Broccoli(BrassicaoleraceaL.)tissueheldinacontrolledatmosphere(CA;10%carbondioxideand5%oxygen)senescesmoreslowlythantissueheldinair.CA-treatedbroccolitissuesloselesswaterandsolublesugars,havelowerproteaseactivity,andhavenosignificantlossofcolor(hueangle,chlorophyllcontent)for96hafterharvest(20°C,dark)comparedtotissueheldinairthatstartstosenesceandyellowafter48h.ThecurrentstudyexamineddifferentialgeneexpressioninbroccolitissuesinresponsetopostharvestCAtreatment.ThisgeneticanalysiswasundertakentoidentifyCA-responsivegenesthatmayactassignalingelementsandrepresspostharvestsenescenceprocesses.CA-responsivegeneswithup-anddown-regulatedexpression(comparedtoaircontrols)wereisolatedaftera6hCAtreatmentbydifferentialdisplay-polymerasechainreaction.ThecandidateCA-responsivegenesincludedanumberofnovelgeneswithoutpreviouslyassignedfunctions,andgenesofknownfunctionpreviouslyfoundtoberegulatedbystress(e.g.dehydration,saltstress,lowtemperature,andsugarstarvation).

CysteineproteaseinhibitorsdelayedthesenescenceofSandersoniaaurantiacaHook.flowers.TepalfADIngandwiltingoccurredlaterinthe2,2´-dipyridyl-treatedflowers,andtheseflowershadagreatersolubleproteincontentandlessactiveendoproteasescomparedwithcontrolflowersthatwereheldinwater.Biochemicalanalysisrevealedthepresenceofseveralprotease-activebandsinthesolubleproteinfractionSandersoniatepals.Activityofthepolypeptidesincreasedasflowersenescenceprogressed.WesternanalysiswithanantibodyraisedagainstthecastorbeancysteineproteinaseidentifiedhomologousproteinsinSandersoniaflowers(ca46,41and31kDa).ThreeCDNAsencodingcysteineproteinaseswereisolatedfromSandersoniatepals(PRT5,PRT15andPRT22).Expressionofallthreeincreasedintepalsassenescenceprogressed.mRNAsforPRT5weredetectedonlyinsenescingflowertissue,whereasPRT15andPRT22wereexpressedinleaf,stemandroottissue.PRT5hassignificanthomologytoC-terminusKDELproteins,whichhavearoleinthedegradationofplantcellcontentsduringprogrammedcelldeath.PRT15ismostsimilartocysteineproteinaseswithalongC-terminalextension,whereasPRT22ishomologoustostress-inducedcysteineproteinases.

Thepotentialforenzymaticsolubilizationofbrewers’spentgrainbycarbohydrasesandproteaseswasexaminedoverabroadpHrange(pH3.2−11.2).EnzymesfromTrichoderma(Depol686)weremostefficientatalowerpH,whileenzymesfromtheHumicolapreparation(Depol740)werethebestperformeroverthewholerange.Profilingofkeyglycosidehydrolase,esteraseandproteaseactivitiesacrossthepHrangedemonstratedthatsolubilizationofspentgrainbytheTrichodermenzymescorrespondedtotherangeofmaximumactivities.ThiswasnotthecasewiththeHumicolaenzymes,wheremaximumsolubilizationofthesubstrateoccurredatpH9.1,atwhichpHthedeterminedactivitieswerelow.ProteaseactivityinDepol740wasassociatedwithahighsolubilization,butinhibitionofproteolyticactivityresultedinonlya5%decreaseinspentgrainsolubilization.Theseresultssuggestthatwhileenzymescanbeusedtoexploitagro-industrialsbyproduct,theuseofhighpHincreasestheextentofhydrolysisandanunidentifiedfactorproducedbyHumicolaimprovestheenzyme-catalyzedsolubilizationoflignocellulosicmaterial.

TheimpactofmicrobialproteasesonthereleaseofcarbohydratesfromBSGwasstudied.Theproteaseswereabletoreleasethenon-cellulosicglucose,aportionofferuloylatedarABInoxylanandover50%oftheproteinfrombrewers"spentgrain(BSG)after24hhydrolysis.Thenon-cellulosicglucosewasderivedfromresidualstarch-derivedproductspersistinginBSGaftermashing.Theproteasesdidnotcleavethehydroxycinnamateesterlinkagespresentonthearabinoxylanbackbone,andthusdonotbehaveasferuloylesterases.However,thematerialsolubilisedfromspentgrainbytheproteasescontainedupto198µgboundferulicacid/gextract,whichrepresented8.6%ofthetotalferulicacidpresentinBSG.Theseresultssuggestthataportionofwater-extractableferuloylatedarabinoxylanandstarchistrappedwithintheBSGmatrixbyaproteinaceousbarrier.

Theworldwidecontaminationofcereals,oilseeds,andothercropsbymycotoxin-producingmouldsisasignificantproblem.Mycotoxinshaveadverseeffectsonhumansandanimalsthatresultinillnessesandeconomiclosses.Reductionoreliminationofmycotoxincontaminationinfoodandfeedisanimportantissue.Thisstudyaimedtoscreensoilbacteriafordegradationofzearalenone(ZEN).ApurecultureofstrainCK1isolatedfromsoilsamplesshowedmostcapableofdegradationofZEN.Usingphysiological,biochemical,and16SrRNAgenesequenceanalysismethods,CK1wasidentifiedasBacilluslicheniformis.Additionof2ppmofZENinLuria–Bertani(LB)medium,B.licheniformisCK1decreased95.8%ofZENafter36hofincubation.InZEN-contaminatedcornmealmedium,B.licheniformisCK1decreasedmorethan98%ofZENafter36hofincubation.Inaddition,B.licheniformisCK1wasnon-hemolytic,non-enterotoxinproducing,anddisplayedhighlevelsofextracellularxylanase,cellulase,andproteaseactivities.ThesefindingssuggestthatB.licheniformisCK1couldbeusedtoreducetheconcentrationsofZENandimprovethedigestibilityofnutrientsinfeedstuffssimultaneously.

Analeurain-likeprotein,BoCP5,isup-regulatedduringharvest-inducedsenescenceinbroccolifloretandleaftissue.BoCP5ismostcloselyrelatedtoanArabidopsisprotein(91%,AAF43041)andhas71%identitytobarleyaleurain(P05167).ThemRNAforthisgeneaccumulateswithin6hafterharvestinbroccoliflorets,anditsexpressionisreducedintissuethathasbeenheldinsenescence-delayingtreatments(e.g.water,sucrosefeeding,controlledatmosphere).Thegeneisalsoexpressedinleavesduringaging-relatedandharvest-inducedsenescence.Analysisofproteinbandsthatcross-reactwithantibodiesraisedtothebacterialBoCP5fusionprotein,revealedprominentimmunoreactivebandsatca.26,28,31,and38kDinflorettissue.The31kDbandwasabsentinproteinextractsfromleaftissue.Agrobacterium-mediatedtransformationwasusedtoproducetransgenicbroccoliplantswithdown-regulatedBoCP5.AreductioninthepostharvestexpressionofBoCP5inflorettissuewasachievedforfourtransgeniclinesinthecurrentstudy.Inthreeoftheselinespostharvestfloretsenescence(yellowing)wasdelayed,andfloretscontainedsignificantlygreaterchlorophylllevelsduringpostharveststorageat20°Cthanwild-typeplants.Line4showedthegreatestdown-regulationofBoCP5,andinthislinepostharvestproteaseactivityremainedatpre-harvestlevels,andtheyieldofsolubleproteinsextractedfromfloretsafterharvestwassignificantlygreaterthanthatofwild-typetissue.

Crudeproteolyticenzymewasextractedfrompapayalatexusingtwosolvents,waterandphosphatebufferpH6.Theyieldofextractedenzymeusingwaterasasolventwassimilartothatusingphosphatebuffer.Followingthesolventextraction,theextractedenzymewasprecipitatedin45wt%saturatedammoniumsulfatesolution.Theyieldandactivityofprecipitatedenzymeconsiderablydecreased.Crudeproteolyticenzymeextractedusingwaterasanextractingliquidwas,therefore,selectedtouseingelatinproductionfromrawhidehydrolysis,comparingtotheuseofcommercialpapain.Theeffectsofhydrolysisconditionsongelatinrecoveryandpropertiesofobtainedgelatinwereinvestigated.Theoptimumconditionsfortheactivitiesofbothcrudeextractedenzymeandcommercialpapainwereat75°CandpH7.Atthiscondition,thehighestpercentagesofgelatinrecoverywereobtainedfromrawhidehydrolysisreactions.Thegelatinrecoveryandgelstrengthofgelatinobtainedfromcrudeextractedenzymeandcommercialpapainhydrolysisweresimilar.Thisprovedthatcrudeextractedenzymefrompapayalatexcouldbeeffectivelyusedingelatinproduction,insteadoftheuseofcommercialpapain,withacomparativelylowcost.

Harvest‐inducedsenescenceofbroccoliresultsintissuewiltingandsepalchlorosis.Assenescenceprogresses,chlorophyllandproteinlevelsinflorettissuesdeclineandendo‐proteaseactivity(measuredwithazo‐casein)increases.Proteaseactivityincreasedfrom24hafterharvestfortissuesheldinairat20°C.Activitywaslowerinflorettissuesfrombranchletsthathadbeenheldinsolutionsofsucrose(2%w/v)orunderhighcarbondioxide,lowoxygen(10%CO₂,5%O₂)conditions.Fourprotease‐activeproteinbandswereidentifiedinsenescingflorettissuebyzymography,andtheuseofchemicalinhibitorsofproteaseactionsuggeststhatsome44%ofproteaseactivityinsenescingflorettissue72hafterharvestisduetotheactionofcysteineandserineproteases.FourputativecysteineproteasecDNAshavebeenisolatedfrombroccoliflorettissue(BoCP1,BoCP2,BoCP3,BoCP4).ThecDNAsaremostsimilar(73–89%attheaminoacidlevel)todehydration‐responsivecysteineproteasespreviouslyisolatedfromArabidopsisthaliana(RD19,RD21).ThemRNAsencodedbythebroccolicDNAsareexpressedinflorettissueduringharvest‐inducedsenescencewithmRNAaccumulatingwithin6hofharvestforBoCP1,12hofharvestforBoCP4andwithin24hofharvestforBoCP2andBoCP3.InductionofthecDNAsisdifferentiallydelayedwhenbroccolibranchletsareheldinsolutionsofwaterorsucrose.Inaddition,theexpressionofBoCP1andBoCP3isinhibitedintissueheldinatmospheresofhighcarbondioxide/lowoxygen(10%CO₂,5%O₂).TheputativecysteineproteasemRNAsareexpressedbeforemeasurableincreasesinendo‐proteaseactivity,lossofprotein,chlorophyllortissuechlorosis.

WereportontheproductionandselectionoftransgenicBrassicaoleraceavar.Italicalineswithadownregulatedsolubleacidinvertase(BoINV2).Explantsofbroccoli(cv.Triathlon)weretransformedwithanantisenseconstructofBoINV2underthecontrolofanAsparagusofficinalis-derivedharvest-inducedpromoterusingAgrobacteriumtumefaciens-mediatedtransformation.BoINV2isupregulatedinwild-typebroccoliflorettissueafterharvest.TransgenicbroccolilinesshowedreducedBoINV2mRNAaccumulationimmediatelyafterharvestcomparedwithwild-type.DownregulationofBoINV2hadnosignificantimpactontheexpressionofasecondbroccoliacidinvertasegene(BoINV1),butplantswithdownregulatedBoINV2alsohadlowerexpressionofasenescence-associatedcysteineprotease(BoCP5)comparedwithwild-type.ThetotalsolublesugarlevelsinflorettissueofantisenseBoINV2linesweregreaterthanwild-typetissueafterharvest(upto1.5timeshigher).Solubleproteincontentofwild-typetissuedecreasedfrom48hafterharvestwithanincreaseinproteaseactivity.Incomparison,twoantisenseBoINV2linesretainedat-harvestlevelsofsolubleproteinuntil72and96hafterharvestandhadlowerpostharvestendoproteaseactivitycomparedwithwild-type.AntisenseBoINV2linesalsohadaslowerrateoffloretsepalchlorosisafterharvestcomparedwithwild-type.

Thecurrentstudyevaluatedtheeffectofsolubledietarycelluloseongrowth,survivalanddigestiveenzymeactivityinthreeendemic,Australianfreshwatercrayfishspecies(redclaw:Cheraxquadricarinatus,marron:C.tenuimanus,yabby:C.destructor).Separateindividualfeedingtrialswereconductedforlate-stagejuvenilesfromeachspeciesinanautomatedrecirculatingfreshwater,culturesystem.Animalswerefedeitheratestdiet(TD)thatcontained20%solublecelluloseorareferencediet(RD)substitutedwiththesameamountofcornstarch,overa12-weekperiod.RedclawfedwithRDshowedsignificantlyhigher(P<0.05)=""specific=""growth=""rates=""(sgr)=""compared=""with=""animals=""fed=""the=""td,=""while=""sgr=""of=""marron=""and=""yabby=""fed=""the=""two=""diets=""were=""not=""significantly=""different.=""expressed=""cellulase=""activity=""levels=""in=""redclaw=""were=""not=""significantly=""different=""between=""diets.=""marron=""and=""yabby=""showed=""significantly=""higher=""cellulase=""activity=""when=""fed=""the=""rd="">P<0.05).=""amylase=""and=""protease=""activity=""in=""all=""three=""species=""were=""significantly=""higher=""in=""the=""animals=""fed=""with=""rd="">P<0.05).=""these=""results=""indicate=""that=""test=""animals=""of=""all=""three=""species=""appear=""to=""utilize=""starch=""more=""efficiently=""than=""soluble=""dietary=""cellulose=""in=""their=""diet.=""the=""inclusion=""of=""20%=""soluble=""cellulose=""in=""diets=""did=""not=""appear,=""however,=""to=""have=""a=""significant=""negative=""effect=""on=""growth=""rates.="">

BACKGROUND:MpAPr1,encodinganacidproteasefromthewineyeastMetschnikowiapulcherrimaIWBTY1123,waspreviouslyisolatedandshowntodisplaypotentialactivityagainstcaseinandgrapeproteins.However,itscharacterisationremainedpartial.RESULTS:MpAPr1wasclonedintothepGAPZαAvectorandtransformedintoKomagataellapastorisX33forheterologousexpression.Afterverificationofactivity,theenzymepropertieswerecharacterised.ProteaseactivitywithintheconcentratedsupernatantwasretainedoverapHrangeof3.0to5.0andbetween10°Cto50°C.Optimalconditionsforproteaseactivitywerefoundat40°CandpH4.5.ActivitywasmostlyunaffectedbythepresenceofmetalionswiththeexceptionofCu₂₊andNi₂₊.FurThermore,proteolyticactivitywasretainedinthepresenceofsugarandethanol.pHandtemperatureconditionsforMpAPr1expressioninK.pastoriswereoptimised.Purificationwasachievedbymeansofcationexchangechromatographyandkineticparameters(K_mandV_maxweredetermined.MpAPr1activityagainstgrapeproteinswasconfirmed,buttheextentofthedegradationwasdependentonthenatureoftheseproteinsandtheenvironmentalconditions.CONCLUSION::Overall,theresultssuggestthatMpAPr1couldbeappliedinfoodbiotechnologyprocessessuchaswinemaking.

Thesynergyofendopeptidasesandexopeptidasesisthekeyforanefficienthydrolysisofproteins.FlavourzymeissoldasacommercialpeptidasepreparationfromAspergillusoryzaethatexhibitsvariousendo-andexopeptidaseactivitiesand,therefore,generatesproteinhydrolysateswithhighdegreesofhydrolysis.Themanufacturer(Novozymes)standardizestheenzymepreparationforonepeptidaseactivity,determinedwiththeMarkersubstrateH-Leu-pNA.However,sevenpeptidasesofFlavourzymewererecentlyidentifiedandpurified,andthesignificantcontributionofsixofthemtowheatglutenhydrolysiswasdemonstrated.Theknowledgeaboutthebatch-to-batchvariationandstoragestabilityoftheFlavourzymepreparationregardingtheotherpeptidaseactivitiesarestillunclear,andthisisimportantinformationfortheusageoftheenzymepreparationtogainreproducIBLeproteinhydrolysisprocesses.Inthepresentstudy,wetested12Flavourzymebatchesfortheactivityofthesevenpeptidases.Theimpactofthestoragetimeonthepeptidaseactivitiesandthemagnitudeofthebatch-to-batchvariationwereinvestigated.IncontrasttotheactivitydeterminedwithH-Leu-pNAasasubstrate,thevariationsoftheotherpeptidaseactivitieswerenoticeable.Thevariationoftheendopeptidaseactivitywasmostdistinctandtheactivitydecreasedduringthestoragetimeofthepreparation.ThevariationoftheFlavourzymecompositionalsoaffectedthereproducibilityofacaseinbatchhydrolysisprocess,whichshouldbetakenintoaccountforanyfutureresearchandindustrialapplication.