TheD-GluconicAcid/D-Glucono-δ-lactonetestkitissuitableforthespecificmeasurementandanalysisofD-gluconicacid/D-gluconolactoneinfoodsandbeverages.ExtendedcofactorsstABIlity.Dissolvedcofactorsstablefor>1yearat4oC.Suitableformanual,auto-analyserandmicroplateformats.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.InteractionofNectarin4withafungalproteintriggersamicrobialsurveillanceanddefensemechanisminnectar.Harper,A.D.,Stalnaker,S.H.,Wells,L.,Darvill,A.,Thornburg,R.&York,W.S.(2010).Phytochemistry,71(17-18),1963-1969.LinktoArticleReadAbstractUnderstandingthebiochemicalmechanismsbywhichplantsrespondtomicrobialinfectionisafundamentalgoalofplantscience.Extracellulardermalglycoproteins(EDGPs)arewidelyexpressedinplanttissuesandhavebeenimplicatedinplantdefenseresponses.AlthoughEDGPsareknowntointeractwithfungalproteins,thedownstreameffectsoftheseinteractionsarepoorlyunderstood.Togaininsightintothesephenomena,weusedtobaccofloralnectarasamodelsystemtoidentifyamechanismbywhichtheEDGPknownasNectarinIV(NEC4)functionsaspathogensurveillancemolecule.OurdatademonstratesthattheinteractionofNEC4withafungalendoglucanase(XEG)promotesthecatalyticactivityofNectarinV(NEC5),whichcatalyzestheconversionofglucoseandmolecularoxygentogluconicacidandH2O2.SignificantlyenhancedNEC5activitywasobservedwhenXEGwasaddedtonectarornectarinsolutionsthatcontainNEC4.ThisresponsewasalsoobservedwhenthepurifiedNEC4:XEGcomplexwasaddedtoNEC4-depletednectarinsolutions,whichdidnotrespondtoXEGalone.TheseresultsindicatethatformationoftheNEC4:XEGcomplexisakeystepleadingtoinductionofNEC5activityinfloralnectar,resultinginanincreaseinconcentrationsofreactiveoxygenspecies(ROS),whichareknowntoinhibitmicrobialgrowthdirectlyandactivatesignaltransductionpathwaysthatinduceinnateimmunityresponsesintheplant.TheLysRtranscriptionfactor,HexS,isrequiredforglucoseinhibitionofprodigiosinproductionbySerratiamarcescens.Stella,N.A.,Fender,J.E.,Lahr,R.M.,Kalivoda,E.J.&Shanks,R.M.(2012).AdvancesinMicrobiology,2(4).LinktoArticleReadAbstractGenerationofmanyusefulmicrobe-derivedsecondarymetabolites,includingtheredpigmentprodigiosinofthebacteriumSerratiamarcescens,isinhibitedbyglucose.Inapreviousreport,ageneticapproachwasusedtodeterminethatglucosedehydrogenaseactivity(GDH)isrequiredforinhibitingprodigiosinproductionandtranscriptionoftheprodigiosinbiosyntheticoperon(pigA-N).However,thetranscriptionfactor(s)thatregulatethisprocesswerenotcharacterized.HerewetestedthehypothesisthatHexS,aLysR-familytranscriptionfactorsimilartoLrhAofEscherichiacoli,isrequiredforinhibitionofprodigiosinbygrowthinglucose.WeobservedthatmutationofthehexSgeneinS.marcescensallowedtheprecociousproductionofprodigiosininglucose-richmediumconditionsthatcompletelyinhibitedprodigiosinproductionbythewildtype.Unlikepreviouslydescribedmutantsabletogenerateprodigiosininglucose-richmedium,hexSmutantsexhibitedGDHactivityandmediumacidificationsimilartothewildtype.GlucoseinhibittionofpigAexpressionwasshowntobedependentuponHexS,suggestingthatHexSisakeytranscriptionfactorinsecondarymetaboliteregulationinresponsetomediumpH.Thesedatagiveinsightintotheprodigiosinregulatorypathwayandcouldbeusedtoenhancetheproductionofsecondarymetabolites.ModelingofContinuousGluconicAcidProductionbyFermentation.Fatmawati,A.&Agustriyanto,R.(2010).ScienceJournal.1(1),82-89.LinktoArticleReadAbstractThebatchfermentationkineticofgluconicacidproductionhasbeenstudied.ThecontinuousfermentationprocessofglucosebyAspergillusnigertoproducegluconicacidundertheinfluenceofinletsubstrateconcentrationandhydraulicretentiontimehasalsobeeninvestigated.Thefermentationwasmodeledtobecarriedoutinacontinuousstirredtankreactor.Theresultsshowedthatatthestudiedinletglucoseconcentrationof150,200,and250g/l,thehydraulicretentiontimeresultedintheincreasingamountofcellandgluconicacidconcentrationbutdecreasingglucoseconcentrationattheoutletstreamofthereactor.Themodelresultsalsosuggestedthatthepossiblerangeofhydraulicretentiontimefortheinletsubstrateconcentrationof150,200,and250g/lwere3-13,8-12,and7-11h,respectively.Thereforetherecommendedvaluesofhydraulicretentiontimewere13,12and11hfortheinletsubstrateconcentrationof150,200,and250g/l,respectively.Geneticdiversityofphosphate-solubilizingpeanut(ArachishypogaeaL.)associatedbacteriaandmechanismsinvolvedinthisability.Anzuay,M.S.,Frola,O.,Angelini,J.G.,Ludueña,L.M.,Fabra,A.&Taurian,T.(2013).Symbiosis,60(3),143-154.LinktoArticleReadAbstractInthisstudy,attemptsweremadetoanalyzemechanismsinvolvedinthebacterialphosphate-solubilizingabilityofpeanutisolates.Bacteriaweretaxonomicallyidentifiedbyanalysisof16SrDNAsequence.LevelsofsolublePreleasedbytheisolatesinunbufferedorbufferedwithTris–HClorMESNBRIP-BPBmediumaswellastheproductionofD-gluconicacidweredeterminedintheirculture.PresenceoftwoofthegenesencodingthecofactorPQQofGDHenzymewasanalyzedinthegenomeofthisbacterialcollection.16SrDNAsequenceanalysisindicatedthatisolatesbelongtogeneraSerratia,Enterobacter,Pantoea,Acinetobacter,BacillusandEnterococcus.Allbacteriashowedabilitytosolubilizetricalciumphosphateeitherinunbufferedorbufferedmedium.Nevertheless,additionofbuffersolutionsreducedlevelsofPiliberatedbytheisolates.AlthoughalmostallisolatesproduceddetectableamountsofD-gluconicacid,nocorrelationwithlevelsofPsolublereleasedwereobserved.ThepresenceofpqqEandpqqCgeneswasdetectedonlyinGramnegativebacteria.Itwasconcludedfromthisstudythatthemechanisminvolvedinphosphatesolubilizationisorganicacidsproductionand,presenceofpqqgenesinallGramnegativebacteriaanalyzedencouragestoconfirmtheirroleinbacterialphosphatesolubilizingabilityaswelltoidentifygenesinvolvedinthisPGPtraitinGrampositivebacteria.Aerobicdeconstructionofcellulosicbiomassbyaninsect-associatedStreptomyces.Takasuka,T.E.,Book,A.J.,Lewin,G.R.,Currie,C.R.&Fox,B.G.(2013).ScientificReports,3.LinktoArticleReadAbstractStreptomycesarebestknownforproducingantimicrobialsecondarymetabolites,buttheyarealsorecognizedfortheircontributionstobiomassutilization.Despitetheirimportancetocarboncyclinginterrestrialecosystems,ourunderstandingofthecellulolyticabilityofStreptomycesiscurrentlylimitedtoafewsoil-isolates.Here,wedemonstratethebiomass-deconstructingcapabilityofStreptomycessp.SirexAA-E(ActE),anaerobicbacteriumassociatedwiththeinvasivepine-boringwoodwaspSirexnoctilio.Whengrownonplantbiomass,ActEsecretesasuiteofenzymesincludingendo-andexo-cellulases,CBM33polysaccharide-monooxygenases,andhemicellulases.Genome-widetranscriptomicandproteomicanalyses,andbiochemicalassayshaverevealedthekeyenzymesusedtodeconstructcrystallinecellulose,otherpurepolysaccharides,andbiomass.Themixtureofenzymesobtainedfromgrowthonbiomasshasbiomass-degradingactivitycomparabletoacellulolyticenzymecocktailfromthefungusTrichodermareesei,andthusprovidesacompellingexampleofhighcellulolyticcapacityinanaerobicbacterium.Serratiamarcescensquinoproteinglucosedehydrogenaseactivitymediatesmediumacidificationandinhibitionofprodigiosinproductionbyglucose.Fender,J.E.,Bender,C.M.,Stella,N.A.,Lahr,R.M.,Kalivoda,E.J.&Shanks,R.M.(2012).AppliedandEnvironmentalMicrobiology,78(17),6225-6235.LinktoArticleReadAbstractSerratiamarcescensisamodelorganismforthestudyofsecondarymetabolites.Thebiologicallyactivepigmentprodigiosin(2-methyl-3-pentyl-6-methoxyprodiginine),likemanyothersecondarymetabolites,isinhibitedbygrowthinglucose-richmedium.WhereaspreviousstudiesindicatedthatthisinhibitoryeffectwaspHdependentanddidnotrequirecyclicAMP(cAMP),thereisnoinformationonthegenesinvolvedinmediatingthisphenomenon.Hereweusedtransposonmutagenesistoidentifygenesinvolvedintheinhibitionofprodigiosinbyglucose.Multiplegeneticlociinvolvedinquinoproteinglucosedehydrogenase(GDH)activitywerefoundtoberequiredforglucoseinhibitionofprodigiosinproduction,includingpyrroloquinolinequinoneandubiquinonebiosyntheticgenes.UponassessingwhethertheenzymaticproductsofGDHactivitywereinvolvedintheinhibitoryeffect,weobservedthatD-glucono-1,5-lactoneandD-gluconicacid,butnotD-gluconate,wereabletoinhibitprodigiosinproduction.ThesedatasupportamodelinwhichtheoxidationofD-glucosebyquinoproteinGDHinitiatesareductioninpHthatinhibitsprodigiosinproductionthroughtranscriptionalcontroloftheprodigiosinbiosyntheticoperon,providingnewinsightintothegeneticpathwaysthatcontrolprodigiosinproduction.Strainsgeneratedinthisreportmaybeusefulinlarge-scaleproductionofsecondarymetabolites.Applyingsystemsbiologytoolstostudyn‐butanoldegradationinPseudomonasputidaKT2440.Vallon,T.,Simon,O.,Rendgen‐Heugle,B.,Frana,S.,Mückschel,B.,Broicher,A.,Siemann-Herzberg,M.,Pfannenstiel,J.,Hauer,B.,Huber,A.,Breuer,M.&Breuer,M.(2015).EngineeringinLifeSciences,15(8),760-771.LinktoArticleReadAbstractTosmoothentheprocessofn-butanolformationinPseudomonasputidaKT2440,detailedknowledgeoftheimpactofthisorganicsolventoncellphysiologyandregulationisofoutmostimportance.Here,weconductedadetailedsystemsbiologystudytoelucidatecellularresponsesatthemetabolic,proteomic,andtranscriptionallevel. PseudomonasputidaKT2440wascultivatedinmultiplechemostatfermentationsusingn-butanoleitherassolecarbonsourceortogetherwithglucose.PseudomonasputidaKT2440revealedmaximumgrowthrates(µ)of0.3 h-1withn-butanolassolecarbonsourceandof0.4 h-1usingequalC-molaramountsofglucoseandn-butanol.WhileC-molespecificsubstrateconsumptionandbiomass/substrateyieldsappearedequalatthesegrowthconditions,thecellularphysiologywasfoundtobesubstantiallydifferent:adenylateenergychargelevelsof0.85werefoundwhenn-butanolservedassolecarbonsource(similartoglucoseassolecarbonsource),butwerereducedto0.4when n-butanolwascoconsumedatstablegrowthconditions.FurThermore,characteristicmaintenanceparameterschangedwithincreasing n-butanolconsumption.13Cfluxanalysisrevealedthatcentralmetabolismwassplitintoaglucose-fueledEntner–Doudoroff/pentose-phosphatepathwayandann-butanol-fueledtricarboxylicacidcyclewhenbothsubstrateswerecoconsumed.Withthehelpoftranscriptomeandproteomeanalysis,thedegradationpathwayofn-butanolcouldbeunraveled,thusrepresentinganimportantbasisforrenderingP.putidaKT2440fromann-butanolconsumertoaproducerinfuturemetabolicengineeringstudies.RapidAssessmentofGrayMold(Botrytiscinerea)InfectioninGrapesUsingBiosensorsSystem.Cinquanta,L.,Albanese,D.,DeCurtis,F.,Malvano,F.,Crescitelli,A.&DiMatteo,M.(2015).AmericanJournalofEnologyandViticulture,66(4).LinktoArticleReadAbstractBotrytiscinereaisresponsibleforthegraymolddisease,whichcausesconsiderableeconomiclossesforwinemakers.Itsevaluationinwinegrapesiscommonlyperformedthroughvisualestimation,whichwasdemonstratedtobepronetoassessorbias.RapidandsimpleenzymaticcarbonscreenprintedamperometricbiosensorswerehereusedtoevaluategluconicacidandglycerolcontentonwinegrapesatdifferentB.cinereainfectiondegrees.Thelowerconcentrationsmeasurablebyscreen-printedamperometricbiosensorswere3mg/Lforgluconicacid(correspondingtoaninfectiondegreelowerthan1%)and35mg/Lforglycerol;theresponsetimeswithaflowrateof0.5mL/minwereinarangeof0.5to2mininthelinearranges.Thisstudydemonstratestheeffectivenessofthebiosensorsforrapidanalysisofgluconicacidandglycerolingrapes,confirmingtheirhighcorrelationwithB.cinereadegreeofinfection(R2 =0.98).Thus,thebiosensordevelopedtomeasuregluconicacidingrapes(ormust),wasmoreprecise,andgaveafasterresponsethanmethodsthatcurrentlyexistallowingthepercentageofinfectionofgrapeberriesbyB.cinereatobeevaluated.Revalorizationofstrawberrysurplusesbybio-transformingitsglucosecontentintogluconicacid.Cañete-Rodríguez,A.M.,Santos-Dueñas,I.M.,Jiménez-Hornero,J.E.,Torija-Martínez,M.J.,Mas,A.&García-García,I.(2016).FoodandBioproductsProcessing,99,188-196.LinktoArticleReadAbstractModernsocietiesproducemassivesurplusesoffood,by-productsandwastesthatincreasetheinterestfortheirrevalorization.ThisworkexaminestheuseofacultureofGluconobacterjaponicusCECT8443,withoutpHcontrol,toconvertselectivelytheglucosecontentofindustriallypasteurizedstrawberrypuréeintogluconicacidforthedevelopmentofnewbeverages.However,dependingontheinitialconcentrationofglucose,themicroorganismcouldtransformtheacidformedintoothercompounds;forthisreason,inthisworktheeffectofinitialsugarconcentrationonthepreservationoftheacidwasinvestigated.Theresultsshowthatthegluconicacidformedinstrawberrypuréecontainingnoaddedsugarsstartedtodisappearafterglucosedepletion,buttheacidconcentrationremainedconstantifsugar-enrichedpuréewasused.Theuseofthisindustrialsubstrateresultedinthepresenceofyeastsandhenceinsomefructoseuptake;however,thefructoseconsumptionwasnegligibleuntilafter20–30 h.Theuseoffoodby-productsisanexcellentopportunitynotonlytorecovervaluablecompoundsbutforthedevelopmentofnewchemicalandbiotechnologicalapproachesfortheirrevalorization.Thisstrategyshouldimproveregionaleconomiesandcontributetoasustainablemanagementoftheseunderexploitedresources.AnapproachforestimatingthemaximumspecificgrowthrateofGluconobacterjaponicusinstrawberrypuréewithoutcellconcentrationdata.Cañete-Rodríguez,A.M.,Santos-Dueñas,I.M.,Jiménez-Hornero,J.E.,Torija-Martínez,M.J.,Mas,A.&García-García,I.(2016).BiochemicalEngineeringJournal,105,314-320.LinktoArticleReadAbstractTheestimationofthemaximumspecificgrowthrate(µmax)fornon-readilyculturablebacteria,growingoncomplexmediacontainingsUSPendedsolids,isadifficulttaskconsideringtheimportantproblemsinobtainingreliablemeasuresofcellconcentration.AnexampleofthissituationcanbeacultureofGluconobacterjaponicusgrowinginstrawberrypuréeforproducinggluconicacid.BasedonthedependencybetweenenergyrequirementsofthegenusGluconobacterandsubstrateuptakeaswellasitsconstantrelationshipbetweengluconicacidproductionandtotalsubstrateuptake,thetotalsubstrateconcentrationprofileduringtheexponentialgrowthphasecouldbeusedforestimatingµmaxwithoutcellconcentrationmeasures.Inthiscase,thehighselectivityofthestrainforglucoseincomparisontofructoseresultedinnofructoseconsumptionduringthebatch;so,justusingtheglucoseconcentrationsdataduringtheexponentialphaseallowustoobtainanestimationofµmax.Additionally,aroughestimationoftheapparentandstoichiometricyieldsofcellonglucoseisalsopossible.UV-methodforthedeterminationofD-GluconicAcidandD-Glucono-δ-lactoneinfoodstuffs,beveragesandothermaterialsPrinciple:             (gluconatekinase)(1)D-Gluconate+ATP→gluconate-6-phosphate+ADP             (gluconate-6-phosphatedehydrogenase)(2)Gluconate-6-phosphate+NADP+→ribulose-5-phosphate+                                    NADPH+CO2+H+                         (pH11)(3)D-Glucono-δ-lactone+H2O→D-gluconateKitsize:  * 60assays(manual)/600(microplate)                                          /600(auto-analyser)* Thenumberofmanualtestsperkitcanbedoubledifallvolumesarehalved. ThiscanbereadilyaccommodatedusingtheMegaQuantTM WaveSpectrophotometer(D-MQWAVE).Method:                          Spectrophotometricat340nmReactiontime:                 ~6minDetectionlimit:                0.792mg/LApplicationexamples:Wine,meat,processedmeat(e.g.additives),fruitjuice,dairyproducts,pharmaceuticals,paperandothermaterials(e.g.biologicalcultures,samples,etc.)Methodrecognition:   MethodsbasedonthisprinciplehavebeenacceptedbyISO,DINandGOSTAdvantagesAllreagentsstablefor>2yearsafterpreparation Verycompetitiveprice(costpertest) Veryrapidreaction Mega-Calc™softwaretoolisavailablefromourwebsiteforhassle-freerawdataprocessing Standardincluded Extendedcofactorsstability Suitableformanual,microplateandauto-analyserformats

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