Team:BGU ISRAEL/Measurements
Measurements
One of the major parts of our project was to increase the PET-degradation efficiency of an existing PET degrading enzyme- LC-Cutinase. In order to detect an improvement in its efficiency, we needed to develop a 'toolkit' that would help us fully characterize the wild type and mutated proteins, in a way that could allow us to compare results and understand the effect of our mutations on the protein.
The algorithm that we used to mutate our enzyme is set to increase the stability of proteins. However, mutations in proteins' structure can have several effects, for example, on the protein structure, catalytic abilities, or even expression levels. We needed to use existing tests and create new ones to test each of these possibilities.
We started with an existing p-NP Butyrate degradation test. In Suliman, et al. (2012) work on LC-Cutinase, it was used as a substrate to test the catalytic constants, optimal pH and Tm of the enzyme, and we thought it would be an easy starting point to compare both between our wild type protein and the one used in the article, and between different mutated variants.
Our results showed improved pNP-B degradation ability with two of our enzymes. Once we acquired those results, we wanted to know whether the mutated proteins have improved catalytic abilities, or they are more abundant in the bacterial supernatant due to increased expression levels or stability. That is why we wanted to purify our enzyme from the supernatant in order to quantify the amount of different variants being expressed by a constant number of bacteria.
In order to do that, we used cation exchange chromatography to purify our enzyme. After purification, we measured the
different concentration of all variants and wild type. We've managed to achieve good results in purifying only a few of our
variants, but it thought us that the expression levels are indeed increased from the wild type enzyme. However, as you can see
in our results page, after concentration measurements, we repeated the pNP-B tests with identical amounts of enzymes in each
sample (wild type, codon optimized and variants) and we still received increased activity levels in the two previously improved variants.
We speculated that by preforming mutations we've managed to both increase stability and activity.
Another way to identity our enzymes was to use mass spectrometry on purified protein and managed to identify a few of our variants. We used this method to confirm and support the previous results.
The final aspect and the most important one we wanted to address was the PET degradation ability of LC-Cutinase and its
variants. First, we used a scanning electron microscope because we wanted to see if and how the LC-Cutinase degrades
the PET polymer.
Moreover, It was important for us to compare between the enzymes' abilities to degrade pNP-B and PET, since they can be
very different. Because of that, we developed two assays- Terephthalic acid detection assay,
and Examining the utilization of PET by E. coli expressing LC-Cutinase- you can find more about those
tests in the Experiment and Result pages.
In conclusion, all of these tests combined helped us understand the effect of our mutations on LC-Cutinase catalytic activity, expression levels or stability, and PET degradation ability. To further explore our mutants, we would have liked to repeat these tests several times in order to receive results regarding the entire protein library we built. This 'toolkit' is also useful for us in case we want to create new mutates with combined mutations, and to compare them to the previous mutants. We would also have liked to perform stability tests under different conditions such as temperature and pH levels.
Address:
Ben-Gurion University of the Negev
Ben Gurion 1, Beer Sheva 8410501, Israel
Mail: igembgu2016@gmail.com
Connect With Us!
