黑麦草玉米谷蛋白水解产物中分离的一种五肽的生物活性

黑麦草玉米谷蛋白水解产物中分离的一种五肽的生物活性BioactivityofaPentapeptideIsolatedfromCornGlutenHydrolysateonLoliumperenneL.anaturalherbicideCornglutenhydrolysate(CGH)FivedipeptidesgreaterisolateandidentifybioactivityPetridishbioassaySephadexG-15gelfiltrationandtwo-stepC18reversedphasehighperformanceliquidchromatographyapentapeptide+0.5mg/mLofthepentapeptide50%ofrootlengthintheperennialryegrassbioassayinhibitCornglutenmeal(CGM)isabyproductofcorn(ZeamaysL.)fromthewet-millingprocessandhasbeenpatentedasanaturalpreemergenceherbicide.Itinhibitsrootgrowthofgerminatingseedsbutdoesnotdamageplantsthathaveformedamaturerootsystem.BecauseCGMcontainsapproximately10%nitrogenbyweight,itisusefulasanaturalpreemergenceherbicideandfertilizerforvariousplantproductionsystems.Cornglutenhydrolysate(CGH),awater-solublematerialderivedfromtheactionofbacterialproteinase,wasfoundmoreherbicidallyactiveagainstthreegrassspeciesthanCGMbothinthesoilandPetridishbioassays.ItalsohasagreaternitrogencontentandcouldpotentiallybeusedtofortifytheCGMtoimproveitasanaturalweedandfeedmaterial.Itshighwatersolubilitymakesitsprayableandeasytoapplyasaherbicideinsomecropproductionsystems.Itisalsousefulasasourcefortheisolationandidentificationofthebioactivecompounds.MaterialsandMethodsPerennialRyegrassBioassays1mltestsolutionPetridishwithfiterpaper10seedsCoveredSealedwithParafilmincubatedinagrowthchamber90μmol/s/m2，25/15℃day/night,an8hphotoperiodafter7days:Thelongestrootandshootofeachseedlingweremeasured.7measurementsfoundtohaverootlengthswithin±1S.DThesevenmeasurementsaveragerootlengthofthecontrol（receivedD.D.H2Oonly）Thevaluewasexpressedasthepercentageofthecontrollength.×100ExtractionandPurificationGlutenhydrolysatepowderD.D.H2Oa10%solution0.2μmmembranefilterfiltratedesignatedasGH-fSephadexG-15gelfiltrationcolumn:calibratedwithbluedextranandNaClaqueoussolutionelutedwithD.D.H20ataflowrateof1.6mL/min.aGilsonmodel201fractioncollector｛collectEightyfractionswith270dropsaYSIconductivitybridgeConductivity30mlGH-fwasloadedontothecolumnelutedwithD.D.H20atrateof16mL/minSelectedtubesbioassayedfortheirbioactivityTubes50-56werefoundtocontaintheroot-andshoot-inhibitingcompound(s)freezedriedandsubjectedtothefollowingpurificationsteps12mgof53GH-g15-eHPLC0-25%MeOHin50min,25-100%MeOHin25min,maintainedat100%MeOH15min,and100-0%MeOHinthenext10minatflowrateof4mL/min.UVabsorbanceat280nmcollectedat10-minintervalsforthefirst50minandat25minintervalsforthenext50min.SevenfractionssubjectedtotheperennialryegrassbioassayFourfractionswerefoundtohavegreaterthan90%rootinhibition.Thethirdfractionwaschosenforfurtherpurification12mgofthebioactive53GH-g15-ewasinjectedontoasemipreparativeDeltaPakwithaMeOHinwaterlineargradientoffrom0to15%in30minataflowrateof4mL/min.ThesamplewasmonitoredbyUVabsorbanceat214nm.Fivefractionswerecollectedbasedonthefivemajorpeakswithretentiontimebetween19and29minonthechromatogram.OnepeakwasidentifiedasbioactiveandsubjectedtofurtherpurificationbyusingaC18RPnarrowbore.withalinearABgradientfrom5to25%Bin40min.B:0.08%TFAinacetonitrilerate:200μL/minEluentA:0.1%trifiuoroaceticacidinD.D.H20PeptidesequencingAAcompositionThemolecularweightAmajorpeakValidationoftheInhibitoryActivityofthePureCompoundToconfirmthebioactivityoftheisolatedcompound,peptidewiththesamesequenceasthebioactivepeakisolatedfromCGHwassynthesizedbytheProteinFacilityatIowaStateUniversityandwastestedforitsinhibitoryactivity.Anaqueoussolutioninaconcentrationseriesof0,0.5,1.0,1.5,2.0,and4.0mg/mLwaspreparedandsubjectedtotheperennialryegrassbioassay.StatisticalAnalysisleastsignificantdifference(LSD)ResultsandDiscussionThebioassayresultsofthefractionssuggestedthatthereareprobablyatleasttwoothergroupsofcompoundsinCGHhydrolysatewhichhaverootinhibitingactivity.AllsixbioactivepeptideswereprovedtoinhibitrootformationataconcentrationlowerthanthatofeitherCGMorCGH,andtheyweredemonstratedasnaturallyoccurring,growthregulatingcompounds.Theyhavepotentialforuseincombinationwithnontoxic,compatiblecarriersorforthefortificationoftheCGMorCGHtoincreasetheireffectivenessaspreemergenceherbicides.FieldstudiesusingCGMandCGHtodevelopasnaturalpreemergenceherbicidesinturfandstrawberryplotsareunderway.