I have isolated a bacterium that displays the ability to grow on benzoic acid as the sole source of carbon and energy. Using crude enzyme extracts I have been doing an assay for catechol 1,2-dioxygenase activity using phosphate buffer (20 mM, pH 7), 0.5 mM substrate and about 0.45 mg of extract in a final reaction volume of 1 ml. Activity is observed by measuring the increase in absorbance at 260 nm due to the formation of cis,cis-muconate. Activities recorded have been very low. For example, 0.440 nmol/min/mg protein upon the addition of catechol to the reaction. Can anyone offer me any advice regarding any errors that I might be making in the assay that may account for these low activities. Any advice would be much appreciated!!!
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