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Abstract

Itaconic Acid

The expression of GRE3 has been visualized via SDS-PAGE. Positive clones were grown at 37° celsius until an OD of 0,5. Afterwards the cells were induced utilizing 20µl of 1M IPTG for 10h at 28° celsius. Finally the cells were lysated with heat and the suspension was put on the PAGE.

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GRE3 assay

To prove the enzymatic activity of the aldose reductase GRE3 in dependance of NADPH we designed an applicable assay as following. We used a spectral analysis with a wavelength of 340nm to make the conversion from NADPH to NADP⁺ observable. A drop in the curve of the absorption spectrum therefore shows that NADPH is being converted to NADP+ i.e. the enzyme works. Spectral analysis was performed with 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 described below:

First Na₂HPO₄ was adjusted to a pH of 7.0 to function as a buffer. The final concentration of Na₂HPO₄ was 0,1M. The assay system contained 0,1mM NADPH and 8 µl were added per well. As a possible blank wells with just NADPH (8 µl to 192 µl of buffer solution) were provided. In addition we added blanks containing just xylitol (0,1M) as well as one containing just NADP+ (0,1mM). The negative control contained a purified TES protein fraction from disrupted BL21 cells. The assay mixture included 154 µl buffer solution, 8 µl xylose, 8 µl NAPDH, and 30 µl of different protein amounts each, ranging from 5µl to 30 µl (in six steps; protein concentration unknown because purification was not performed, just a lysis of the cells with TES). All samples were prepared on ice. (In hindsight the possible blank with just NADPH appears to be a non-optimal solution because the auto catalyzation of this chemical likely happens just a few minutes into the assay.)
The 96 well microplate was loaded as depicted in the picture below:
<img src=""> 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. Figure 5 96-well microplate layout Figure 6 The plot on the right hand side is showing the change in absorption of NADPH at 340nm in solution for the two different kinds of samples in correlation to the kinetic cycles i.e time.

• The 'gre' curve shows the enzymatic activity of GRE3. The curve drops later than the 'k' curve because the active conversion of xylose to xylitol in dependance of NADPH happens at a much quicker rate than the auto catalyzation of NADPH itself.
• The 'k' curve shows the negative control containing just NADPH without GRE3.

<img src="" width="466px" height="400px">

The code utilized to render the plots is embedded below.

<script src="http://pastebin.com/embed_js.php?i=9xmJJWgr"></script>

Ethylene Glycol

Xylitol