TheD-GlucoseHK(Regular)testkitisahighpurityreagentforthemeasurementandanalysisofD-glucoseinplantandfoodproducts.CanbeusedincombinationwithotherMegazyme"sproductsthatrequireglucosedetermination.ExtendedcofactorsstABIlity.Dissolvedcofactorsstablefor>1yearat4oC.Suitableformanual,auto-analyserandmicroplateformats.Measurementoftotalstarchincerealproductsbyamyloglucosidase-alpha-amylasemethod:collaborativestudy.McCleary,B.V.,Gibson,T.S.&Mugford,D.C.(1997).JournalofAOACInternational,80,571-579.LinktoArticleReadAbstractAnAmericanAssociationofCerealChemists/AOACcollaborativestudywasconductedtoevaluatetheaccuracyandreliabilityofanenzymeassaykitprocedureformeasurementoftotalstarchinarangeofcerealgrainsandproducts.Thefloursampleisincubatedat95degreesCwithThermostablealpha-amylasetocatalyzethehydrolysisofstarchtomaltodextrins,thepHoftheslurryisadjusted,andtheslurryistreatedwithahighlypurifiedamyloglucosidasetoquantitativelyhydrolyzethedextrinstoglucose.Glucoseismeasuredwithglucoseoxidase-peroxidasereagent.Thirty-twocollaboratorsweresent16homogeneoustestsamplesas8blindduplicates.Thesesamplesincludedchickenfeedpellets,whitebread,greenpeas,high-amylosemaizestarch,whitewheatflour,wheatstarch,oatbran,andspaghetti.Allsampleswereanalyzedbythestandardprocedureasdetailedabove;4samples(high-amylosemaizestarchandwheatstarch)werealsoanalyzedbyamethodthatrequiresthesamplestobecookedfirstindimethylsulfoxide(DMSO).Relativestandarddeviationsforrepeatability(RSD(r))rangedfrom2.1to3.9%,andrelativestandarddeviationsforreproducibility(RSD(R))rangedfrom2.9to5.7%.TheRSD(R)valueforhighamylosemaizestarchanalyzedbythestandard(non-DMSO)procedurewas5.7%;thevaluewasreducedto2.9%whentheDMSOprocedurewasused,andthedeterminedstarchvaluesincreasedfrom86.9to97.2%.Grapeandwineanalysis:Oenologiststoexploitadvancedtestkits.Charnock,S.C.&McCleary,B.V.(2005).RevuedesEnology,117,1-5.LinktoArticleReadAbstractItiswithoutdoubtthattestingplaysapivotalrolethroughoutthewholeofthevinificationprocess.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.LinktoArticleReadAbstractManyoftheenzymatictestkitsareofficialmethodsofprestigiousorganisationssuchastheAssociationofOfficialAnalyticalChemicals(AOAC)andtheAmericanAssociationofCerealChemists(AACC)inresponsetotheinterestfromoenologists.Megazymedecidedtouseitslonghistoryofenzymaticbio-analysistomakeasignificantcontributiontothewineindustry,bythedevelopmentofarangeofadvancedenzymatictestkits.Thistaskhasnowbeensuccessfullycompletedthroughthestrategicandcomprehensiveprocessofidentifyinglimitationsofexistingenzymaticbio-analysistestkitswheretheyoccurred,andthenusingadvancedtechniques,suchasmolecularBIOLOGy(photo1),torapidlyovercomethem.Noveltestkitshavealsobeendevelopedforanalytesofemerginginteresttotheoenologist,suchasyeastavailablenitrogen(YAN;seepages2-3ofissue117article),orwherepreviouslyenzymesweresimplyeithernotavailable,orweretooexpensivetoemploy,suchasforD-mannitolanalysis.Measurementofcarbohydratesingrain,feedandfood.McCleary,B.V.,Charnock,S.J.,Rossiter,P.C.,O’Shea,M.F.,Power,A.M.&Lloyd,R.M.(2006).JournaloftheScienceofFoodandAgriculture,86(11),1648-1661.LinktoArticleReadAbstractProceduresforthemeasurementofstarch,starchdamage(gelatinisedstarch),resistantstarchandtheamylose/amylopectincontentofstarch,β-glucan,fructan,glucomannanandgalactosyl-sucroseoligosaccharides(raffinose,stachyoseandverbascose)inplantmaterial,animalfeedsandfoodsaredescribed.Mostofthesemethodshavebeensuccessfullysubjectedtointerlaboratoryevaluation.Allmethodsarebasedontheuseofenzymeseitherpurifiedbyconventionalchromatographyorproducedusingmolecularbiologytechniques.Suchmethodsallowspecific,accurateandreliablequantificationofaparticularcomponent.Problemsincalculatingtheactualweightofgalactosyl-sucroseoligosaccharidesintestsamplesarediscussedindetail.AdvancesinmoleculartoolsfortheuseofZygosaccharomycesbailiiashostforbiotechnologicalproductionsandconstructionofthefirstauxotrophicmutant.Dato,L.,Branduardi,P.,Passolunghi,S.,Cattaneo,D.,Riboldi,L.,Frascotti,G.,Valli,M.&Porro,D.(2010).FEMSYeastResearch,10(7),894-908.LinktoArticleReadAbstractThenonconventionalyeastZygosaccharomycesbailiihasbeenproposedasanewhostforbiotechnologicalprocessesduetoconvenientpropertiessuchasitsresistancetohighsugarconcentrations,relativelyhightemperaturesandespeciallytoacidicenvironments.WedescribeaseriesofnewexpressionvectorsspecificforZ.bailiiandtheresultingimprovementsinproductionlevels.ByexploitingthesequencesoftheendogenousplasmidpSB2,2μm-likemulticopyvectorswereobtained,givingafivefoldincreaseinproduction.Aspecificintegrativevectorwasdevelopedwhichledto100%stabilityintheabsenceofselectivepressure;amultiple-integrationvectorwasconstructed,basedonanrRNAgeneunitportionclonedandsequencedforthispurpose,drivingtheinsertionofupto80copiesoftheforeignconstruct.Moreover,weshowtheconstructionofthefirststableauxotrophicmutantofZ.bailii,obtainedbytargetedgenedeletionappliedtoZbLEU2.ThedevelopmentofmoleculartoolsfortheZ.bailiimanipulationhasnowreachedalevelthatmaybecompatiblewithitsindustrialexploitation;theproductionoforganicacidsisaprominentfieldofapplication.InfluenceofdifferentcarbonsourcesonbacterialcelluloseproductionbyGluconacetobacterxylinusstrainATCC53524.Mikkelsen,D.,Flanagan,B.M.,Dykes,G.A.&Gidley,M.J.(2009).JournalofAppliedMicrobiology,107(2),576-583.LinktoArticleReadAbstractAims:TodeterminetheeffectofcarbonsourcesoncelluloseproducedbyGluconacetobacterxylinusstrainATCC53524,andtocharacterizethepurityandstructuralfeaturesofthecelluloseproduced.MethodsandResults:ModifiedHestrinSchrammmediumcontainingthecarbonsourcesmannitol,glucose,glycerol,fructose,sucroseorgalactosewereinoculatedwithGa.xylinusstrainATCC53524.Platecountsindicatedthatallcarbonsourcessupportedgrowthofthestrain.Sucroseandglycerolgavethehighestcelluloseyieldsof3•83and3•75gl-1respectivelyafter96hfermentation,primarilyduetoasurgeincelluloseproductioninthelast12h.Mannitol,fructoseorglucoseresultedinconsistentratesofcelluloseproductionandyieldsof>2•5gl-1.Solidstate13CCP/MASNMRrevealedthatirrespectiveofthecarbonsource,thecelluloseproducedbyATCC53524waspureandhighlycrystalline.Scanningelectronmicrographsillustratedthedenselypackednetworkofcellulosefibreswithinthepelliclesandthatthedifferentcarbonsourcesdidnotmarkedlyalterthemicro-architectureoftheresultingcellulosepellicles.Conclusions:TheproductionrateofbacterialcellulosebyGa.xylinus(ATCC53524)wasinfluencedbydifferentcarbonsources,buttheproductformedwasindistinguishableinmolecularandmicroscopicfeatures.SignificanceandImpactoftheStudy:OurstudiesforthefirsttimeexaminedtheinfluenceofdifferentcarbonsourcesontherateofcelluloseproductionbyGa.xylinusATCC53524,andthemolecularandmicroscopicfeaturesofthecelluloseproduced.GalactomannanhydrolysisandmannosemetabolisminCellvibriomixtus.Centeno,M.S.,Guerreiro,C.I.P.D.,Dias,F.M.V.,Morland,C.,Tailford,L.E.,Goyal,A.,Prates,J.A.M.,Ferreira,L.M.A.,Caldeira,R.M.H.,Mongodin,E.F.,Nelson,K.E.,Gilbert,H.J.&Fontes,C.M.(2006).FEMSMicrobiologyLetters,261(1),123-132.LinktoArticleReadAbstractGalactomannanhydrolysisresultsfromtheconcertedactionofmicrobialendo-mannanases,manosidasesandα-galactosidasesandisamechanismofintrinsicbiologicalimportance.HerewereporttheidentificationofageneclusterintheaerobicsoilbacteriumCellvibriomixtusencodingenzymesinvolvedinthedegradationofthispolymericsubstrate.Thefamily27α-galactosidase,termedCmAga27A,preferentiallyhydrolysegalactosecontainingpolysaccharides.Inaddition,wehavecharacterizedanenzymewithepimeraseactivity,whichmightberesponsiblefortheconversionofmannoseintoglucose.Theroleoftheidentifiedenzymesinthehydrolysisofgalactomannanbyaerobicbacteriaisdiscussed.SensorcombinationandchemometricvariableselectionforonlinemonitoringofStreptomycescoelicolorfed-batchcultivations.Ödman,P.,Johansen,C.L.,Olsson,L.,Gernaey,K.V.&Lantz,A.E.(2010).AppliedMicrobiologyandBiotechnology,86(6),1745-1759.LinktoArticleReadAbstractFed-batchcultivationsofStreptomycescoelicolor,producingtheantibioticactinorhodin,weremonitoredonlinebymultiwavelengthfluorescencespectroscopyandoff-gasanalysis.Partialleastsquares(PLS),locallyweightedregression,andmultilinearPLS(N-PLS)modelswerebuiltforpredictionofbiomassandsubstrate(casaminoacids)concentrations,respectively.Theeffectofcombinationoffluorescenceandgasanalyzerdataaswellasofdifferentvariableselectionmethodswasinvestigated.Improvedpredictionmodelswereobtainedbycombinationofdatafromthetwosensorsandbyvariableselectionusingageneticalgorithm,intervalPLS,andtheprincipalvariablesmethod,respectively.Astepwisevariableeliminationmethodwasappliedtothethree-wayfluorescencedata,resultinginsimplerandmoreaccurateN-PLSmodels.Thepredictionmodelswerevalidatedusingleave-one-batch-outcross-validation,andthebestmodelshadrootmeansquareerrorofcross-validationvaluesof1.02gl-1biomassand0.8gl-1totalaminoacids,respectively.Thefluorescencedatawerealsoexploredbyparallelfactoranalysis.Theanalysisrevealedfourspectralprofilespresentinthefluorescencedata,threeofwhichwereidentifiedaspyridoxine,NAD(P)H,andflavinnucleotides,respectively.Sealingabilityofanewpolydimethylsiloxane-basedrootcanalfillingmaterial.Özok,A.R.,vanderSluis,L.W.M.,Wu,M.K.&Wesselink,P.R.(2008).JournalofEndodontics,34(2),204-207.LinktoArticleReadAbstractWetestedthenullhypothesisthatthereisnodifferenceinthesealingabilityofGuttaFlow,RoekoSeal,andAH26inrootcanals.SixtyextractedmandibularpremolarswerefilledwithAH26(lateralcompaction),RoekoSeal,orGuttaFlow(modifiedsingle-cone).Thesealingabilityoftherootcanalfillingswasmeasuredweekly(4weeks)byusingaglucosepenetrationmodel.Kruskal-Wallistestrevealedsignificantdifferencesinglucosepenetrationbetweentheexperimentalgroupsatweeks1,2,3,and4.WhereasGuttaFlowshowedthehighestamountofleakageatalltimes,AH26showedthelowest.TherewasnosignificantdifferencebetweenRoekoSeal-filledandAH26-filledrootcanalsthroughouttheexperimentalperiod.AH26showedbettersealingabilityinrootcanalsthanGuttaFlow.Adiposetriglyceridelipaseplaysakeyroleinthesupplyoftheworkingmusclewithfattyacids.Schoiswohl,G.,Schweiger,M.,Schreiber,R.,Gorkiewicz,G.,Preiss-Landl,K.,Taschler,U.,Zierler,K.A.,Radner,F.P.W.,Eichmann,T.O.,Kienesberger,P.C.,Eder,S.,Lass,A.,Haemmerle,G.,Alsted,T.J.,Kiens,B.,Hoefler,G.,Zechner,R.&Zimmermann,R.(2010).JournalofLipidResearch,51(3),490-499.LinktoArticleReadAbstractFAsaremobilizedfromtriglyceride(TG)storesduringexercisetosupplytheworkingmusclewithenergy.Micedeficientforadiposetriglyceridelipase(ATGL-ko)exhibitdefectivelipolysisandaccumulateTGinadiposetissueandmuscle,suggestingthatATGLdeficiencyaffectsenergyavailabilityandsubstrateutilizationinworkingmuscle.Inthisstudy,weinvestigatedtheeffectofmoderatetreadmillexerciseonbloodenergymetabolitesandliverglycogenstoresinmicelackingATGL.BecauseATGL-komiceexhibitmassiveaccumulationofTGintheheartandcardiomyopathy,wealsoinvestigatedamousemodellackingATGLinalltissuesexceptcardiacmuscle(ATGL-ko/CM).IncontrasttoATGL-komice,thesemicedidnotaccumulateTGintheheartandhadnormallifeexpectancy.ExerciseexperimentsrevealedthatATGL-koandATGL-ko/CMmiceareunabletoincreasecirculatingFAlevelsduringexercise.ThereducedavailabilityofFAforenergyconversionledtorapiddepletionofliverglycogenstoresandhypoglycemia.Together,ourstudiessuggestthatATGL-komicecannotadjustcirculatingFAlevelstotheincreasedenergyrequirementsoftheworkingmuscle,resultinginanincreaseduseofcarbohydratesforenergyconversion.Thus,ATGLactivityisrequiredforproperenergysupplyoftheskeletalmuscleduringexercise.Experimentalevidenceforasignificantcontributionofcellulosetoindooraerosolmassconcentration.Cerqueira,M.,Marques,D.,Caseiro,A.&Pio,C.(2010).AtmosphericEnvironment,44(6),867-871.LinktoArticleReadAbstractAnapartmentbedroomlocatedinaresidentialareaofAveiro(Portugal)wasselectedwiththeaimofcharacterizingthecellulosecontentofindooraerosolparticles.Twosetsofsamplesweretaken:(1)PM10collectedsimultaneouslyinindoorandoutdoorair;(2)PM10andPM2.5collectedsimultaneouslyinindoorair.Theaerosolparticleswereconcentratedonquartzfibrefilterswithlow-volumesamplersequippedwithsizeselectiveinlets.Thefilterswereweighedandthenextractedforcelluloseanalysisbyanenzymaticmethod.Theaverageindoorcelluloseconcentrationwas1.01±0.24μgm-3,whereastheaverageoutdoorcelluloseconcentrationwas0.078±0.047μgm-3,accountingfor4.0%and0.4%,respectively,ofthePM10mass.Thecorrespondingaverageratiobetweenindoorandoutdoorcelluloseconcentrationswas11.1±4.9,indicatingthatcelluloseparticlesweregeneratedindoors,mostlikelyduetothehandlingofcotton-madetextilesasaresultofroutinedailyactivitiesinthebedroom.Indoorcelluloseconcentrationsaveraged1.22±0.53μgm-3intheaerosolcoarsefraction(determinedfromthedifferencebetweenPM10andPM2.5concentrations)andaveraged0.38±0.13μgm-3intheaerosolfinefraction.Theaverageratiobetweenthecoarseandfinefractionsofcelluloseconcentrationsintheindoorairwas3.6±2.1.Thisratioisinlinewiththeprimaryoriginofthisbiopolymer.ResultsfromthisstudyprovidethefirstexperimentalevidenceinsupportofasignificantcontributionofcellulosetothemassofsUSPendedparticlesinindoorair.EffectofHXT1andHXT7hexosetransporteroverexpressiononwild-typeandlacticacidproducingSaccharomycescerevisiaecells.Rossi,G.,Sauer,M.,Porro,D.&Branduardi,P.(2010).MicrobialCellFactories,9(1),15.LinktoArticleReadAbstractBackground:Sinceaboutthreedecades,Saccharomycescerevisiaecanbeengineeredtoefficientlyproduceproteinsandmetabolites.Evenrecognizingthatinbaker"syeastonedeterminingstepfortheglucoseconsumptionrateisthesugaruptake,thisfacthasneverbeenconceivedtoimprovethemetabolite(s)productivity.Inthisworkwecomparedtheethanoland/orthelacticacidproductionfromwildtypeandmetabolicallyengineeredS.cerevisiaecellsexpressinganadditionalcopyofonehexosetransporter.Results:DifferentS.cerevisiaestrains(wildtypeandmetabolicallyengineeredforlacticacidproduction)weretransformedwiththeHXT1ortheHXT7geneencodingforhexosetransporters.DataobtainedsuggestthattheoverexpressionofanHxttransportermayleadtoanincreaseinglucoseuptakethatcouldresultinanincreasedethanoland/orlacticacidproductivities.Asaconsequenceoftheincreasedproductivityandofthereducedprocesstiming,ahigherproductionwasmeasured.Conclusions:Metabolicpathwaymanipulationforimprovingthepropertiesandtheproductivityofmicroorganismsisawellestablishedconcept.Ahighproductionreliesonamulti-factorialsystem.Weshowedthatbymodulatingthefirststepofthepathwayleadingtolacticacidaccumulationanimprovementofabout15%inlacticacidproductioncanbeobtainedinayeaststrainalreadydevelopedforindustrialapplication.Constructionandcharacterizationofthreelactatedehydrogenase-negativeEnterococcusfaecalisV583mutants.Jönsson,M.,Saleihan,Z.,Nes,I.F.&Holo,H.(2009).AppliedandEnvironmentalMicrobiology,75(14),4901-4903.LinktoArticleReadAbstractTherolesofthetwoldhgenesofEnterococcusfaecaliswerestudiedusingknockoutmutants.Deletionofldh-1causesametabolicshiftfromhomolacticfermentationtoethanol,formate,andacetoinproduction,withahighlevelofformateproductionevenunderaerobicconditions.Ldh-2playsonlyaminorroleinlactateproduction.UV-methodforthedeterminationofD-Glucoseinfoodstuffs,beveragesandothermaterialsPrinciple:             (hexokinase)(1)D-Glucose+ATP→G-6-P+ADP   (glucose-6-phosphatedehydrogenase)(2)G-6-P+NADP+→gluconate-6-phosphate+NADPH+H+Kitsize:                         (K-GLUHK-110A)* 110assays(manual)/1100(microplate)                                         /1000(auto-analyser)or                                         (K-GLUHK-220A)* 220assays(manual)/2200(microplate)                                          /2000(auto-analyser)* Thenumberofmanualtestsperkitcanbedoubledifallvolumesarehalved. ThiscanbereadilyaccommodatedusingtheMegaQuantTM WaveSpectrophotometer(D-MQWAVE).Method:                          Spectrophotometricat340nmReactiontime:                 ~5minDetectionlimit:                0.66mg/LApplicationexamples:Wine,beer,fruitjuices,softdrinks,milk,jam,dieteticfoods,bakeryproducts,candies,fruitandvegetables,tobacco,cosmetics,pharmaceuticals(e.g.infusions),feed,paper(andcardboard)andothermaterials(e.g.biologicalcultures,samples,etc.)Methodrecognition:   MethodsbasedonthisprinciplehavebeenacceptedbyAOAC,EN,NEN,NF,DIN,GOST,OIV,IFU,AIJNandMEBAKAdvantagesVerycompetitiveprice(costpertest) Allreagentsstablefor>2yearsafterpreparation Rapidreaction Mega-Calc™softwaretoolisavailablefromourwebsiteforhassle-freerawdataprocessing Standardincluded Extendedcofactorsstability Suitableformanual,microplateandauto-analyserformats

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