Enzymatic activity, visual detection

All ee screening systems using UV/Vis spectroscopy as a detection system have a number of advantages, including the possibility of visual pre-screening for activity on microtiter plates in some cases. Moreover, if a reliable UV/Vis signal arises as a consequence of an enzymatic reaction, commercially available UV/Vis plate readers can be used to screen thousands of mutant enzymes catalyzing the reactions of interest in the wells of microtiter plates. 

The zones or spots of separated compormds are generally visualized by detecting dehydrogenase activity with tetrazolium salt-based reagents. The metabolically active bacteria convert the tetrazolium salt into red formazan dye (2,3,5-triphenyl-2H-tetra-zolium chloride, tetrazolium red). A number of tetrezolium salts were evaluated as potential substrates for the enzymatic reaction, but p-iodonitrote-trazolium violet, tetranitro blue tetrazolium, and MTT were found to be suitable substrates. The equations below show the principle of the reaction with tetrazolium salt, 3-[4,5-dimethylthiazol-2-yl]-2,5 dip-henyltetrazolium bromide (MTT, tetrazolium dye). 

---