TheD-GluconicAcid/D-Glucono-δ-lactonetestkitissuitableforthespecificmeasurementandanalysisofD-gluconicacid/D-gluconolactoneinfoodsandbeverages.
Grapeandwineanalysis:Oenologiststoexploitadvancedtestkits.
Charnock,S.C.&McCleary,B.V.(2005).RevuedesEnology,117,1-5.
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Itiswithoutdoubtthattestingplaysapivotalrolethroughoutthewholeofthevinificationprocess.Toproducethebestposs
IBLequalitywineandtominimiseprocessproblemssuchas“stuck”fermentationortroublesomeinfections,itisnowrecognisedthatifpossibletestingshouldbeginpriortoharvestingofthegrapesandcontinuethroughtobottling.Tr
ADItionalmethodsofwineanalysisareoftenexpensive,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,suchasmolecular
BIOLOGy(
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.
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Understandingthebiochemicalmechanismsbywhichplantsrespondtomicrobialinfectionisafundamentalgoalofplantscience.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).
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Generationofmanyusefulmicrobe-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.
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Thebatchfermentationkineticofgluconicacidproductionhasbeenstudied.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.
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Inthisstudy,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.
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Streptomycesarebestknownforproducingantimicrobialsecondarymetabolites,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.
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Serratiamarcescensisamodelorganismforthestudyofsecondarymetabolites.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.
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Tosmoothentheprocessof
n-butanolformationin
PseudomonasputidaKT2440,detailedknowledgeoftheimpactofthisorganicsolventoncellphysiologyandregulationisofoutmostimportance.Here,weconductedadetailedsystemsbiologystudytoelucidatecellularresponsesatthemetabolic,proteomic,andtranscriptionallevel. PseudomonasputidaKT2440wascultivatedinmultiplechemostatfermentationsusing
n-butanoleitherassolecarbonsourceortogetherwithglucose.
PseudomonasputidaKT2440revealedmaximumgrowthrates(µ)of0.3 h
-1with
n-butanolassolecarbonsourceandof0.4 h
-1usingequalC-molaramountsofglucoseand
n-butanol.WhileC-molespecificsubstrateconsumptionandbiomass/substrateyieldsappearedequalatthesegrowthconditions,thecellularphysiologywasfoundtobesubstantiallydifferent:adenylateenergychargelevelsof0.85werefoundwhen
n-butanolservedassolecarbonsource(similartoglucoseassolecarbonsource),butwerereducedto0.4when n-butanolwascoconsumedatstablegrowthconditions.Fur
Thermore,characteristicmaintenanceparameterschangedwithincreasing n-butanolconsumption.
13Cfluxanalysisrevealedthatcentralmetabolismwassplitintoaglucose-fueledEntner–Doudoroff/pentose-phosphatepathwayandan
n-butanol-fueledtricarboxylicacidcyclewhenbothsubstrateswerecoconsumed.Withthehelpoftranscriptomeandproteomeanalysis,thedegradationpathwayof
n-butanolcouldbeunraveled,thusrepresentinganimportantbasisforrendering
P.putidaKT2440froman
n-butanolconsumertoaproducerinfuturemetabolicengineeringstudies.
RapidAssessmentofGrayMold(Botrytiscinerea)InfectioninGrapesUsingBiosensorsSystem.
Cinquanta,L.,Albanese,D.,DeCurtis,F.,Malvano,F.,Crescitelli,A.&DiMatteo,M.(2015).AmericanJournalofEnologyandViticulture,66(4).
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Botrytiscinereaisresponsibleforthegraymolddisease,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.
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Modernsocietiesproducemassivesurplusesoffood,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.
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Theestimationofthemaximumspecificgrowthrate(µ
max)fornon-readilyculturablebacteria,growingoncomplexmediacontainings
USPendedsolids,isadifficulttaskconsideringtheimportantproblemsinobtainingreliablemeasuresofcellconcentration.Anexampleofthissituationcanbeacultureof
Gluconobacterjaponicusgrowinginstrawberrypuréeforproducinggluconicacid.Basedonthedependencybetweenenergyrequirementsofthegenus
Gluconobacterandsubstrateuptakeaswellasitsconstantrelationshipbetweengluconicacidproductionandtotalsubstrateuptake,thetotalsubstrateconcentrationprofileduringtheexponentialgrowthphasecouldbeusedforestimatingµ
maxwithoutcellconcentrationmeasures.Inthiscase,thehighselectivityofthestrainforglucoseincomparisontofructoseresultedinnofructoseconsumptionduringthebatch;so,justusingtheglucoseconcentrationsdataduringtheexponentialphaseallowustoobtainanestimationofµ
max.Additionally,aroughestimationoftheapparentandstoichiometricyieldsofcellonglucoseisalsopossible.
