Wereporthereinthedevelopmentofanovelassayprocedureforthemeasurementofβ-glucanaseandlichenase(EC3.2.1.73)incrudeenzymeextracts.Twoassayformatsbasedona)adirectcleavageorb)anenzymecoupledsubstratewereinitiallyinvestigated.The‘directcleavage’substrate,namely4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-3¹-cellotriosyl-β-glucopyranoside(MBG4),wasfoundtobethemoregenerallyapplicablereagent.Thissubstratewasfullycharacterisedusingacrudemaltβ-glucanaseextract,abacteriallichenase(Bacillussp.)andanon-specificendo-1,3(4)-β-glucanasefromClostridiumThermocellum(EC3.2.1.6).Standardcurveswerederivedthatallowtheassayabsorbanceresponsetobedirectlyconvertedtoβ-glucanase/lichenaseactivityonbarleyβ-glucan.ThespecificityofMBG4wasconfirmedbyanalysingtheactionofcompetingglycosylhydrolasesthataretypicallyfoundinmaltonthesubstrate.Manualandautomatedassayformatsweredevelopedfortheanalysisofa)β-glucanaseinmaltflourandb)lichenaseenzymeextractsandtherepeatABIlityoftheseassayswasfullyinvestigated.

AProcedurehasbeendevelopedfortheassayofmaltβ-glucanase[a(1→3)(1→4)-β-D-glucanase]whichemploysassubstrate,barleyβ-glucandyedwithRemazolbrilliantBlueandchemicallymodifiedwithcarboxymethylgroupstoincreasesolubility.Thedescribedassayproceduretogetherwithamodifiedextractionformatallowsanalysisofuptotenmaltsamplesinlessthan80min.Also,theprocedureisspecificforenzymesactiveonbarleyβ-glucan,isaccurateandreliable,andcanbereADIlyappliedtotheanalysisofβ-glucanaseinmalt,greenmaltandwort.

Asimpleprocedurefortheassayof(1→3)(1→4)-β-D-glucanase(lichenase)hasbeendeveloped.Thisassayemploysassubstratebarley(1→3)(1→4)-β-D-glucandyedwithRemazolbrilliantBlueRandchemicallymodifiedwithcarboxymethylgroupstoincreasesolubility.Preparationofthissubstraterequiredthedevelopmentofanimprovedprocedurefortheextractionandpurificationofbarleyβ-glucan.AssaysbasedontheuseofthedescribedchromogenicsubstrateatpH6•5aresensitiveandspecificforenzymesactiveonbarleyβ-glucan.

Brewing,theoldestapplicationofbio-technologyisnowamixoftradeartandmodernscience.Thisarticledescribesnewapplicationsofenzymechemistrytotrouble-shootinginbeerproduction.

Aprocedurerecentlydescribedfortheassayofmaltβ-glucanase,whichemploysadye-labelledandchemically-modifiedbarleyβ-glucansubstrate,hasbeenimprovedbychangingtheprecipitantsolutionusedtoterminatethereaction.Thenewprecipitantsolutioncontains0•4%(w/v)zincacetateand4%(w/v)sodiumacetatedissolvedin80%(v/v)aqueousmethylcellosolve.Withthisprecipitanttheprocedurecanbedirectlyappliedtotheassayofcellulaseactivity,andwithminormodification,totheassayoflichenaseactivity.

β-Glucanasefrombarleymaltisknowntobethermolabilebutimportantinthemashingprocess.Therefore,thepotentialofincreasingthethermostabilityofβ-glucanaseinACESbuffer(0.1M,pH5.6)byhighhydrostaticpressurehasbeeninvestigated.Inactivationoftheenzymeaswellaschangesoftheconversionrateinresponsetocombinedpressure-temperaturetreatmentsintherangeof0.1–900MPaand30–75°Cwereassessedbyanalyzingthekineticrateconstants.Asignificantstabilizationofβ-glucanaseagainsttemperature-inducedinactivationwasdetectedat400MPa.Withincreasingpressureupto600MPathecatalyticactivityofβ-glucanasewasprogressivelydecelerated.However,fortheoveralldepolymerizationreactionofβ-glucansinACESbuffer(0.1M,pH5.6)amaximumwasidentifiedat215MPaand55°Cyieldingapproximately2/3higherdegradationofβ-glucanafter20minascomparedtothemaximumatambientpressure(45°C).

Maltingistheidealstagetodealwithβ-glucans.Theirhydrolysisisveryimportantasthediffusionofbothhormonesandhydrolyticenzymesintheendospermofgerminatedgraindependonit.Ahighmaltβ-glucanaseactivityisnotaguaranteeofanextensivehydrolysisofβ-glucans.WhenBacillussubtilisisusedtocontrolmouldgrowth,redsorghummaltβ-glucanaseactivity(measuredusingcarboxymethylcelluloseasthesubstrate)wasimprovedwithoutsignificantlyaffectingthehydrolysisofmaltβ-glucans.Thus,inordertoreducetheresidualβ-glucanscontent,soakingin0.2%NaOHwascombinedwithabiocontrol.Soakingin0.2%NaOHisrecognizedascapableofimprovinggrainhydrationbyopening-uptheendospermcellwalls.Thecombineduseof0.2%NaOHwithB.subtilis-basedbiocontroltreatmentsduringredsorghummalting,leadstomaltwithincreasedβ-glucanaseactivityandasignificantreductionofresidualβ-glucanswhencomparedwiththe16hbiocontrolsteepingwithoutpriorsteepingin0.2%NaOH.β-glucanaseactivityincreaseswithincreasedgerminationtemperatureandtimewhile,conversely,theresidualβ-glucanscontentofthemaltsdecreases.Indeed,whilethelevelofβ-glucanasewasnotvastlydifferentbetweenthemaltsobtainedaftersteepingindistilledwaterandthoseobtainedafter8hsteepingin0.2%NaOHfollowedby8hresteepingindistilledwater(NaOH+H₂Otreatment),theirresidualβ-glucanslevelsdiffersignificantly.B.subtilis-basedtreatmentleadstomaltwithimprovedβ-(1-3)-andβ-(1-4)-glucanaseactivitieswithoutsignificantlyimprovedmaltβ-(1-3),(1-4)-glucanaseactivity.Whilemaltsobtainedafter84hgerminationweren"tsignificantlydifferentintermsofmaltβ-(1-3),(1-4)-glucanaseactivitiesforallsteepingtreatments,theuseof0.2%NaOHsteepingpriortoresteepingledtomaltswithimprovedβ-glucanscontent.Combiningthesteepingindilutealkalineandbiocontrolenablestakingadvantageofthedilutealkalineeffectonresidualβ-glucanscontent,dueprobablytotheopening-upofthecellwallsandtheimprovementofwateruptake,andthatofthebiocontrol(improvementofβ-glucanasesynthesis).

Retinol-bindingprotein(RBP)functionsasatransporterforretinol(vitaminA)inplasmainhighereukaryotes.WehavesuccessfullyexpressedhumanRBPinSaccharomycescerevisiae,anditssecretionwasfoundtobeinducedbyretinolalsointhislowereukaryote.Reducedinductionofsecretionbyretinolinatemperature-sensitivesec18-1mutantthatisblockedinsecretionattherestrictedtemperaturesuggeststhatasinmammaliancells,RBPcanbereleasedfromtheendoplasmicreticulumuponadditionofretinol.Thus,themolecularmechanisminvolvedinretinol-dependentsecretionofRBPappearstobeconservedinyeast,andthispointstoyeastasaputativemodelsystemforstudyingretinol-regulatedsecretionofRBP.RBPpurifiedfromyeastwasfoundtobeindistinguishablefromRBPpurifiedfromhumanplasmainseveralfunctionalassays.

1.Adietwithadditiontonormalbarleyofmaltfromtransgenicbarleyexpressingaproteinengineered,thermotolerantBacillus(1,3-1,4)-β-glucanaseduringgerminationhaspreviouslybeendemonstratedtoprovideabroilerchickenweightgaincomparabletomaizediets.Italsoreduceddramaticallythenumberofbirdswithadheringstickydroppings,butdidnotentirelyeliminatestickydroppings.Oneoftheobjectivesofthebroilerchickentrialsreportedherewastodetermineifhigherconcentrationsoftransgenicmaltcouldalleviatethestickydroppings.2.Anotheraimwastoinvestigatethefeasibilityofusingmaturetransgenicgraincontainingthethermotolerant(1,3-1,4)-β-glucanaseasfeedadditionandtocomparedietscontainingtransgenicgraintoadietwiththerecommendedamountofacommercialβ-glucanase-basedproduct.3.Inclusionof75or151g/kgtransgenicmaltcontaining4·7or98mg/kgthermotolerant(1,3-1,4)-β-glucanasewith545or469g/kgnon-transgenicbarleyinsteadofmaizeyieldedaweightgaininCornishCrossbroilerchickensindistinguishablefrompresentlyusedmaizediets.Thegeneencodingtheenzymeisexpressedinthealeuronewithabarleyα-amylasegenepromoterandtheenzymeissynthesisedwithasignalpeptideforsecretionintotheendospermofthemaltinggrain.4.Equalweightgainwasachieved,whenthefeedincluded39g/kgtransgenicbarleygrain[containing66mg/kgthermotolerant(1,3-1,4)-β-glucanase]and581g/kgnon-transgenicbarleyinsteadofmaize.Inthiscase,thegeneencodingtheenzymehasbeenexpressedwiththeD-hordeingene(Hor3-1)promoterduringgrainmaturation.Theenzymeissynthesisedasaprecursorwithasignalpeptidefortransportthroughtheendoplasmicreticulumandtargetedintothestoragevacuoles.Depositionoftheenzymeintheprolaminstorageproteinbodiesoftheendospermprotectsitfromdegradationduringtheprogrammedcelldeathoftheendosperminthefinalstagesofgrainmaturationandprovidesextraordinaryheatstability.Thelargeamountofhighlyactive(1,3-1,4)-β-glucanaseinthematuregrainallowedthereductionofthetransgenicgrainingredientto0·2g/kgdiet,thusmakingtheingredientcomparabletothatofthetracemineralsaddedtostandarddiets.5.Adirectcomparisonusingtransgenicgrainsupplementatthelevelof1g/kgoffeedwiththestandardrecommendedadditionofthecommercialenzymepreparationAvizyme1100^®at1g/kgyieldedequalweightgain,feedconsumptionandfeedefficiencyinbirdsfedabarley-baseddiet.6.Theproductionofstickydroppingscharacteristicofbroilersfedonbarleydietswasavoidedwithall9experimentaldietsandreducedtothelevelobservedwithastandardmaizedietbysupplementationWithtransgenicbarley.7.Theexcellentgrowthandnormalsurvivalofthe400broilerstestedonbarleydietssupplementedwithtransgenicgrainormaltshowedthegrainandmaltnottobetoxic.8.Thebarleyfeedwithaddedtransgenicgrainormaltcontainingthermotolerant(1,3-1,4)-β-glucanaseprovidesanenvironmentallyfriendlyalternativetoenzymeadditives,asitusesphotosyntheticenergyforproductionoftheenzymeinthegrainandthusavoidsuseofnon-renewableenergyforfermentation.Thedepositionoftheenzymeintheproteinbodiesofthegraininthefieldmakescoatingproceduresforstabilisationofenzymeactivitysuperfluous.9.Barleyfeedwiththesmallamountoftransgenicgrainasadditivetonormalbarleyprovidesanalternativeforbroilerfeedinareaswheregrainmaizecannotbegrownforclimaticreasonsorbecauseofunsuitablesoilandthushastobeimported.

Enzymaticmodificationoftheendospermofmaltingbarleyisamainfeatureofthemaltingprocess.Unevenenzymaticmodificationoftheendosperm(heterogeneity)cancausebrewhouseproblems.Althoughthereisageneralcorrelationbetweenendospermmodification,beta-glucanbreakdownandendo-beta-glucanasedevelopment,itisbasedonaverageresultsfromsampleanalysesandmayconcealheterogeneity.Theprimaryaimofthisworkwastouseindividualgrainanalysestoinvestigatefactorsthatcontrolendospermmodificationandbeta-glucanbreakdown.Intermsofbeta-glucanbreakdownandphysicalmodification,thebarleyvarietyChariotmaltedfasterthanDecanter.However,bothvarietiesshowedsimilardistributionofendo-beta-glucanaseinindividualgrainsduringmalting.Furtherworkonindividualgrainsshowedthatthecorrelationbetweenbeta-glucanbreakdownandendo-beta-glucanaseactivitywasnotsignificant.Surprisinglybeta-glucanbreakdowndidnotalwayscorrelatewiththephysicalmodificationoftheendosperm.Boththesefindingssuggestthatsampleanalysesofbeta-glucanlevelsandmaltbeta-glucanaseactivitiesarenotreliableindicatorsofthedegreesofwhichmaltsamplesaremodifiedduringmalting.Sincethedistributionofbeta-glucaninindividualgrainsoftheunmaltedbarleyvarietieswassimilar,thetotalbeta-glucanlevelsoftheoriginalbarleydidnotdeterminetherateatwhichbeta-glucanwasbroken-downduringmalting.Althoughproteinstudiesareatapreliminarystage,therateofproteinbreakdownwasnotcorrelatedwiththerateatwhichbeta-glucanwasbrokendowninthemaltinggrain.ItispossIBLethatthephysico-chemicalpropertiesofendospermstorageproteinsmaylimittherateofbeta-glucanbreakdownduringmalting.

ThegenecbhBfromthecellulolyticbacteriumCellulomonasfimiencodesapolypeptideof1090aminoacids.CellobiohydrolaseB(CbhB)is1037aminoacidslong,withacalculatedmolecularmassof109765Da.Theenzymecomprisesfivedomains:anN-terminalcatalyticdomainof643aminoacids,threefibronectintypeIIIrepeatsof97aminoacidseach,andaC-terminalcellulose-bindingdomainof104aminoacids.Thecatalyticdomainbelongstofamily48ofglycosylhydrolases.CbhBhasaverylowactivityonCM-cellulose.ViscometricanalysisofCM-cellulosehydrolysisindicatesthattheenzymeisanexoglucanase.Cellobioseisthemajorproductofhydrolysisofcellulose.IncommonwithtwootherexoglycanasesfromC.fimi,CbhBhaslowbutdetectableendoglucanaseactivity.CbhBisthesecondexo-cellobiohydrolasefoundinC.fimi.Therefore,thecellulasesystemofC.fimiresemblesthoseoffungiincomprisingmultipleendoglucanasesandcellobiohydrolases.

1,3-1,4-β-glucanasewasanimportantbiotechnologicalaidinthebrewingindustry.Inapreviousresearch,aBacillusBglTOmutant(BglTO)withhightolerancetowardshightemperatureandlow-pHconditionswasconstructedandexpressedinEscherichiacoli.However,E.coliwasnotasuitablehostforenzymeproductioninfoodindustry.Therefore,thepresentworkaimedtoachievethehigh-levelexpressionofBglTOinBacillussubtilisWB600andtotestitseffectinCongressmashing.Theβ-glucanasemutantwassuccessfullyexpressedinB.subtilisWB600andfavorableplasmidsegregationandstructuralstabilitywereobserved.Themaximalextracellularactivityofβ-glucanaseinrecombinantB.subtilisWB600reached4840.4UmL⁻¹aftercultivationconditionoptimization,whichwas1.94-foldhigherthanthatbeforeoptimization.ThefermentationcapacityofrecombinantB.subtilisreached242.02UmL⁻¹h⁻¹,whichwasthehighestamongallreportedβ-glucanases.TheadditionofBglTOinCongressmashingsignificantlyreducedthefiltrationtimeandviscosityofmashby29.7%and12.3%,respectively,whichwassuperiortotwocommercialenzymes.ThesefavorablepropertiesindicatedthatB.subtilisWB600wasasuitablehostforproductionofBglTO,whichwaspromisingforapplicationinthebrewingindustry.