Catechol 1,2 Dioxygenase Activity in Bacterial Isolate - (Jan/09/2011 )

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!!!

How do you know the bacterium is indeed growing on the benzoic acid as the sole carbon source?

pito on Sun Jan 9 19:30:53 2011 said:

Bacterium has been grown in a mineral salts medium containing benzoic acid as the sole source of carbon and energy. Growth has been observed by measuring the increase in optical density at 600 nm, increase in cell numbers and by protein determination.

So you are sure that it works , the problem is that its going slow?
(you need to be 100% sure that they grow indeed on the benzoic acid and not a c source they have from another petri dish or medium you grow them in before putting them on a benzoic acid medium).

This can be due the fact they do not grown well on benzoic acid as the sole carbon source.
They might need to adjust.

+ why dont you test them growing on a good (rich medium) with benzoic acid as the c source but for the rest a good medium (I am thinking you use a very minimal medium?)


And is it aereated ? You are are keeping the pH constant?