Megazyme/Amyloglucosidase Assay Reagent /RAMGR3/200 assays per kit / 4 vials

Anenzyme-linkedassayforthemeasurementofamyloglucosidaseincommercialenzymemixturesandcrudeculturefiltratesisdescribed.Amethodforthesynthesisofthesubstrateemployed,p-nitrophenylβ-D-maltoside,isalsodescribed.Thesubstrateisusedinthepresenceofsaturatinglevelsofβ-glucosidase.WitharangeofAspergillussp.culturefiltrates,anexcellentcorrelationwasfoundforvaluesobtainedwiththisassayandaconventionalassayemployingmaltoseassubstratewithmeasurementofreleasedglucose.

Theroleofstarch-degradingmycelialfungi,andthealcoholproductionandethanoltoleranceoftheyeastsisolatedfromselectedVietnamesetraditionalricewinestarterswereexamined,andoptimumconditionsfortheseessentialstepsinricewinefermentationweredetermined.OfpureisolatesfromVietnamesericewinestarters,mouldstrainsidentifiedasAmylomycesrouxii,Amylomycesaff.rouxii,RhizopusoligosporusandRhizopusoryzae,weresuperiorinstarchdegradation,glucoseproductionandamyloglucosidaseactivityduringthesaccharificationofpurpleglutinousrice.A.rouxiiwasabletoproduceupto25%w/wglucosewithanamyloglucosidaseactivityupto0.6Ug^-1offermentedmouldedmass.FiveyeastisolatesidentifiedasSaccharomycescerevisiaewereselectedfortheirsuperioralcoholproductivity.Theywereabletodepletearelativelyhighinitialpercentageofglucose(20%w/v),forming8.8%w/vethanol.TheethanoltoleranceofS.cerevisiaeinchallengetestswas9–10%w/v,and13.4%w/vasmeasuredinfed-batchfermentations.Optimumconditionsforthesaccharificationwere:incubationfor2dat34°C,ofsteamedriceinoculatedwith5logcfug^-1;forthealcoholicfermentation4dat28.3°C,ofsaccharifiedriceliquidinoculatedwith5.5logcfumL^-1.

Currentantifoulingtechnologiesforshiphullsarebasedonmetalssuchascuprousoxideandco-biocideslikezincpyrithione.Duetothepersistentadverseenvironmentaleffectsofthesebiocides,enzyme-basedantifoulingpaintsareproposedasabio-based,non-accumulatingalternative.Here,ahydrogenperoxide-producingsystemcomposedofhexoseoxidase(HOX,EC1.1.3.5),glucoamylase(GA,EC3.2.1.3)andstarchistestedforthechemicalandphysicalfunctionalitiesnecessaryforsuccessfulincorporationintoamarinecoating.Theactivityandstabilityoftheenzymesinseawaterwasevaluatedatdifferenttemperatures,andpaintcompatibilitywasassessedbymeasuringthedistributionandactivityoftheenzymesincorporatedintoprototypecoatingformulations.WeusedabiomimeticencapsulationprocedureforHOXthroughpolyethylenimine-templatedsilicaco-precipitation.Theco-precipitationandformulationofapowderformixingintoamarinepaintwasperformedinaone-stepeconomicalandgentleformulationprocess,inwhichsilicaco-precipitatedHOXwascombinedwithGAandstarchtoformtheantifoulingsystem.Silicaco-precipitationsignificantlyimprovedthestabilityandperformanceoftheantifoulingsysteminmarine-likeconditions.Forexample,encapsulationofHOXresultedin46%higheractivityatpH8,anditsstabilityinartificialseawaterincreasedfromretainingonly3.5%activityafter2weekstoretaining55%activityafter12weeks.Acoatingcomprisingthefullenzymesystemreleasedhydrogenperoxideatratesexceedingatargetof36nmolcm^-2d^-1for3monthsinalaboratoryassay,andhadpotentialforprolongedactionthroughincorporationinaself-polishingcoating.

Asafirststepinthedevelopmentofdefinedfungalstartergranulesforcontrolledwinemakingfrompurpleglutinousrice,theinteractionofmouldsandyeastsisolatedfromVietnamesericewinestartersandtheeffectofsomerepresentativeorientalherbsonthegrowthofmouldsandyeastswereexamined.AmylomycesrouxiiandSaccharomycescerevisaewereshowntobecompatibleinmixedcultures,andtheherbs“TieuHoi”(Fennel:FoeniculumvulgareMiller)and“DinhHuong”(Clove:SyzygiumaromaticumL.)whichareusedassupplementaryingredientsbysomelocalstarterproducers,wereobservedtostimulatethemouldandyeastgrowth.Basedontraditionalstartermanufacturingmethodsandmodifiedonthebasisofoptimizationexperiments,alaboratory-scalemanufacturingprocessfordefinedmixed-culturestartergranuleswasestablished.Inaccordancewiththenationalstandardmethod,thewineproducedwithnewexperimentalstartergranuleswasfoundtohavesuperiorflavourandoverallacceptability,comparedwithlocalcommercialricewines.Industrialrelevance.OneofthemajorproblemsfacedbycommercialbrewersofricewineinVietnam,isthevariablequalityandperformanceofthetraditionalstartertabletsthatarecommonlyused.Therelevanceofthepresentpaperisthatastable,granulatedstarterhasbeendeveloped,containingadefinedmixtureofmouldandyeastcultures.Thishasproventobeshelfstableformorethan3months,producingaverywellacceptedqualityofwine.

Theactivitylevelsofamylolyticenzymesandβ-glucanaseinmaltspreparedfromfourtropicalcerealgrainswereassessedtoestablishtherelativeusefulnessofthesemaltsforproductionofglucosesyrups.Ricemaltshowedthehighestactivityfortheamylolyticenzymes,whilstmilletandsorghummaltswererichestinβ-glucanaseactivity.Optimumamylolyticenzymedevelopmentinricemaltsoccurredbetween9–13days;and11daysforoptimumβ-glucanasedevelopmentinmilletmalt.β-Amylasewasthepredominantenzymeinallthecerealmaltsexceptmaize,forwhichthepredominantenzymewasα-amylase.Optionsforoptimisingtheproductionofenzymesfromthethreecereals,andtheirpotentialforuseintheproductionofglucosesyrupsarediscussed.

Thereisanincreasingdemandforbiotech-basedproductionofrecombinantproteinsforuseaspharmaceuticalsinthefoodandfeedindustryandinindustrialapplications.YeastSaccharomycescerevisiaeisamongpreferredcellfactoriesforrecombinantproteinproduction,andthereisincreasinginterestinimprovingitsproteinsecretioncapacity.Duetothecomplexityofthesecretorymachineryineukaryoticcells,itisdifficulttoapplyrationalengineeringforconstructionofimprovedstrains.Hereweusedhigh-throughputmicrofluidicsforthescreeningofyeastlibraries,generatedbyUVmutagenesis.Severalscreeningandsortingroundsresultedintheselectionofeightyeastcloneswithsignificantlyimprovedsecretionofrecombinantα-amylase.Efficientsecretionwasgeneticallystableintheselectedclones.Weperformedwhole-genomesequencingoftheeightclonesandidentified330mutationsintotal.Geneontologyanalysisofmutatedgenesrevealedmanybiologicalprocesses,includingsomethathavenotbeenidentifiedbeforeinthecontextofproteinsecretion.Mutatedgenesidentifiedinthisstudycanbepotentiallyusedforreversemetabolicengineering,withtheobjectivetoconstructefficientcellfactoriesforproteinsecretion.Thecombineduseofmicrofluidicsscreeningandwhole-genomesequencingtomapthemutationsassociatedwiththeimprovedphenotypecaneasilybeadaptedforotherproductsandcelltypestoidentifynovelengineeringtargets,andthisapproachcouldbroadlyfacilitatedesignofnovelcellfactories.