HighpuritydyedandcrosslinkedinsolubleAZCL-Amyloseforidentificationofenzymeactivitiesinresearch,microbiologicalenzymeassaysandinvitrodiagnosticanalysis.
Dyedandcrosslinkedamylose.Substratefortheassayofα-amylase.
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.
Purificationandcharacterisationoftwoextremelyhalotolerantxylanasesfromanovelhalophilicbacterium.
Wejse,P.L.,Ingvorsen,K.&Mortensen,K.K.(2003).Extremophiles,7(5),423-431.
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Thepresentworkreportsforthefirsttimethepurificationandcharacterisationoftwoextremelyhalotolerantendo-xylanasesfromanovelhalophilicbacterium,strainCL8.Purificationofthetwoxylanases,Xyl1and2,wasachievedbyanionexchangeandhydrophobicinteractionchromatography.Theenzymeshadrelativemolecularmassesof43kDaand62kDaandpIof5.0and3.4respectively.StimulationofactivitybyCa+2,Mn+2,Mg+2,Ba+2,Li+2,NaN3,andisopropanolwasobserved.TheKmandVmaxvaluesdeterminedforXyl1with4-O-methyl-D-glucuronoxylanare5mg/mland125,000nkat/mgrespectively.ThecorrespondingvaluesforXyl2were1mg/mland143,000nkat/mgprotein.Xylobioseandxylotriosewerethemajorendproductsforbothendoxylanases.ThexylanaseswerestableatpH4–11showingpHoptimaaroundpH6.Xyl1showsmaximalactivityat60°C,Xyl2at65°C(at4MNaCl).Thexylanasesshowedhightemperaturestabilitywithhalf-livesat60°Cof97minand192minrespectively.Bothxylanasesshowedoptimalactivityat1MNaCl,butsubstantialactivityremainedforbothenzymesat5MNaCl.
Productionofcyclomaltononaose(δ-cyclodextrin)bycyclodextringlycosyltransferasesfromBacillusspp.andbacterialisolates.
Larsen,K.L.,Christensen,H.J.S.,Mathiesen,F.,Pedersen,L.H.&Zimmermann,W.(1998).AppliedMicrobiologyandBiotechnology,50(3),314-317.
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Theconversionofsolublestarchtocyclomaltohexaose(α-CD),cyclomaltoheptaose(β-CD),cyclomaltooctaose(γ-CD)andcyclomaltononaose(δ-CD)bycyclodextringlycosyltransferases(E.C.2.4.1.19)fromBacillusspp.andbacterialisolateswasstudied.Theresultsshowthatδ-CDwasformedbyalltheenzymesinvestigatedintherangeof5%–11.5%ofthetotalamountofα-,β-,γ-,andδ-CDproduced.
AssessmentofBacilluslicheniformisα-amylaseasacandidateenzymeforgeneticengineeringofmaltingbarley.
Vickers,J.E.,Hamilton,S.E.,Jersey,J.D.,Henry,R.J.,Marschke,R.J.&Inkerman,P.A.(1996).JournaloftheInstituteofBrewing,102(2),75-78.
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Bacilluslicheniformisα-amylase,athermostablestarch-degradingenzyme,hasbeenassessedasacandidateenzymeforthegenetictransformationofmaltingbarley.Thetemperatureoptimum,pHoptimumandthermostabilityofB.licheniformisα-amylasewerecomparedwiththoseofbarleyα-amylase.ThebacterialenzymehasahigherpHoptimum(~9),ahighertemperatureoptimum(~90°C)andmuchhigherthermostabilityatelevatedtemperaturesthanthebarleyenzyme.ThespecificactivityofthebacterialenzymeunderconditionsofpHandtemperaturerelevanttothebrewingprocess(pH5.5,65°C)is~1.5-foldhigherthanthatofthebarleyenzyme.Measurementsofα-amylaseactivityduringamicro-mashshowedthatthebacterialenzymeisatleastasstableasthebarleyenzymeundertheseconditions,andthatalevelofexpressionforthebacterialenzymecorrespondingto~0.5%oftotalmaltproteinwouldapproximatelydoubletheα-amylaseactivityinthemash.B.licheniformisα-amylaseactivitywasrapidlyeliminatedbyboilingfollowingmashingaswouldoccurduringbrewing.Thecombinedresultssuggestthatbarleyexpressingthebacterialenzymemaybeusefulinthebrewingprocess.
Hydrolysisofstarchesandfloursbysorghummaltamylasesfordextrinsproduction.
Ba,K.,Aguedo,M.,Tine,E.,Paquot,M.,Destain,J.&Thonart,P.(2013).EuropeanFoodResearchandTechnology,236(5),905-918.
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Cornandwheatstarchesaswellaswheatandcassavaflourswerehydrolyzedusingsorghummaltat65°Cfor6h.Duringthesereactions,dextroseequivalent(DE)valueswerefollowedunderthreeconcentrationsofsorghummaltandcalciumchloride.WheatflourpresentedthehighestDEvaluesandcassavaflourhadthehighesthydrolysisyield.Thus,differentdextrinswereproducedinapilotplantandwereanalyzedbyHPSECandHPAEC-PADfortheirmolecularweightdistributionandoligosaccharidescomposition,respectively.Theresultsindicatedthatoligosaccharideswithbroadmolecularweightdistributionswerepresentinthedextrinsproducedandthattheproportionofmaltosewasveryhigh.
Taxonomicandfunctionaldiversityofpseudomonadsisolatedfromtherootsoffield‐growncanola.
Misko,A.L.&Germida,J.J.(2002).FEMSMicrobiologyEcology,42(3),399-407.
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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),17-22.
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Asemi-qualitativescreeningbasedonproteaseandamylaseactivityevaluationinabasalagarmediumsupplementedwithinsolublechromogenicsubstratesbasedonAZCL(Azurine-Crosslinkedwithamyloseorcasein)usingaplateassaywasusedforselectingthepolarstreptomycetesabletoproducecoldactivesandalkalineamylasesandproteases.ThistechniqueprovidesaspecificandrapidsimultaneousdetectionofhighactivehydrolaseproducingstrainsbasedonthevisiblesolubilizationofsmallparticlesofAZCLandtheformationofhaloesonplates.Ithasagreatpotentialinincreasingtheefficacyofscreeningstreptomycetesabletoproducehydrolyticenzymes.Thisstudyrevealedthepotentialoftheselectedstreptomycetesisolatedfrompolarsoilstobiosynthesizeamylasesandproteasescold-adaptedatlowtemperatures(from5to20°C)andalkalinepHvalues(8to9).
Rapiddetectionofmalto‐oligosaccharide‐formingbacterialamylasesbyhighperformanceanion‐exchangechromatography.
Duedahl‐Olesen,L.,Larsen,K.L.&Zimmermann,W.(2000).LettersinAppliedMicrobiology,30(4),312-316.
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Highperformanceanion-exchangechromatographywithpulsedamperometricdetectionwasappliedfortherapidanalysisofmalto-oligosaccharidesformedbyextracellularenzymepreparationsfrom49starch-degradingbacterialstrainsisolatedfromsoilandcompostsamples.Malto-oligosaccharide-formingamylases,indicatedbyapredominantformationofmaltohexaosefromstarch,wereproducedbyenzymepreparationsfromfouroftheisolatesgrowingatpH7·0and10.
Characterisationofthreestarchdegradingenzymes:Thermostableβ-amylase,maltotetraogenicandmaltogenicα-amylases.
Derde,L.J.,Gomand,S.V.,Courtin,C.M.&Delcour,J.A.(2012).FoodChemistry,135(2),713-721.
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Maltogenicα-amylasefromBacillusstearothermophilus(BStA)iswidelyusedasbreadcrumbanti-firmingenzyme.Amaltotetraose-formingα-amylasefromPseudomonassaccharophila(PSA)wasrecentlyproposedasalternative,hencetheneedtocomparebothexo-actingenzymeswithsomeendo-actioncomponent.Apurelyexo-actingthermostableβ-amylasefromClostridiumthermosulfurogenes(CTB)wasincludedforreferencepurposes.Undertheexperimentalconditionsused,temperatureoptimaoftheenzymesarerathersimilar(60–65°C),buttemperaturestabilitydecreasedintheorderBStA,PSAandCTB.Theactionoftheenzymesondifferentsubstratesandtheirimpactontherheologicalbehaviourofmaizestarchsuspensionsdemonstratedthat,whileCTBactsexclusivelythroughanexo-actionmechanism,BStAdisplayedlimitedendo-actionwhichbecamemorepronouncedathighertemperatures.PSAhasmoresubstantialendo-actionthanBStA,whichisrathertemperatureindependent.Thisisimportantfortheirimpactinprocessessuchasbreadmaking,wheretemperatureisgraduallyincreased.
Insightintothedistributionofarabinoxylans,endoxylanases,andendoxylanaseinhibitorsinindustrialwheatrollermillstreams.
Dornez,E.,Gebruers,K.,Wiame,S.,Delcour,J.A.&Courtin,C.M.(2006).JournalofAgriculturalandFoodChemistry,54(22),8521-8529.
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Togaininsightintothedistributionofarabinoxylans(AX),endoxylanases,andendoxylanaseinhibitorsinindustrialwheatrollermilling,allstreams,thatis,54flourfractions,4branfractions,andthegerm,wereanalyzedforash,starch,andproteincontents,α-amylaseactivitylevels,total(TOT-AX)andwater-extractablearabinoxylan(WE-AX)contents,endoxylanaseactivitylevels,andendoxylanaseinhibitor(TAXIandXIP)contents.Ingeneral,branfractionsweresignificantlyricherinTOT-AXandWE-AXcontents,endoxylanaseactivitylevels,andendoxylanaseinhibitorcontentsthangermand,evenmoreso,thanflourfractions.Inthe54differentflourfractions,minimalandmaximalvaluesforTOT-AXandWE-AXcontentsdifferedbyca.2-fold,whereastheydifferedbyca.15-foldforendoxylanaseactivitylevels.Thelatterwerepositivelycorrelatedwithashandnegativelycorrelatedwithstarchcontent,suggestingthattheendoxylanaseactivityinflourisstronglyinfluencedbythelevelofbrancontamination.TAXIcontentsintheflourfractionsvariedca.4-foldandwerestronglycorrelatedwithbran-relatedparameterssuchasashcontentandenzymeactivitylevels,whereasXIPcontentsvariedca.3-foldandwerenotcorrelatedwithanyoftheparametersmeasuredinthisstudy.Theresultscanbevaluableinblendingandoptimizingwheatflourfractionstoobtainflourswithspecifictechnologicalandnutritionalbenefits.
AnewthermoactivepullulanasefromDesulfurococcusmucosus:cloning,sequencing,purification,andcharacterizationoftherecombinantenzymeafterexpressioninBacillussubtilis.
Duffner,F.,Bertoldo,C.,Andersen,J.T.,Wagner,K.&Antranikian,G.(2000).JournalofBacteriology,182(22),6331-6338.
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ThegeneencodingathermoactivepullulanasefromthehyperthermophilicanaerobicarchaeonDesulfurococcusmucosus(apuA)wasclonedinEscherichiacoliandsequenced.apuAfromD.mucosusshowed45.4%pairwiseaminoacididentitywiththepullulanasefromThermococcusaggregansandcontainedthefourregionsconservedamongallamylolyticenzymes.apuAencodesaproteinof686aminoacidswitha28-residuesignalpeptideandhasapredictedmassof74kDaaftersignalcleavage.TheapuAgenewasthenexpressedinBacillussubtilisandsecretedintotheculturefluid.ThisisoneofthefirstreportsonthesuccessfulexpressionandpurificationofanarchaealamylopullulanaseinaBacillusstrain.Thepurifiedrecombinantenzyme(rapuDm)iscomposedoftwosubunits,eachhavinganestimatedmolecularmassof66kDa.Optimalactivitywasmeasuredat85°CwithinabroadpHrangefrom3.5to8.5,withanoptimumatpH5.0.Divalentcationshavenoinfluenceonthestabilityoractivityoftheenzyme.RapuDmwasstableat80°Cfor4handexhibitedahalf-lifeof50minat85°C.Byhigh-pressureliquidchromatographyanalysisitwasobservedthatrapuDmhydrolyzedα-1,6glycosidiclinkagesofpullulan,producingmaltotriose,andalsoα-1,4glycosidiclinkagesinstarch,amylose,amylopectin,andcyclodextrins,withmaltotrioseandmaltoseasthemainproducts.SincethethermoactivepullulanasesknownsofarfromArchaeaarenotactiveoncyclodextrinsandareinfactinhibitedbythesecyclicoligosaccharides,theenzymefromD.mucosusshouldbeconsideredanarchaealpullulanasetypeIIwithawidersubstratespecificity.
Anexceptionallycold-adaptedalpha-amylasefromametagenomiclibraryofacoldandalkalineenvironment.
Vester,J.K.,Glaring,M.A.&Stougaard,P.(2015).AppliedMicrobiologyandBiotechnology,99(2),717-727.
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Acold-activeα-amylase,Amy13C6,identifiedbyafunctionalmetagenomicsapproachwasexpressedinEscherichiacoliandpurifiedtohomogeneity.SequenceanalysisshowedthattheAmy13C6amylasewassimilartoα-amylasesfromtheclassClostridiaandrevealedclassicalcharacteristicsofcold-adaptedenzymes,asdidcomparisonofthekineticparametersKmandKcattoamesophilicα-amylase.Amy13C6wasshowntobeheat-labile.Temperatureoptimumwasat10–15°C,andmorethan70%oftherelativeactivitywasretainedat1°C.ThepHoptimumofAmy13C6wasatpH8–9,andtheenzymedisplayedactivityintwocommercialdetergentstested,suggestingthattheAmy13C6α-amylasemaybeusefulasadetergentenzymeinenvironmentallyfriendly,low-temperaturelaundryprocesses.
Postharvestneedleabscissionresistanceofbalsamfir(Abiesbalsamea)ismodifiedbyharvestdate.
MacDonald,M.T.,Lada,R.R.,Veitch,R.S.,Thiagarajan,A.,&Adams,A.D.(2014).CanadianJournalofForestResearch,44(11),1394-1401.
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EarlierharvestdateshavebecomenecessaryforCanadianChristmastreeproducerstomeetinternationaldemand,thoughbyharvestingthesetreesearlytheymayexperiencepoorneedleretention.Theobjectiveofthisstudyistounderstandtheeffectofharvestdateonneedleretentionandlinkthosechangestocoldacclimation.Inoneexperiment,balsamfirtreeswithvariedneedleabscissionresistance(NAR)werecollectedinOctoberandinJanuaryandmonitoredforneedleretention.Thiswasrepeatedfor3years.Inasecondexperiment,45brancheswerecollectedeachmonthfromSeptembertoJanuaryandmonitoredforneedleretention,xylempressure,membraneinjury,capacitance,andaccumulationofgalactose,raffinose,andabscisicacid.High-NARtreeshadlittleimprovementinneedleretentionfromOctobertoJanuary,whereaslow-NARtreeshadsignificantlyimprovedneedleretentionfromOctobertoJanuary.BetweenSeptemberandJanuary,therewasan85%increaseinraffinose,147%increaseingalactose,80%increaseinabscisicacid,and62%decreaseinstemcapacitance.Earlyharvestwasnotdetrimentalforalltrees,anditappearsthatcoldacclimationislinkedtopostharvestneedleabscission,thoughcoldacclimationdoesnotadequatelyexplaindifferencesbetweenNARclasses.
Multipleanalysesofmicrobialcommunitiesappliedtothegutofthewood-feedingtermiteReticulitermesflavipesfedonartificialdiets.
Tarayre,C.,Bauwens,J.,Mattéotti,C.,Brasseur,C.,Millet,C.,Massart,S.,Destain,J.,Vandenbol,M.,De Pauw,E.,Haubruge,E.,Francis,F.,Thonart,P.,Portetelle,D.&Francis,F.(2015).Symbiosis,65(3),143-155.
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ThepurposeofthisworkwastheobservationofthedifferencesbetweenthemicrobialcommunitieslivinginthegutofthetermiteReticulitermesflavipesfedondifferentdiets.Thetermiteswerefedonpoplarwood(originaldiet)andartificialdietsconsistingofcrystallinecellulose(withandwithoutlignin),α-cellulose(withandwithoutlignin)andxylan.Thetermiteswerethendissectedandtheprotistcommunitieswereanalyzedthroughmicroscopy,leadingtotheconclusionthatprotistspeciesarestronglyinfluencedbydiets.BIOLOGECOMicroplates®wereusedtoassessthemetabolicpropertiesofthedifferenttypesofconsortia,highlightingstrongdifferencesonthebasisofprincipalcomponentanalysisandcalculationofsimilarityrates.Themicroorganismswerecultivatedinliquidmediacorrespondingtotheartificialdietsbeforebeingcharacterizedthroughametageneticanalysisofgutmicrobiota(16SribosomalDNA).Thisanalysisidentifiedseveralphyla:Acidobacteria,Actinobacteria,Bacteroidetes,Cyanobacteria,Fibrobacteres,Firmicutes,Nitrospirae,OP9,Planctomycetes,Proteobacteria,Spirochaetes,TM6,Tenericutes,VerrucomicrobiaandWS3.TheOTUswerealsodeterminedandconfirmedtheabundanceofProteobacteria,Bacteroidetes,FirmicutesandVerrucomicrobia.Itwaspossibletoisolateseveralstrainsfromtheliquidmedia,andonebacteriumandseveralfungiwerefoundtoproduceinterestingenzymaticactivities.ThebacteriumChryseobacteriumsp.XAvLWproducedα-amylase,β-glucosidase,endo-1,4-β-D-glucanase,endo-1,4-β-D-xylanaseandfilterpaper-cellulase,whilethefungiSarocladiumkilienseCTGxxylandTrichodermavirensCTGxAviLgeneratedthesameactivitiesaddedwithendo-1,3-β-D-glucanase.
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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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.
