Checking the Functionality of the Synthetic Amylase with Starch Agarose Plate.

In order to test our synthetic amylases in another way, we put together a starch agarose plate (gelatinized algae material mixed with a starch solution).

Preparation of a starch argarose plate (according to Dr. Eduard Tauratzhofer, sugar research, Tulln):

One needs 11,5 g nutrient agar, 10 g potato starch and 500 ml distilled water. The mixture is autoclaved and finally put in a sterile Petri dish before it is left to cool in a clean-box

The probe solution was put into 3-4 holes that were made in the agarose plate with the help of an apple corer. After 5 hours of incubation at 37°C, the plate was colored with Gram’s iodine reagent. The iodine connected itself to the starch complexes, giving the starch-rich areas a dark violet color. After being given a short time to set in, the reagent was poured away and cleaned off with distilled water.

Circular "rings" of various sizes depending on the relative activity of the applied amylase samples remained colorless around the holes, since the amylase had already deconstructed the starches into glucose in these areas. One can tell if and how active the individual amylase samples were in respect to one another with the naked eye due to the sizes of the rings. In addition to the synthetic amylase sample, we also tested and compared a commercial variant that is used in industrial starch conversion.

A few rings that were too small or too large made it necessary to repeat the experiment with an optimized dilution.

The results are further proof the amylase 2.0 functions well.

In this photo, the colorless (and therefore starch-free) rings around the holes are identifiable. The upper-left hole was used as a control so that we could be sure it was really our amylase actively working in the samples and that there was no contamination. This plate shows a very happy picture, since each hole was filled with a different sample (LBE, M9E, LBN) of our synthetics amylase and every one of them showed quite a bit of activity.

This photo shows the clear difference between the different concentrations of the same sample (LBE). The sample in the upper hole was more heavily diluted as the lower ones. The right hole without a ring was not filled and served as a control.

In this picture it is easy to tell what effects the temperature has on the efficiency of the enzymes. The left plate was incubated at 37 °C, the right one at only 20 °C. Samples with various levels of distillation were pipetted into all of the holes. The corresponding holes in each plate were given the same concentration. The upper right Loch nicht befüllt, da wir aus vorherigen Versuchen bereits wussten, dass diese Verdünnung bei gegebener Temperatur einen zu großen Kreis bildet. Dieser hätte wiederum die Auswertung der anderen Proben auf derselben Platte gestört. Es ist gut zu erkennen, dass die Ringe der linken, wärmer inkubierten Platte deutlich größer sind. Das heißt, bei 37°C ist die Amylase deutlich aktiver als bei 20°C.