Permeabilization of gramnegative bacteria with KPi/hexane for the release of Lasparaginase相关交流-蚂蚁淘在线

Source:HikmetGeckil"sLab

Abstract:Thisisafast,efficientandreproducIBLerecoveryprocedureforperiplasmicL-asparaginasefromdistinctlyrelatedgram-negativebacteria,Escherichiacoli,EnterobacteraerogenesandPseudomonasaeruginosa.Asthemethodusesinexpensiveorganicsolventhexaneandanaqueoussaltsolution,itisalsohighlycost-effectiveincomparisonwiththecurrentlyavailabletechniquesusedforthereleaseofthisenzyme.Ashexaneisahighlywaterimmiscibleorganicsolvent,itcanberemovedeasilyfromthetopoftheaqueousphasebyasimpleevaporation.Enzymerecoveriesupto3-foldwithrespecttosonicationcanbeachievedwiththissystem.

Harvest(10,000rpmfor5min)24hgrown(inLBpH7.5,37^oC,200rpm)Escherichiacoli,EnterobacteraerogenesorPseudomonasaeruginosacells.
Washoncewith0.05Mpotassiumphosphate(KPi)bufferpH8.6,andresUSPendedtoA₆₀₀=5.0inthesamebufferwith1%hexane.
Incubatethesuspenssionatroomtemperaturefor1hour,brieflywortexingforevery10min.
Leavethetubecapsopenfor5mininordertoevaporatevolatileupperphase(hexane)priortoanalysisofL-asparaginaseactivityinthecell-freeaqueousphase.
Preparecellsuspensionsalsoindistilledwateror0.05MKPiwithnohexaneascontrols.
MeasuretheenzymeactivityutilizingtheNesslerizationreaction.ThismethodutilizesthedeterminationofammonialiberatedfromL-asparagineintheenzymereactionbytheNesslerreaction.L-asparaginasecatalyzesthehydrolysisofL-asparaginetoL-asparticacidandammonia.
Startthereactionbyadding0.1mlpermeABIlizedcellsuspensionor0.1mlsupernatantofcellsuspensionsfromaqueousphaseintothepre-warmed0.9ml0.01ML-asparaginepreparedin0.05MTris-HClbuffer,pH8.6andincubatefor30minat37⁰C.
Stopthereactionbytheadditionof0.1ml1.5MTCA.
Centrifugethereactionmixtureatroomtemperature(10,000rpmfor5min)toremovetheprecipitate.
Determinetheammoniareleasedinthesupernatantcolorimetrically(A₄₈₀)byadding0.25mlNesslerreagentintotubescontaining0.5mlsupernatantand1.75mldH₂O.
Vortexthecontentandleaveatroomtemperaturefor10min.
ReadtheA₄₈₀valuesagainsttheblanksthatreceivedTCAbeforeextractaddition.

Notes:OneL-asparaginaseunit(U)isdefinedastheamountofenzymethatliberates1mmoleofammoniaperminat37⁰C.Specificactivityisexpressedasunitspermilligramofproteinreleased.Theammoniaconcentrationproducedinthereactioncanbedeterminedfromthestandardcurveobtainedwithammoniumsulfateasthestandard.Thelimitofdetectionofammoniabythismethodisabout10mM.Determinethetotalproteincolorimetricallybyanystandardmethodavailable,usingbovineserumalbuminoranyothercommerciallyavailableproteinasthestandard.

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Permeabilization&nbsp;of&nbsp;gramnegative&nbsp;bacteria&nbsp;with&nbsp;KPi/hexane&nbsp;for&nbsp;the&nbsp;release&nbsp;of&nbsp;Lasparaginase

Permeabilization of gramnegative bacteria with KPi/hexane for the release of Lasparaginase