Due to time constraints, we have not yet obtain the targeted enzyme using a sequential directed evolution system. Structure-based rational design has also been available to test only one. The competition is not the end of our project, and we will still continue the directed evolution of TbsADH afterwards.
On the one hand, we will continue the design of the enzyme by continuous directed evolution and rational design. On the other hand, we will work on the optimization of the reaction system, including but not limited to the search for a suitable phase transfer catalyst or the synergistic reaction between the two phases by changing the reaction conditions, and the replacement of the NADPH cycle. It is well known that complete oxidation of glucose provides 24 electrons, but glucose dehydrogenase only provides two. This is a waste of both raw material and reducing power. We are designing reaction systems that use electrodes to reduce NADP+ in order to improve the utilization of all substrates.
In the discussion section we also mentioned the optimal temperature of glucose dehydrogenase, and we also hope to modify GDH by directed evolution to improve its thermal stability so that it can be better adapted to TbsADH to increase the rate of chemical reactions. We think such an attempt is meaningful and it has great application for practical production.