Figure 1 Reaction scheme of the itaconic acid producing operon (only cadA). The substrate for the reaction is cis-aconitate. Cis-aconitate is metabolized to itaconic acid in 1 step by decarboxylation.

cadA Assay

The decarboxylic activity of cadA was shown via a pH indicator assay. As a byproduct of the catalytic conversion of cis-aconitate to itaconic acid carbon dioxide is released into the assay mixture. Here it forms carbonic acid and thereby lowers the pH value of the mixture. This is visualized through the indicator bromothymol blue (BTB) which changes its configuration state depending on the surrounding pH-Value (pH 6.0 - 7.6). That change of configuration can be shown via a photometric analysis in a TECAN® Infinite 200 PRO microplate reader. The resulting data sheets are then put into a plotting script written in R and exported as a ggplot.

The assay was performed as following:
First Na₂HPO₄ was adjusted to a pH of 7.0 to function as a buffer. The final concentration of Na₂HPO₄ was 5mM. The assay system contained 10% (v/v) indicator stock (BTB) and 20 ul were added per well. As a negative control a purified TES protein fraction from disrupted BL21 cells was used. In a range from 0mM to 1mM (0,2mM steps; total of 6 reactions) cis-aconitate (substrate), dissolved in Na₂HPO₄-buffer solution was added per well to enable the enzymatic conversion. To put the turnover into relation with the maximum turnover we added extra rows that contained equal amounts of substrate and product (itaconic acid). Finally a row containing just itaconic acid was put at the bottom to be able to use the resulting values as a blank for the assay. All samples were prepared on ice.

The 96 well microplate was loaded as depicted in the picture below:

	The assay was run for 200 kinetic cycles, each 30 secs long and with 25 photo pulses per cycle. The reader was heated to the appropriate temperature of 37° celsius. Absorbance was measured at the absorption maximum of BTB which in this case is 620nm.
Figure 5 96-well microplate layout
Figure 6 The plot on the right hand side is showing the change in absorption of the bromothymol blue in solution for the three different kinds of samples in correlation to the kinetic cycles i.e time. The 'p' curve shows the absolute conversion of educt to product (product curve). The 'ca' curve shows the enzymatic activity of cadA. The activity explains the steady rise of the curve which comes from the acidification of the assay mixture in those wells. Because of the conversion from cis-aconitate to itaconic acid and the byproduct carbon dioxide (which forms bicarbonate acid) and lowers the pH The 'k' curve shows the negative control containing TES protein fraction without cadA. The sudden drop of the 'p' curve in the beginning of the measurement could be explained through various reasons. One could be that the bubbles that formed in the wells when adding the chemicals could interfere with the light. Also the different substances were added on ice so when the plate was put in the heated reader water in the air might have condensated at the bottom of the wells and hinder the light beam. The resulting vibrations of shutting doors too harshly might also interfere with the plate moving mechanism inside the reader and cause malfunctioning.

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