Engineering success
In order to solve the problem of extended half-life and improved enzyme activity of the laccases, we carried out extensive research on possible ways to overcome this problem. This is important because the enzyme should be as stable as possible in order to ensure the maintenance of the enzymes activity and stability, for example in the vicinity of a sewage treatment plant. As the conditions there are rather harsh, it is important to extensively investigate possible modifications in order to create the best possible conditions for the enzyme and the Mesostructured cellular silica foam (MCF).
Idea
The first idea was to introduce a poly-lysine tag at the C-terminus across the active side. Our research had shown that this tag could extend the enzyme's activity by two days. This would meet the criteria of higher activity and stability. In order to decide which length this poly-lysine-tag should have, various criteria had to be considered. First, the poly-lysine-tag has to be long enough, so the immobilization happens via this tag, but not too long, to have any impact on the catalytic activity and the stability of the enzyme. By using modelling approaches and integrating our research, we decided to use two different lengths of the poly-lysine-Tag: 6x lysine and 3x lysine.
Engineering
To add the poly-lysine-tag to our laccase, primers were designed to introduce this modification. By using different primer designs with different lengths of the poly-lysine-tag, differences in activity and stability could be experimentally determined. Due to COVID-19 and the resulting time restrictions we could not perform any tests. However, in theory, we would perform different experiments with the enzyme immobilized on the Mesostructured cellular silica foam (MCF) regarding pH value and temperature over time. On the basis of the results one could then also see whether the presumed improvements occur at all with these modifications or whether further research is needed, and the old thesis must be dismissed. An effective experiment would be an ABTS assay under different pH and temperature conditions. If a positive result occurred, the various modifications could then be used to determine the most effective length of the poly-lysine-tag and to what extent this contributes to improved activity and stability. The results can then be used to learn and draw conclusions about the ideal length of the poly-lysine-tag and, if necessary, the length needs to be adjusted, and additional experiments must be conducted (i.e. with a shortened or lengthened poly-Lysine-Tag).
