TheAceticAcid(AcetateKinaseManualFormat)testkitissuitableforthemeasurementandanalysisofaceticacidinfoodandbeverages.
Thisrapidandreliablemanualaceticacidkitissimpletoperform(onlytwoabsorbancereadingsrequired),andbecauseatrueend-pointismeasured,doesnotinvolvecomplicatedcalculationslikeotherkits.Thisproductisverystablebothduringstorageanduse(>2years),hasextendedlinearity(comparedtoACSbasedkits),containsPVPtopreventtannininhibition,andisperformedatarelativelylowpH(7.4),thusminimisingesterhydrolysisrelatedinterference.Thismethodissuitableforthemeasurementofaceticacid/acetateinfoods,beveragesandothermaterials.
Grapeandwineanalysis:Oenologiststoexploitadvancedtestkits.
Charnock,S.C.&McCleary,B.V.(2005).RevuedesEnology,117,1-5.
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Itiswithoutdoubtthattestingplaysapivotalrolethroughoutthewholeofthevinificationprocess.Toproducethebestpossiblequalitywineandtominimiseprocessproblemssuchas“stuck”fermentationortroublesomeinfections,itisnowrecognisedthatifpossibletestingshouldbeginpriortoharvestingofthegrapesandcontinuethroughtobottling.Traditionalmethodsofwineanalysisareoftenexpensive,timeconsuming,requireeitherelaborateequipmentorspecialistexpertiseandfrequentlylackaccuracy.However,enzymaticbio-analysisenablestheaccuratemeasurementofthevastmajorityofanalytesofinteresttothewinemaker,usingjustonepieceofapparatus,thespectrophotometer(seepreviousissueNo.116foradetailedtechnicalreview).Grapejuiceandwineareamenabletoenzymatictestingasbeingliquidstheyarehomogenous,easytomanipulate,andcangenerallybeanalysedwithoutanysamplepreparation.
Megazyme“advanced”winetestkitsgeneralcharacteristicsandvalidation.
Charnock,S.J.,McCleary,B.V.,Daverede,C.&Gallant,P.(2006).ReveuedesOenologues,120,1-5.
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ManyoftheenzymatictestkitsareofficialmethodsofprestigiousorganisationssuchastheAssociationofOfficialAnalyticalChemicals(AOAC)andtheAmericanAssociationofCerealChemists(AACC)inresponsetotheinterestfromoenologists.Megazymedecidedtouseitslonghistoryofenzymaticbio-analysistomakeasignificantcontributiontothewineindustry,bythedevelopmentofarangeofadvancedenzymatictestkits.Thistaskhasnowbeensuccessfullycompletedthroughthestrategicandcomprehensiveprocessofidentifyinglimitationsofexistingenzymaticbio-analysistestkitswheretheyoccurred,andthenusingadvancedtechniques,suchasmolecularbiology(photo1),torapidlyovercomethem.Noveltestkitshavealsobeendevelopedforanalytesofemerginginteresttotheoenologist,suchasyeastavailablenitrogen(YAN;seepages2-3ofissue117article),orwherepreviouslyenzymesweresimplyeithernotavailable,orweretooexpensivetoemploy,suchasforD-mannitolanalysis.
ThemetabolicpotentialofEscherichiacoliBL21indefinedandrichmedium.
Li,Z.,Nimtz,M.&Rinas,U.(2014).MicrobialCellFactories, 13(1),45.
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Background:Theproteomereflectstheavailablecellularmachinerytodealwithnutrientsandenvironmentalchallenges.ThemostcommonE.colistrainBL21growingindifferent,commonlyemployedmediawasevaluatedusingadetailedquantitativeproteomeanalysis.Results:ThepresenceofpreformedbiomassprecursormoleculesinrichmediasuchasLuriaBertanisupportedrapidgrowthconcomitanttoacetateformationandapparentlyunbalancedabundancesofcentralmetabolicpathwayenzymes,e.g.highlevelsoflowerglycolyticpathwayenzymesaswellaspyruvatedehydrogenase,andlowlevelsofTCAcycleandhighlevelsoftheacetateformingenzymesPtaandAckA.Theproteomeofcellsgrowingexponentiallyinglucose-supplementedmineralsaltmediumwasdominatedbyenzymesofaminoacidsynthesispathways,containedmorebalancedabundancesofcentralmetabolicpathwayenzymes,andalowerportionofribosomalandothertranslationalproteins.Entryintostationaryphaseledtoareconstructionofthebacterialproteomebyincreasinge.g.theportionofproteinsrequiredforscavengingrarenutrientsandgeneralcellprotection.Thisproteomicreconstructionduringentryintostationaryphasewasmorenoticeableincellsgrowinginrichmediumastheyhaveagreaterreservoirofrecyclableproteinsfromthetranslationalmachinery.Conclusions:TheproteomiccomparisonofcellsgrowingexponentiallyindifferentmediareflectedtheantagonisticandcompetitiveregulationofcentralmetabolicpathwaysthroughtheglobaltranscriptionalregulatorsCra,Crp,andArcA.Forexample,theproteomeofcellsgrowingexponentiallyinrichmediumwasconsistentwithadominatingroleofphosphorylatedArcAmostlikelyaresultfromlimitationsinreoxidizingreducedquinonesintherespiratorychainunderthesegrowthconditions.Theproteomicalterationsofexponentiallygrowingcellsintostationaryphasecellswereconsistentwithstringent-likeandstationaryphaseresponsesandadominatingcontrolthroughDksA-ppGppandRpoS.
Comparativetranscriptomicprofileanalysisoffed-batchculturesexpressingdifferentrecombinantproteinsinEscherichiacoli.
Sharma,A.K.,Mahalik,S.,Ghosh,C.,Singh,A.B.&Mukherjee,K.J.(2011).AMBExpress,1(1),33.
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Thereisaneedtoelucidatetheproductspecificfeaturesofthemetabolicstressresponseofthehostcelltotheinductionofrecombinantproteinsynthesis.Forthis,themethodofchoiceistranscriptomicprofilingwhichprovidesabetterinsightintothechangestakingplaceincomplexglobalmetabolicnetworks.Thetranscriptomicprofilesofthreefed-batchculturesexpressingdifferentproteinsviz.recombinanthumaninterferon-beta(rhIFN-β),XylanaseandGreenFluorescenceProtein(GFP)werecomparedpostinduction.Weobservedadepressioninthenutrientuptakeandutilizationpathways,whichwascommonforallthethreeexpressedproteins.ThusglyceroltransportersandgenesinvolvedinATPsynthesisaswellasaerobicrespirationwereseverelydown-regulated.Ontheotherhandtheaminoaciduptakeandbiosynthesisgenesweresignificantlyrepressedonlywhensolubleproteinswereexpressedunderdifferentpromoters,butnotwhentheproductwasexpressedasaninclusionbody(IB).HighlevelexpressionundertheT7promoter(rhIFN-βandxylanase)triggeredthecellulardegradationmachineryliketheosmoprotectants,proteasesandmRNAdegradationgeneswhichwerehighlyup-regulated,whilethistrendwasnottruewithGFPexpressionunderthecomparativelyweakerarapromoter.Thedesignofabetterhostplatformforrecombinantproteinproductionthusneedstotakeintoaccountthespecificnatureofthecellularresponsetoproteinexpression.
OrganiccarbonsupplementationofsterilizedmunicipalwastewaterisessentialforheterotrophicgrowthandremovingammoniumbythemicroalgaChlorellaVulgaris.
Perez‐Garcia,O.,Bashan,Y.&EstherPuente,M.(2011).JournalofPhycology,47(1),190-199.
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HeterotrophicgrowthofthemicroalgaChlorellavulgarisBeij.insyntheticaswellassterilizedmunicipalwastewaterofanonindustrializedcitywasmeasured.Thecitywastewatercontainedhighlevelsofammoniumandnitrate,mediumlevelsofphosphate,andlowlevelsofnitriteandorganicmoleculesandcouldnotsupportheterotrophicgrowthofC.vulgaris.Evaluationof11knowncarbonsourcesforthismicroalgathatwereaddedtostandardsyntheticwastewatercontainingthesamelevelsofnitrogenandphosphorusasthemunicipalwastewaterrevealedthatthebestcarbonsourcesforheterotrophicgrowthwereNa-acetateandD-glucose.Theseprovidedthehighestgrowthratesandthelargestremovalofammonium.Growthincreasedwithconcentrationofthesupplementtoanoptimumat0.12MNa-acetate.Thiscarbonsourcewasconsumedcompletelywithin10dofincubation.HigherconcentrationsinhibitedthegrowthofC.vulgaris.Themicroalgalpopulationsunderheterotrophicgrowthconditionswereonelevelofmagnitudehigherthanthatunderautotrophicgrowthconditionsthatservedasacomparison.Nogrowthoccurredinthedarkintheabsenceofacarbonsource.Na-acetatewassuperiortoD-glucose.Inmunicipalwastewater,whenNa-acetateorD-glucosewasadded,C.vulgarissignificantlyenhancedammoniumremovalunderheterotrophicconditions,anditscapacitywasequaltoammoniumremovalunderautotrophicgrowthconditions.ThisstudyshowedthatsterilizedwastewatercanbetreatedbyC.vulgarisunderheterotrophicconditionsifsupplementedwiththeappropriateorganiccarbonsourceforthemicroalgae.
Large-scalemetabolomeanalysisandquantitativeintegrationwithgenomicsandproteomicsdatainMycoplasmapneumonia.
Maier,T.,Marcos,J.,Wodke,J.A.H.,Paetzold,B.,Liebeke,M.,Gutiérrez-Gallego,R.&Serrano,L.(2013).MolecularBioSystems,9(7),1743-1755.
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Systemsmetabolomics,theidentificationandquantificationofcellularmetabolitesandtheirintegrationwithgenomicsandproteomicsdata,promisesvaluablefunctionalinsightsintocellularbiology.However,technicalconstraints,samplecomplexityissuesandthelackofsuitablecomplementaryquantitativedatasetspreventedaccomplishingsuchstudiesinthepast.Here,wepresentanintegrativemetabolomicsstudyofthegenome-reducedbacteriumMycoplasmapneumoniae.Weexperimentallyanalyseditsmetabolomeusingacross-platformapproach.Weexplainintracellularmetabolitehomeostasisbyquantitativelyintegratingourresultswiththecellularinventoryofproteins,DNAandothermacromolecules,aswellaswithavailablebuildingblocksfromthegrowthmedium.Wecalculatedinvivocatalyticparametersofglycolyticenzymes,makinguseofmeasuredreactionvelocities,aswellasenzymeandmetabolitepoolsizes.Aquantitative,inter-speciescomparisonofabsoluteandrelativemetaboliteabundancesindicatedthatmetabolicpathwaysareregulatedasfunctionalunits,therebysimplifyingadaptiveresponses.Ouranalysisdemonstratesthepotentialfornewscientificinsightbyintegratingdifferenttypesoflarge-scaleexperimentaldatafromasinglebiologicalsource.
Declineinmammarytranslationalcapacityduringintravenousglucoseinfusionintolactatingdairycows.
Curtis,R.V.,Kim,J.J.M.,Bajramaj,D.L.,Doelman,J.,Osborne,V.R.&Cant,J.P.(2014).JournalofDairyScience,97(1),430-438.
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Theobjectiveofthisstudywastodetermineeffectsofglucoseonmilkproteinyieldandmammarymammaliantargetofrapamycin(mTOR)activityindairycattleinearlylactation.Eightmultiparouscowsat73±8dinmilkwererandomlyassignedto2treatmentsinacrossoverdesignfortwo6-dperiods.Treatmentswerejugularinfusionofeithersaline(Sal)or896g/dglucose(Glc).Allcowswerefedatotalmixedrationwith42%neutraldetergentfiber,hadfreeaccesstowater,andweremilkedtwiceaday.Withineachperiod,bloodsamplesweretaken(d5)andmammarytissuewascollectedbybiopsy(d6)fromeachhindquarterforWesternblotanalysis.InadditiontoSalandGlctreatments,ond6,rapamycindissolvedin50%dimethylsulfoxidewasadministeredviatheteatcanalsintotheleftquarters,withacontrolsolutionadministeredintotherightquarters.RapamycinhadnoeffectonmilkproteinyieldsorphosphorylationstateofmTORsignalingproteins.InfusionsofGlcsignificantlyincreasedmilkyieldbutonlytendedtoincreasemilkproteinyields.MilkfattendedtobedecreasedincowsinfusedwithGlc,whereaslactoseyieldsweresignificantlyincreased.Glucoseinfusiondidnotincreaseplasmaglucoselevels,butinsulinandnonessentialAAconcentrationsincreasedby21and16%,respectively,branched-chainAAconcentrationsdecreased24%,andessentialAAconcentrationstendedtodecreaseby14%.InfusionofGlcsignificantlydecreasedabundancesofbothphosphorylatedandtotalribosomalS6kinase1(S6K1)inmammarytissueby27and11%,respectively.Abundanceofphosphorylatedeukaryoticinitiationfactor4E-bindingprotein1(4EBP1)decreasedsignificantlyby25%,whereastotal4EBP1exhibitedatendencytodecreaseby16%.WeconcludethatthemTORsignalingpathwayisnottheonlyregulatorofmilkproteinsynthesis.DecreasesinessentialAAconcentrationsinplasmasuggestthatproteinsynthesiswasstimulatedinnonmammarytissuesofthebody,presumablyskeletalmuscle.
AnewfamilyofcarbohydrateesterasesisrepresentedbyaGDSLhydrolase/acetylxylanesterasefromGeobacillusstearothermophilus.
Alalouf,O.,Balazs,Y.,Volkinshtein,M.,Grimpel,Y.,Shoham,G.&Shoham,Y.(2011).JournalofBiologicalChemistry,286(49),41993-42001.
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Acetylxylanesteraseshydrolyzetheesterlinkagesofacetylgroupsatpositions2and/or3ofthexylosemoietiesinxylanandplayanimportantroleinenhancingtheaccessibilityofxylanasestothexylanbackbone.ThehemicellulolyticsystemofthethermophilicbacteriumGeobacillusstearothermophilusT-6comprisesaputativeacetylxylanesterasegene,axe2.ThegeneproductbelongstotheGDSLhydrolasefamilyanddoesnotsharesequencehomologywithanyofthecarbohydrateesterasesintheCAZyDatabase.Theaxe2geneisinducedbyxylose,andthepurifiedgeneproductcompletelydeacetylatesxylobioseperacetate(fullyacetylated)andhydrolyzesthesyntheticsubstrates2-naphthylacetate,4-nitrophenylacetate,4-methylumbelliferylacetate,andphenylacetate.ThepHprofilesforKcatandKcat/Kmsuggesttheexistenceoftwoionizablegroupsaffectingthebindingofthesubstratetotheenzyme.UsingNMRspectroscopy,theregioselectivityofAxe2wasdirectlydeterminedwiththeaidofone-dimensionalselectivetotalcorrelationspectroscopy.Methyl2,3,4-tri-O-acetyl-β-D-xylopyranosidewasrapidlydeacetylatedatposition2oratpositions3and4togiveeitherdiacetylormonoacetylintermediates,respectively;methyl2,3,4,6-tetra-O-acetyl-β-D-glucopyranosidewasinitiallydeacetylatedatposition6.Inbothcases,thecompletehydrolysisoftheintermediatesoccurredatamuchslowerrate,suggestingthatthepreferredsubstrateistheperacetatesugarform.Site-directedmutagenesisofSer-15,His-194,andAsp-191resultedincompleteinactivationoftheenzyme,consistentwiththeirroleasthecatalytictriad.Overall,ourresultsshowthatAxe2isaserineacetylxylanesteraserepresentinganewcarbohydrateesterasefamily.
Microbialcarbohydrateesterasesdeacetylatingplantpolysaccharides.
Biely,P.(2012).BiotechnologyAdvances,30(6),1575-1588.
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Severalplantpolysaccharidesarepartiallyesterifiedwithaceticacid.Oneoftherolesofthismodificationisprotectionofplantcellwallsagainstinvadingmicroorganisms.Acetylationofglycosylresiduesofpolysaccharidespreventshydrolysisoftheirglycosidiclinkagesbythecorrespondingglycosidehydrolases.Inthiswaytheacetylationalsorepresentsanobstacleofenzymaticsaccharificationofplanthemicellulosestofermentablesugarswhichappearstobeahottopicofcurrentresearch.Wecaneliminatethisobstaclebyalkalineextractionorpretreatmentleadingtosaponificationofesterlinkages.However,thistaskhasbeenaccomplishedinadifferentwayinthenature.Theacetylgroupsbecametargetsofmicrobialcarbohydrateesterasesthatevolvedtoovercomethecomplexityoftheplantcellwallsandthatcooperatewithglycosidehydrolasesinplantpolysaccharidedegradation.Thisarticleconcentratesonenzymesdeacetylatingplanthemicellulosesexcludingpectin.Theyarecurrentlygroupedinatleast8families,specificallyinCEfamilies1–7and16,originallyassignedasacetylxylanesterases,theenzymesactingonhardwoodacetylglucuronoxylananditsfragmentsgeneratedbyendo-β-1,4-xylanases.Thereareesterasesdeacetylatingsoftwoodgalactoglucomannan,buttheyhavenotbeenclassifiedyet.TheenzymespresentinCEfamilies1–7differinstructureandsubstrateandpositionalspecificity.Therearefamiliesbehavingasendo-typeandexo-typedeacetylates,i.e.esterasesdeacetylatinginternalsugarresiduesofpartiallyacetylatedpolysaccharidesandalsoesterasesdeacetylatingnon-reducingendsugarresiduesinoligosaccharides.Withoneexception,theenzymesofallmentionedCEfamiliesbelongtoserinetypeesterases.CEfamily4harborsenzymesthataremetal-dependentasparticesterases.Three-dimensionalstructureshavebeensolvedformembersofthefirstsevenCEfamilies,however,thereisstillinsufficientknowledgeabouttheirsubstratespecificityandrealphysiologicalrole.CurrentknowledgeoncatalyticpropertiesoftheselectedfamiliesofCEsissummarizedinthisreview.Someofthefamiliesareemergingalsoasnewbiocatalystsforregioselectiveacylationanddeacylationofcarbohydrates.
AssayforpeptidoglycanO-acetyltransferase:Apotentialnewantibacterialtarget.
Moynihan,P.J.&Clarke,A.J.(2013).AnalyticalBiochemistry,439(2),73-79.
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TheO-acetylationofpeptidoglycanoccursattheC-6hydroxylgroupofmuramoylresiduesinmanyhumanpathogens,bothgrampositiveandgramnegative,suchasStaphylococcusaureusandspeciesofCampylobacter,Helicobacter,Neisseria,andBacillus,includingBacillusanthracis.TheprocessisamaturationeventbeingcatalyzedeitherbyintegralmembraneO-acetylpeptidoglycantransferase(Oat)ofgram-positivebacteriaorbyatwo-componentpeptidoglycanO-acetyltransferasesystem(PatA/PatB)ingram-negativecells.Here,wedescribethedevelopmentofthefirstinvitroassayforanypeptidoglycanO-acetyltransferaseusingPatBfromNeisseriagonorrhoeaeasthemodelenzyme.Thisassayisbasedontheuseofchromogenicp-nitrophenylacetateasthedonorsubstrateandchitooligosaccharidesasmodelacceptorsubstratesinplaceofpeptidoglycan.TheidentityoftheO-acetylatedchitooligosaccharideswasconfirmedbymatrix-assistedlaserdesorption/ionizationtime-of-flightmassspectrometry.Ratesoftransacetylationsweredeterminedspectrophotometricallybymonitoringp-nitrophenolreleaseafteraccountingforbothspontaneousandenzyme-catalyzedhydrolysisoftheacetatedonor.Conditionswereestablishedforuseoftheassayinmicrotiterplateformat,anditsapplicabilitywasdemonstratedbydeterminingthefirstMichaelis–MentenkineticparametersforPatB.Theassayisreadilyamenableforapplicationinthehigh-throughputscreeningforpotentialinhibitorsofpeptidoglycanO-acetyltransferasesthatmayprovetobeleadsfornovelclassesofantibiotics.
Discoveryofdiversityinxylanbiosyntheticgenesbytranscriptionalprofilingofaheteroxylancontainingmucilaginoustissue.
Jensen,J.K.,Johnson,N.&Wilkerson,C.G.(2013).FrontiersinPlantScience,4(183).
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Theexactbiochemicalstepsofxylanbackbonesynthesisremainelusive.InArabidopsis,threenon-redundantgenesfromtwoglycosyltransferase(GT)families,IRX9andIRX14fromGT43andIRX10fromGT47,arecandidatesforformingthexylanbackbone.Inotherplants,evidenceexiststhatdifferenttissuesexpressthesethreegenesatwidelydifferentlevels,whichsuggeststhatdiversityinthemakeupofthexylansynthasecomplexexists.Recentlywehaveprofiledthetranscriptspresentinthedevelopingmucilaginoustissueofpsyllium(PlantagoovataForsk).ThistissuewasfoundtohavehighexpressionlevelsofanIRX10homolog,butverylowlevelsofthetwoGT43familymembers.ThiscontrastswithrecentwheatendospermtissueprofilingthatfoundarelativelyhighabundanceoftheGT43familymembers.Wehaveperformedanin-depthanalysisofallGTsgenesexpressedinfourdevelopmentalstagesofthepsylliummucilagenouslayerandinasinglestageofthepsylliumstemusingRNA-Seq.ThisanalysisrevealedseveralIRX10homologs,anexpansioninGT61(homologsofAt3g18170/At3g18180),andseveralGTsfromotherGTfamiliesthatarehighlyabundantandspecificallyexpressedinthemucilaginoustissue.OurcurrenthypothesisisthatthefourIRX10genespresentinthemucilagenoustissueshaveevolvedtofunctionwithouttheGT43genes.ThesefourgenesrepresentsomeofthemostdivergentIRX10genesidentifiedtodate.Conversely,thosepresentinthepsylliumstemareverysimilartothoseinothereudicots.Thissuggeststhesegenesareunderselectivepressure,likelyduetothesynthesisofthevariousxylanstructurespresentinmucilagethathasadifferentbiochemicalrolethanthatpresentinsecondarywalls.ThenumerousGT61familymembersalsoshowawidesequencediversityandmayberesponsibleforthelargernumberofsidechainstructurespresentinthepsylliummucilage.
Structuralchangesandcellularlocalizationofresuscitation-promotingfactorinenvironmentalisolatesofMicrococcusluteus.
Koltunov,V.,Greenblatt,C.L.,Goncharenko,A.V.,Demina,G.R.,Klein,B.Y.,Young,M.&Kaprelyants,A.S.(2010).MicrobialEcology,59(2),296-310.
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Dormancyamongnonsporulatingactinobacteriaisnowawidelyacceptedphenomenon.InMicrococcusluteus,theresuscitationofdormantcellsiscausedbyasmallsecretedprotein(resuscitation-promotingfactor,orRpf)thatisfoundin“spentculturemedium.”RpfisencodedbyasingleessentialgeneinM.luteus.HomologsofRpfarewidespreadamongthehighG+CGram-positivebacteria,includingmycobacteriaandstreptomycetes,andmostorganismsmakeseveralfunctionallyredundantproteins.M.luteusRpfcomprisesalysozyme-likedomainthatisnecessaryandsufficientforactivityconnectedthroughashortlinkerregiontoaLysMmotif,whichispresentinanumberofcell-wall-associatedenzymes.Muralyticactivityisresponsibleforresuscitation.Inthisreport,wecharacterizedanumberofenvironmentalisolatesofM.luteus,includingseveralrecoveredfromamber.Therewassubstantialvariationinthepredictedrpfgeneproduct.Whilethelysozyme-likeandLysMdomainsshowedlittlevariation,thelinkerregionwaselongatedfromtenaminoacidresiduesinthelaboratorystrainstoasmanyas120residuesinoneisolate.ThegenesencodingtheseRpfproteinshavebeencharacterized,andapossiblerolefortheRpflinkerinenvironmentaladaptationisproposed.Theenvironmentalisolatesshowenhancedresistancetolysozymeascomparedwiththelaboratorystrainsandthiscorrelateswithincreasedpeptidoglycanacetylation.Instrainsthatmakeaproteinwithanelongatedlinker,Rpfwasboundtothecellwall,ratherthanbeingreleasedtothegrowthmedium,asoccursinreferencestrains.Thisrpfgenewasintroducedintoalysozyme-sensitivereferencestrain.Bothrpfgeneswereexpressedintransformantswhichshowedaslightbutstatisticallysignificantincreaseinlysozymeresistance.
