Chapter13 Enzyme applications and research methods.ppt

Chapter13EnzymeapplicationsandResearchmethods,Outline,AssayofenzymeactivitiesMethodsforexpressingenzymeactivitiesMethodsfordeterminingenzymeactivitiesSeparationandpurificationofenzymes(canbeskipped)Enzymeengineering(self-study),Enzymeactivity,Enzymeactivity=molesofsubstrateconvertedperunittime=ratereactionvolume.Enzymeactivityisameasureofthequantityofactiveenzymepresentandisthusdependentonconditions,whichshouldbespecified.TheSI(TheInternationalSystem)unitisthekatal,1katal=1mols1,butthisisanexcessivelylargeunit.Amorepracticalandcommonlyusedvalueis1enzymeunit(U)=1molmin1.1U=16.67nanokatalsand1kat=6107U,Specificactivity,Thespecificactivityofanenzymeistheactivityofanenzymepermilligramoftotalprotein(expressedinmolmin1mg1).Specificactivitygivesameasurementoftheactivityoftheenzyme.Itistheamountofproductformedbyanenzymeinagivenamountoftimeundergivenconditionspermilligramoftotalprotein.Specificactivityisequaltotherateofreactionmultipliedbythevolumeofreactiondividedbythemassoftotalprotein.TheSIunitiskatalkg1,butamorepracticalunitismolmg1min1.,Enzymeactivityassay,AllenzymeassaysmeasureeithertheconsumptionofsubstrateorproductionofproductovertimeEnzymeassayconditionsAlargeexcessofthesubstrateInitialvelocityOptimalreactionconditions(optimalpH,optimaltemperature,optimalionicstrength,etc).AssaymethodsDirectassayIndirectassayCoupledassay,Lightabsorption,Lightabsorption,Directassayofcytochromecoxidase,IndirectassayofDihydrooratedehydrogenase,Lightabsorption,CoupledassayofHexosekinase,Enzymepurificationisextractionofasingleenzyme/proteinfrome.g.cells,tissue,etc.whichmaycontainmorethan1000differentproteinsandlotsofotherbiomoleculesKeepstructureandactivityintactEachenzymerequiresaspecificstrategyforpurificationHowispurificationmeasured?,Enzymepurification,SelectthesourcesEstablishtheessaymethodDesignthestrategyPreparingextractsforpurificationDisruptionandhomogenizationofcells,tissue,etc.ClearingofextractsChoosepurificationmethodsDeterminepurity,PurificationSteps,PrecipitationmethodsSeparationbasedonmolecularsizeSeparationbasedonchargeSeparationbasedonspecificinteractionwithotherbiomoleculesSeparationbasedonotherprinciplesExamples:AmmoniumsulfateprecipitationGelfiltrationIon-exchangechromatographyBio-affinitychromatographyHydrophobicinteractionchromatography;hydroxyapatitechromatography,Maintypesofpurificationmethods,DeterminationofspecificactivityPurificationtablePhysicalmethods:SDS-PAGEGelfiltration,Howispurificationmeasured?,Purificationtable,Measureforeachpurificationstep:Thevolumeoftheenzymesolution(ml)Theproteincontentofthesolution(mg.ml-1)Theactivityoftheenzymesolution(U.ml-1)Totalamountofenzyme(U):Activity(U.ml-1)xvolume(ml)Specificactivity(U.mg-1):Activity(U.mg-1)/proteincontent(mg.ml-1)Yield(%):Totalamountofenzymeafterapurificationstep/totalamountofenzymebeforethatstepPurificationfactor:Specificactivityofenzymeafterapurificationstep/specificactivitybeforethatstep,Purificationtable(example),ImmobilizedenzymesArtificialenzymesSite-directedmutagenesisofenzymesHybridenzymesAbzymes,Enzymeengineering,