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商品描述
Highpuritydyed,solubleAZ-Rhamnogalacturonanforthemeasurementofenzymeactivity,forresearch,biochemicalenzymeassaysandinvitrodiagnosticanalysis.
Substrateforthespecificassayofendo-rhamnogalacturonanase.
Pectatelyase10AfromPseudomonascellulosaisamodularenzymecontainingafamily2acarbohydrate-bindingmodule.
Brown,I.E.,Mallen,M.H.,Charnock,S.J.,Davies,G.J.&Black,G.W.(2001).BiochemicalJournal,355(1),155-165.
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Pectatelyase10A(Pel10A)enzymefromPseudomonascelluloseiscomposedof649residuesandhasamolecularmassof68.5kDa.SequenceanalysisrevealedthatPel10Acontainedasignalpeptideandtwoserine-richlinkersequencesthatseparatethreemodules.Sequencesimilaritywasseenbetweenthe9.2kDaN-terminalmoduleofPel10Aandfamily2acarbohydrate-bindingmodules(CBMs).ThisN-terminalmoduleofPel10Awasshowntoencodeanindependentlyfunctionalmodulewithaffinitytocrystallinecellulose.Ahighsequenceidentityof66%wasseenbetweenthe14.2kDacentralmoduleofPel10Aandthefunctionallyuncharacterizedcentralmodulesofthexylan-degrADIngenzymesendoxylanase10B,arABInofuranosidase62Candesterase1D,alsofromP.cellulosa.The35.8kDaC-terminalmoduleofPel10Awasshowntohave30and36%identitieswiththefamily10pectatelyasesfromAzospirillumirakenseandanalkaliphilicstrainofBacillussp.strainKSM-P15,respectively.ThisHis-taggedC-terminalmoduleofthePel10Awasshowntoencodeanindependentcatalyticmodule(Pel10Acm).Pel10Acmwasshowntocleavepectateandpectininanendo-fashionandtohaveoptimalactivityatpH10andinthepresenceof2mMCa2+.Highestenzymeactivitywasdetectedat62°C.Pel10Acmwasshowntobemostactiveagainstpectate(i.e.polygalacturonicacid)withprogressivelylessactivityagainst31,67and89%esterifiedcitruspectins.ThesedatasuggestthatPel10Ahasapreferenceforsequencesofnon-esterifiedgalacturonicacidresidues.Significantly,Pel10AandtheP.celluloserhamnogalacturonanlyase11A,intheaccompanyingarticle[McKie,Vincken,Voragen,vandenBroek,StimsonandGilbert(2001)Biochem.J.355,167–177],arethefirstCBM-containingpectinasesdescribedtodate.
CharacterizationofThermophilichalotolerantAeribacilluspallidusTD1fromTaodamhotspring,Thailand.
Yasawong,M.,Areekit,S.,Pakpitchareon,A.,Santiwatanakul,S.&Chansiri,K.(2011).InternationalJournalofMolecularSciences,12(8),5294-5303.
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ThebacterialstrainTD1wasisolatedfromTaoDamhotspringinThailand.StrainTD1wasGrampositive,rod-shaped,aerobic,motile,andendosporeforming.Thecellwas2.0–40µminlengthandabout0.4µmindiameter.Theoptimumgrowthoccurredat55–60°CandatpH7–8.StrainTD1wasabletogrowonmediumcontainingupto10%NaCl.TheDNAG+Ccontentwas38.9mol%.ThecellularfattyacidcontentwasmainlyC16:0,whichcomprised25.04%ofthetotalamountofcellularfattyacid.16SrDNAshowed99%identitytoAeribacilluspallidusDSM3670T.BayesiantreeanalysisstronglysupportedtheideathatstrainTD1isaffiliatedwithgenusAeribacillus,asAeribacilluspallidusstrainTD1.Althoughthe16SrDNAofA.pallidusstrainTD1issimilartothatofA.pallidusDSM3670T,somephysiologicalpropertiesandthecellularfattyacidprofilesdiffersignificantly.A.pallidusstrainTD1canproduceextracellularpectatelyase,whichhasnotbeenreportedelsewhereforotherbacterialstrainsinthegenusAeribacillus.A.pallidusstrainTD1maybeagoodcandidateasapectatelyaseproducer,whichmayhaveusefulindustrialapplications.
Degradationofcellwallmaterialsfromsweetpotato,cassava,andpotatobyabacterialprotopectinaseandterminalsugaranalysisoftheresultingsolubilizedproducts.
Salvador,L.D.,Suganuma,T.,Kitahara,K.,Fukushige,Y.&Tanoue,H.(2002).JournalofBioscienceandBioengineering,93(1),64-72.
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Cellwallmaterials(CWMs)fromsweetpotato,cassava,andpotatostarchresiduesweredegradedusingacrudeenzymesolutionfromtheculturefiltrateofaBacillussp.isolatedfromsoil,Bacillussp.M4.Thisorganismhasbeenfoundtosecretepolygalacturonicacidlyase(PGL)andglycandepolymeraseactivities,especiallyarabinanase,butcellulaseactivitywasnearlyabsent.SugaranalysisofthesolubilizedproductafterenzymetreatmentatpH7.0revealedthatitismainlycomposedofgalacturonicacid,galactose,andarabinose,thesugarsfoundcommonlyinthepectinfraction.Thissuggestedthepresenceofaprotopectinase(PPase)activityintheculturefiltrate.ThepresenceofEDTAcompletelyinhibitedPGLbutPPaseactivitywasalmostretained,suggestingthatthePGLisnottheprimaryactivityresponsIBLeforpectinsolubilization.ThemodeofactionofthecrudeenzymewasdeterminedbyterminalsugaranalysisusingHPAEC-PADafterhydrolysisofthereducedproducts.Resultsrevealedthatgalactoseisthemainneutralsugaratthereducingterminaloftheproducts,althoughrhamnosewasalsopresentinthehighermolecularweightcomponent.ThissuggestedthatatneutralpH,theprimaryactivityintheculturefiltrateofBacillussp.M4isaB-typePPase,whichattackedthegalactanaswellasrhamnogalacturonanmoietiesoftheprotopectin,resultinginthereleaseofasolublepectinfraction.
Discoveryofpectin-degradingenzymesanddirectedevolutionofanovelpectatelyaseforprocessingcottonfabric.
Solbak,A.,Richardson,T.H.,McCann,R.T.,Kline,K.A.,Bartnek,F.,Tomlinson,G.,Tan,X.,Parra-Gessert,L.,Frey,G.J.,Podar,M.,Luginbu¨hl,P.,Gray,K.A.,Mathur,E.J.,Robertson,D.E.,Burk,M.J.,Hazlewood,G.P.,Short,J.M.&Kerovuo,J.(2005).JournalofBIOLOGicalChemistry,280(10),9431-9438.
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Thereisagrowingneedinthetextileindustryformoreeconomicalandenvironmentallyresponsibleapproachestoimprovethescouringprocessaspartofthepretreatmentofcottonfabric.Enzymaticmethodsusingpectin-degradingenzymesarepotentiallyvaluablecandidatesinthiseffortbecausetheycouldreducetheamountoftoxicalkalinechemicalscurrentlyused.UsinghighthroughputscreeningofcomplexenvironmentalDNAlibrariesmorethan40novelmicrobialpectatelyaseswerediscovered,andtheirenzymaticpropertieswerecharacterized.SeveralcandidateenzymeswerefoundthatpossessedpHoptimaandspecificactivitiesonpecticmaterialincottonfiberscompatiblewiththeiruseinthescouringprocess.However,noneexhibitedthedesiredtemperaturecharacteristics.Therefore,acandidateenzymewasselectedforevolution.UsingGeneSiteSaturationMutagenesisTMtechnology,36singlesitemutantsexhibitingimprovedthermotolerancewereproduced.Acombinatoriallibraryderivedfromthe12bestperformingsinglesitemutantswasthengeneratedbyusingGeneReassemblyTMtechnology.Nineteenvariantswithfurtherimprovedthermotolerancewereproduced.Thesevariantsweretestedforbothimprovedthermotoleranceandperformanceinthebioscouringapplication.Thebestperformingvariant(CO14)containedeightmutationsandhadameltingtemperature16°Chigherthanthewildtypeenzymewhileretainingthesamespecificactivityat50°C.Optimaltemperatureoftheevolvedenzymewas70°C,whichis20°Chigherthanthewildtype.Scouringresultsobtainedwiththeevolvedenzymeweresignificantlybetterthantheresultsobtainedwithchemicalscouring,makingitpossibletoreplacetheconventionalandenvironmentallyharmfulchemicalscouringprocess.
Negativesubtractionhybridization:anefficientmethodtoisolatelargenumbersofcondition-specificCDNAs.
Ray,A.,Macwana,S.,Ayoubi,P.,Hall,L.T.,Prade,R.&Mort,A.J.(2004).BMCGenomics,5(1),22.
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Background:TheconstructionofcDNAlibrariesisausefultooltounderstandgeneexpressioninorganismsunderdifferentconditions,butrandomsequencingofunbiasedcDNAcollectionsislaboriousandcangiverisetoredundantESTcollections.WeaimedtoisolatecDNAsofmessagesinducedbyswitchingAspergillusnidulansfromgrowthonglucosetogrowthonselectedpolysaccharides.Approximately4,700contigsfrom12,320ESTswerealreadyavailablefromacDNAlibraryrepresentingtranscriptsisolatedfromglucose-grownA.nidulansduringasexualdevelopment.OurgoalsweretoexpandthecDNAcollectionwithoutrepeatedsequencingofpreviouslyidentifiedESTsandtofindasmanytranscriptsaspossiblethatarespecificallyinducedincomplexpolysaccharidemetabolism.Results:WehavedevisedaNegativeSubtractionHybridization(NSH)methodandtesteditinA.nidulans.NSHentailsscreeningaplasmidlibrarymadefromcDNAspreparedfromcellsgrownunderaselectedphysiologicalconditionwithlabeledcDNAprobespreparedfromanotherphysiologicalcondition.PlasmidswithinsertsthatfailedtohybridizetocDNAprobesthroughtworoundsofscreening(i.e.negatives)indicatethattheyaretranscriptspresentatlowconcentrationinthelabeledprobepool.Thus,thesetranscriptswillbepredominantlycondition-specific,alongwithsomeraretranscripts.Inascreenfortranscriptsinducedbyswitchingthecarbonsourcefromglucoseto12selectedpolysaccharides,3,532negativeswereisolatedfromapproximately100,000surveyedcoloniesusingthismethod.Negativecloneswereend-sequencedandassembledinto2,039contigs,ofwhich1,722werenotpresentinthepreviouslycharacterizedglucose-growncDNAlibrary.Single-channelmicroarrayhybridizationexperimentsconfirmedthatthemajorityofthenegativesrepresentedgenesthatweredifferentiallyinducedbyaswitchfromgrowthinglucosetooneormoreofthepolysaccharides.Conclusions:TheNegativeSubtractionHybridizationmethoddescribedherehasseveralpracticalbenefits.ThismethodcanbeusedtoscreenanyexistingcDNAlibrary,includingfull-lengthandpooledlibraries,anddoesnotrelyonPCRorsequenceinformation.Inaddition,NSHisacost-effectivemethodfortheisolationofnovel,full-lengthcDNAsfordifferentiallyexpressedtranscriptsorenrichmentofraretranscripts.
