofaneutralserineproteasewithnematicidalactivityfromHirsutellarhossiliensis.
Wang,B.,Wu,W.&Liu,X.(2007).Mycopathologia,163(3),169-176.
LinktoArticle
ReadAbstract
Serineproteaseplaysanimportantroleinfungalinfectiontoinvertebratehosts.Anextracellularprotease(Hnsp)wasdetectedinliquidcultureof
HirsutellarhossiliensisOWVT-1withnematodes(
Panagrellusredivivus)astheuniquenitrogensourceandpurifiedtohomogeneitybyammoniumsulphateprecipitation,anionexchangechromatographyandgelfiltration.Itsmolecularmasswasabout32kDa,andtheoptimalreactionpHvalueandtemperaturewerepH7and40°C,respectively.TheHnspactivitywasstableatpH6–8anddecreasedr
ADIcallyat50°Cfor10min.HnspwashighlysensitivetoinhibitorofPMSFandwelldecomposedthesubstrate
N-succinyl-Ala-Ala-Pro-Phe-
p-nitroanilide,suggestingthatitbelongedtothechymotrypsin/subtilisinofserineproteases.TheN-terminalaminoacidsequenceofHnspwasSVTDQQGADCGLARISHRE,whichshowedhighhomologywithotherserineproteasesfromnematophagousfungi.
ABIlitytokillnematodeanddegradeitscuticle
invitroindicatedthatHnspcouldbeinvolvedintheinfectionofnematode.
EffectofpH,temperatureanddietondigestiveenzymeprofilesinthemudcrab,Scyllaserrata.
Pavasovic,M.,Richardson,N.A.,Anderson,A.J.,Mann,D.&Mather,P.B.(2004).Aquaculture,242(1),641-654.
LinktoArticle
ReadAbstract
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.
Towardsamolecularunderstandingofsymbiontfunction:identificationofafungalgeneforthedegradationofxylaninthefungusgardensofleaf-cuttingants.
Schiøtt,M.,Licht,H.H.D.F.,Lange,L.&Boomsma,J.J.(2008).BMCMicrobiology,8(1),40.
LinktoArticle
ReadAbstract
Background:Leaf-cuttingantsliveinsymbiosiswithafungusthattheyrearforfoodbyprovidingitwithliveplantmaterial.Untilrecentlythefungus"maininferredfunctionwastomakeotherwiseinaccess
IBLecellwalldegradationproductsavailabletotheants,butnewstudieshavesheddoubtonthisidea.Toprovideevidenceforthecellwalldegradingcapacityoftheattineantsymbiont,wedesignedPCRprimersfromconservedregionsofknownxylanasegenes,tobeusedinPCRwithgenomicDNAfromthesymbiontastemplate.Wealsomeasuredxylanase,cellulaseandproteinaseactivitiesinthefungusgardensinordertoinvestigatethedynamicsofdegradationactivities.
Results:Weclonedaxylanasegenefromthemutualisticfungusof
Acromyrmexechinatior,determineditsproteinsequence,andinserteditinayeastexpressionvectortoconfirmitssubstratespecificity.Ourresultsshowthatthefungushasafunctionalxylanasegene.Wealsoshowbylabexperimentsinvivothattheactivityoffungalxylanaseandcellulaseisnotevenlydistributed,butconcentratedinthelowerlayeroffungusgardens,withonlymodestactivityinthemiddlelayerwheregongylidiaareproducedandintermediateactivityinthenewlyestablishedtoplayer.Thisverticaldistributionappearstobenegativelycorrelatedwiththeconcentrationofglucose,whichindicatesadirectlyregulatingroleofglucose,ashasbeenfoundinotherfungiandhasbeenpreviouslysuggestedfortheantfungalsymbiont.
Conclusion:Themutualisticfungusof
Acromyrmexechinatiorhasafunctionalxylanasegeneandisthuspresumablyabletoatleastpartiallydegradethecellwallsofleaves.Thisfindingsupportsasaprotrophicoriginofthefungalsymbiont.Theobserveddistributionofenzymeactivityleadsustoproposethatleaf-substratedegradationinfungusgardensisamulti-stepprocesscomparabletonormalbiodegradationoforganicmatterinsoilecosystems,butwiththecrucialdifferencethatasinglefungalsymbiontrealizesmostofthestepsthatarenormallyprovidedbyaseriesofmicroorganismsthatcolonizefallenleavesinadistinctsuccession.
Influenceofdietaryproteinondigestiveenzymeactivity,growthandtailmusclecompositioninredclawcrayfish,Cheraxquadricarinatus(vonMartens).
Pavasovic,A.,Anderson,A.J.,Mather,P.B.&Richardson,N.A.(2007).AquacultureResearch,38(6),644-652.
LinktoArticle
ReadAbstract
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).
Purification,characterization,andgenecloningofanalkalineserineproteasefromahighlyvirulentstrainofthenematode-endoparasiticfungusHirsutellarhossiliensis.
Wang,B.,Liu,X.,Wu,W.,Liu,X.&Li,S.(2009).MicrobiologicalResearch,164(6),665-673.
LinktoArticle
ReadAbstract
HirsutellarhossiliensisOWVT-1hassubstantialpotentialasabiocontrolagentagainstplant-parasiticnematodes.Serineproteaseshaveemergedasapotentiallyusefulfactorinthenematode–fungusinteractions.Whengrowninliquidculturewiththenematode
Panagrellusredivivusasthesolenitrogensource,anextracellularalkalineprotease(Hasp)wasproducedbytheOWVT-1.ThepurifiedHaspkilledthejuvenilesofthesoybean-cystnematode(
Heteroderaglycines)anddegradedproteinsofthenematodecuticle.ThemolecularmassofHaspwasestimatedtobe33kDa.TheoptimumpHandtemperatureforenzymeactivitywerepH9and75°C.TheaminoacidsequenceobtainedbytheN-terminalsequenceanalysiswasappliedfortheprimerdesigntoisolatethe
HaspCDNAgene,whichconsistsof1170bpopenreadingframe.AnalysisofthecDNAandcorrespondinggenomicsequencerevealedthat
Haspincludedfourexons(279,186,513,and192bp)dividedbythreeintrons(65,99,and93bp).Southernblottingshowedthat
Haspwasasingle-copygeneinthegenome.Thededucedaminoacidsequencewasverysimilartootherserineproteasesofendoparasiticandegg-parasiticfungiofnematodesandofentomopathogenicfungibutwaslesssimilartotheserineproteasesofnematode-trappingfungi.Inaphylogeneticanalysisoftheaminoacidsequencesofserineproteases,theserineproteaseof
H.rhossiliensisOWVT-1clusteredwiththeserineproteasesofparasitesofnematodeeggsratherthanwiththoseofthetrappingfungi.
AnovelantifungalPseudomonasfluorescensisolatedfrompotatosoilsinGreenland.
Michelsen,C.F.&Stougaard,P.(2011).CurrentMicrobiology,62(4),1185-1192.
LinktoArticle
ReadAbstract
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.
Tenacibaculumskagerrakensesp.nov.,amarinebacteriumisolatedfromthepelagiczoneinSkagerrak,Denmark.
Frette,L.,Jørgensen,N.O.G.,Irming,H.&Kroer,N.(2004).InternationalJournalofSystematicandEvolutionaryMicrobiology,54(2),519-524.
LinktoArticle
ReadAbstract
AnumberofbacteriawereisolatedfromseawaterinSkagerrak,Denmark,at30mdepth.Twooftheisolates,strainsD28andD30
T,belongedtothe
Flavobacteriaceaewithinthe
Cytophaga–
Flavobacterium–
Bacteroidesgroup.Sequencingof16SrRNAgenesofthetwostrainsindicatedstronglythattheybelongedtothegenus
Tenacibaculumandthattheyshowedgreatestsimilaritytothespecies
Tenacibaculumamylolyticumand
Tenacibaculummesophilum.DNA–DNAhybridizationvalues,DNAbasecompositionandphenotypiccharacteristicsseparatedtheSkagerrakstrainsfromtheotherspecieswithin
Tenacibaculum.Thus,itisconcludedthatthestrainsbelongtoanovelspecieswithinthegenus
Tenacibaculum,forwhichthename
Tenacibaculumskagerrakensesp.nov.isproposed,withstrainD30
T(=
ATCCBAA-458
T=DSM14836
T)asthetypestrain.
Effectsofsolubledietarycelluloseonspecificgrowthrate,survivalanddigestiveenzymeactivitiesinthreefreshwatercrayfish(Cherax)species.
Dammannagoda,L.K.,Pavasovic,A.,Hurwood,D.A.&Mather,P.B.(2015).AquacultureResearch,46(3),626-636.
LinktoArticle
ReadAbstract
Thecurrentstudyevaluatedtheeffectofsolubledietarycelluloseongrowth,survivalanddigestiveenzymeactivityinthreeendemic,Australianfreshwatercrayfishspecies(redclaw:Cheraxquadricarinatus,marron:C.tenuimanus,yabby:C.destructor).Separateindividualfeedingtrialswereconductedforlate-stagejuvenilesfromeachspeciesinanautomatedrecirculatingfreshwater,culturesystem.Animalswerefedeitheratestdiet(TD)thatcontained20%solublecelluloseorareferencediet(RD)substitutedwiththesameamountofcornstarch,overa12-weekperiod.RedclawfedwithRDshowedsignificantlyhigher(P<0.05)=""specific=""growth=""rates=""(sgr)=""compared=""with=""animals=""fed=""the=""td,=""while=""sgr=""of=""marron=""and=""yabby=""fed=""the=""two=""diets=""were=""not=""significantly=""different.=""expressed=""cellulase=""activity=""levels=""in=""redclaw=""were=""not=""significantly=""different=""between=""diets.=""marron=""and=""yabby=""showed=""significantly=""higher=""cellulase=""activity=""when=""fed=""the=""rd="">P<0.05).=""amylase=""and=""protease=""activity=""in=""all=""three=""species=""were=""significantly=""higher=""in=""the=""animals=""fed=""with=""rd="">P<0.05).=""these=""results=""indicate=""that=""test=""animals=""of=""all=""three=""species=""appear=""to=""utilize=""starch=""more=""efficiently=""than=""soluble=""dietary=""cellulose=""in=""their=""diet.=""the=""inclusion=""of=""20%=""soluble=""cellulose=""in=""diets=""did=""not=""appear,=""however,=""to=""have=""a=""significant=""negative=""effect=""on=""growth=""rates.="">
Taxonomicandfunctionaldiversityofpseudomonadsisolatedfromtherootsoffield‐growncanola.
Misko,A.L.&Germida,J.J.(2002).FEMSMicrobiologyEcology,42(3),399-407.
LinktoArticle
ReadAbstract
Amongthemostimportantrhizospherebacteriaarethepseudomonads,whichareaggressivecolonizersandutilizeawiderangeofsubstratesascarbonsources.Theobjectiveofthisstudywastodetermineifthetaxonomicormetabolicdiversityofpseudomonadsdifferedamongfield-growncanolacultivars.Bacteria(n=2257)wereisolatedfromtherhizosphereandrootinteriorofsixcultivarsoffield-growncanola,includingthreetransgenicvarieties.Thebacteriawereidentifiedbyfattyacidmethylester(FAME)analysis,andabout35%wereidentifiedasPseudomonasspecies.ThemostabundantspecieswerePseudomonasputidaandPseudomonaschlororaphis.DendrogramsbasedonFAMEanalysisrevealedthatmanypseudomonadstrainswerefoundinallofthecanolacultivars.Pseudomonadsofthesamestrainwerefoundinboththerhizosphereandtherootinteriorofcanolaplants,suggestingthatendophyticbacteriawereasubsetoftherhizospherecommunity.Becausemetabolicprofilingprovidesmoreusefulinformationthantaxonomy,P.putidaandP.chlororaphisisolateswerecharacterizedfortheirabilitytoutilizecarbonsubstratesandproduceseveralenzymes.Bacteriaisolatedfromdifferentplantcultivarshaddifferentcarbonutilizationprofiles,butwhenonlycarbonsubstratesfoundinrootexudateswereanalyzed,thecultivareffectwaslesspronounced.ThesecharacterizationsalsodemonstratedthatbacteriathatweredeterminedbyFAMEtobethesamestrainweremetabolicallydifferent,suggestingfunctionalredundancyamongPseudomonasisolates.Theresultsofthisstudysuggestthatpseudomonadswerefunctionallydiverse.Theydifferedintheirmetabolicpotentialamongthecanolacultivarsfromwhichtheywereisolated.Becausebacteriacapableofusingmanysubstratescaneffectivelyadapttonewenvironments,theseresultshaveimplicationsfortheuseofpseudomonadsasbiofertilizers,biologicalcontrolagentsandplantgrowth-promotingbacteriaincanola.
Cold-adaptationandalkalinehydrolyticproprietiesofthepolarstreptomycetespredictiononplateassay,basedoninsolublechromogenicsubstrateswithazurinecross-linked.
Cotarlet,M.,Negoită,T.,Bahrim,G.&Stougaard,P.(2008).AnnalsoftheUniversityDunareadeJosofGalati.FascicleVI--FoodTechnology,1(31).
LinktoArticle
ReadAbstract
Asemi-qualitativescreeningbasedonproteaseandamylaseactivityevaluationinabasalagarmediumsupplementedwithinsolublechromogenicsubstratesbasedonAZCL(Azurine-Crosslinkedwithamyloseorcasein)usingaplateassaywasusedforselectingthepolarstreptomycetesabletoproducecoldactivesandalkalineamylasesandproteases.ThistechniqueprovidesaspecificandrapidsimultaneousdetectionofhighactivehydrolaseproducingstrainsbasedonthevisiblesolubilizationofsmallparticlesofAZCLandtheformationofhaloesonplates.Ithasagreatpotentialinincreasingtheefficacyofscreeningstreptomycetesabletoproducehydrolyticenzymes.Thisstudyrevealedthepotentialoftheselectedstreptomycetesisolatedfrompolarsoilstobiosynthesizeamylasesandproteasescold-adaptedatlowtemperatures(from5to20°C)andalkalinepHvalues(8to9).
Characterizationofanewoxidant-stableserineproteaseisolatedbyfunctionalmetagenomics.
Biver,S.,Portetelle,D.&Vandenbol,M.(2013).SpringerPlus,2(1),410.
LinktoArticle
ReadAbstract
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.
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.
LinktoArticle
ReadAbstract
Chineseliquorisoneoftheworld"sbest-knowndistilledspiritsandisthelargestspiritcategorybysales.Theuniqueandtraditionalsolid-statefermentationtechnologyusedtoproduceChineseliquorhasbeenincontinuoususeforseveralthousandyears.Thediverseanddynamicmicrobialcommunityinaliquorstarteristhemaincontributortoliquorbrewing.However,littleisknownabouttheecologicaldistributionandfunctionalimportanceofthesecommunitymembers.Inthisstudy,metatranscriptomicswasusedtocomprehensivelyexploretheactivemicrobialcommunitymembersandkeytranscriptswithsignificantfunctionsintheliquorstarterproductionprocess.Fungiwerefoundtobethemostabundantandactivecommunitymembers.Atotalof932carbohydrate-activeenzymes,includinghighlyexpressedauxiliaryactivityfamily9and10proteins,wereidentifiedat62°Cunderaerobicconditions.Somepotential
Thermostableenzymeswereidentifiedat50,62,and25°C(maturestage).Increasedcontentandoverexpressedkeyenzymesinvolvedinglycolysisandstarch,pyruvateandethanolmetabolismweredetectedat50and62°C.Thekeyenzymesofthecitratecyclewereup-regulatedat62°C,andtheirabundantderivativesarecrucialforflavorgeneration.Here,themetabolismandfunctionalenzymesoftheactivemicrobialcommunitiesinNFliquorstarterwerestudied,whichcouldpavethewaytoinitiateimprovementsinliquorqualityandtodiscovermicrobesthatproducenovelenzymesorhigh-valueaddedproducts.
