Testing the Functionality of the Synthetic Amylase via Photometric Measurements
In order to determine the amylase activity of our samples, we used a method that was quick and easy to carry out. We got the measurements with the help of a photometer, with which a large amount of samples could be quickly analyzed.
The process is as follows:
200 µl of the prepared TRIS- buffer is pipetted into an Eppendorf tube. 50µl of the fluid to be measured is added. Only 20µl of buffer is used for the required blank sample. 1ml of starch solution (which must be prepared beforehand) is added to all the Eppendorf tubes. Finally, all reactions are put into a heating block at 37 °C for 30 min. The amylase – if it is present and active – breaks down the contained starch into glucose during this time.
After this process, 500 &mircro;l of an iodine solution is added and mixed in well with a vortexer. The iodine builds up in the remaining starch complexes and colors them violet. Higher amylase activity (and the resulting high levels of broken down sugar) leaves the sample with less discoloration. This means: the more active the contained amylase, the lighter the color of the solution. The transparency of the samples can be determined with the photometer. The transparency is increased when starches are broken down.
Depending on where the neutral value is set, the results can be measured in positive or negative values. Only the absolute value is of interest. It is clear that no absolute numbers describing the amylase activity can be produced using this method.
It is, however, ascertainable if the enzyme is working or not. It is also possible to determine a trend in the sample’s activity level and if it is especially high.
Picture 1 (Table 1): This graphic shows the different levels of activity during dialysis (the result of protein cleansing) at different concentrations and temperatures. The higher the concentration of the sample, the higher he activity level of the amylase and the warmer the sample, the more active it was as well. This is clear evidence that our synthetic amylase was working.
Picture 2 (Table 2): the graphic shows how the amylase activity rises during the development of the main culture (the growth of the Bacillus.) The zero-point was the iodine solution in the control sample, so the opacity is reduced with higher levels of amylase activity because it causes starches to be broken down and the iodine can no longer build up in the glucose molecules. The mathematical value of the measurements shows the activity levels.
Picture 3 (Table 3): The graphic compares the amylase activity within the different nutrient mediums (cf. lab protocol) for the Bacillus s. at 37° und 70°C. It is clear that the activity level at 70°C is quite a bit higher but also that a functioning amylase
was produced in all nutrient media.
To be noted: the samples with the synthetic amino acids ethionine (LBE, M9E) and norleucine (M9N) show higher activity than the reference sample with natural methionine (LBM, M9M)
This could be explained by a better functioning synthetic amylase or by higher concentration of the synthetic amylase in the samples. This method of measurement does not give any more information regarding this. But, as all of the samples we given the same beginning concentration level of bacteria and media, in other words they were all handled the same and had the same amount removed in probes, a higher functionality is to be assumed of the synthetics amylase.
These measurements also show that the amylase 2.0 is more active than the natural one: amylase 2.0 is likely to work better.