2.5. Enzymeassays
Cell-free extractswerepreparedfromwashedcellssuspended
in threevolumeof50mMphosphatebufferpH7.0bysonication
(Ultrasonicprocessor,modelXL2010)for5minandcentrifugation
at 12,000g for 45minat4 1C. Theclearsupernatantwasusedfor
further enzymaticstudies.2-Chlorobenzoate-1,2-dioxygenase
activitywasassayedspectrophotometricallyforthedetermination
of theproductcatecholat275nm.Catechol1,2-dioxygenase
activitywasassayedspectrophotometricallybymeasuringthe
increaseinabsorbanceat260nmduetotheformationof cis,cismuconic
acidaccordingto Hayaishietal.(1957). Catechol2,3-
dioxygenaseactivitywasassayedspectrophotometricallybymea-
suring theincreaseinabsorbanceat375nmduetotheformation
2-hydroxymuconicsemialdehydeaccordingto Kim etal.(1992).
Proteinwasdeterminedbythemethodof Lowry etal.(1951) using
bovineserumalbumin(BSA)asastandard.Oneunitofenzyme
activitywasdefined astheamountrequiredtocatalyzethe
formation orconsumptionof1 mmol ofproductorsubstrateper
minute.
2.5. EnzymeassaysCell-free extractswerepreparedfromwashedcellssuspendedin threevolumeof50mMphosphatebufferpH7.0bysonication(Ultrasonicprocessor,modelXL2010)for5minandcentrifugationat 12,000g for 45minat4 1C. Theclearsupernatantwasusedforfurther enzymaticstudies.2-Chlorobenzoate-1,2-dioxygenaseactivitywasassayedspectrophotometricallyforthedeterminationof theproductcatecholat275nm.Catechol1,2-dioxygenaseactivitywasassayedspectrophotometricallybymeasuringtheincreaseinabsorbanceat260nmduetotheformationof cis,cismuconicacidaccordingto Hayaishietal.(1957). Catechol2,3-dioxygenaseactivitywasassayedspectrophotometricallybymea-suring theincreaseinabsorbanceat375nmduetotheformation2-hydroxymuconicsemialdehydeaccordingto Kim etal.(1992).Proteinwasdeterminedbythemethodof Lowry etal.(1951) usingbovineserumalbumin(BSA)asastandard.Oneunitofenzymeactivitywasdefined astheamountrequiredtocatalyzetheformation orconsumptionof1 mmol ofproductorsubstrateperminute.
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