Team:Aachen/Proof Of Concept
Proof of Concept
Feasibility in a nutshell
To demonstrate the actual potential and feasibility of our concept, we went much further than envisioning ideas and researching scientific literature to support them. We started working in the lab to aim for conclusive results, which show the success of the different components of our system. We present to you our proof of concept. We want to showcase an example that affirms one of our many possible applications. Here, M.A.R.S. gets into action and is compared to conventionally used system. Emphasizing the feasibility of our system is the key.
Putting M.A.R.S. to the test
Through our laboratory work and simulation approaches we could proof several parts of our system and verified that our system as a whole works in a real industrial context. First of all, we found an anchor peptide that binds specifically and has a high affinity to the magnetic particles that are one part of our system. These magnetic particles have an average diameter of 3.71 µm and possess a polystyrene surface. The anchor peptide MBP-1 bound so tightly to the magnetic particles that also through several washing steps it stood connected to the polystyrene surface. While testing multiple other peptides, we found out that LCI is the equivalent to MBP-1 respectively to the liposomes. The investigated anchor peptides were fused to an eGFP Protein to offer a visual confirmation of possible binding abilities. Through confocal microscopy we could track the binding process and reliably confirm, that our anchor peptides work as a possible conjunction module. The next step would be the expression of an entire protein, which features an additional linker between those two binding domains, to create a bridge between our ATP Factories and magnetic particles.
Computer simulations for improvements
Using a lipid batch for our liposomes with positively charged lipids alleviates the integration of the bacteriorhodopsin. We simulated a protein integration into both lipid batches in silico with GROMACS. We then proceeded to analyze both generated models in Chimera and demonstrated a better, more realistic integration into the new DOTAP rich lipid batch. While the clash/contact analysis did not provide a quantitative proof for the better bacteriorhodopsin integration, it supported our hypothesis and gave us further insights into the plausible underlying mechanisms.
Specific use cases
Apart from the proof of the different compounds of M.A.R.S. we want to come up with a specific example were our system can be used.
The enzyme carboxylate reductase (CAR) needs NADPH and ATP as cofactors. It´s part of very important enzymatic processes and enzyme cascades to produce pharmaceutical precursors such as R-PAC.
As a first reaction of the specific cascade, on which we set a special focus on, the CAR synthetizes a benzaldehyde out of a carbon acid. In a series of experiments, we tested the normally used regeneration system, whole cells as cofactor regeneration approach and did some negative controls to prove the dependency on ATP of this specific enzyme, too. We could represent the activity of the enzyme under the different conditions in form of kinetic curves. As a result, we saw less main product conversion with the whole cell system that with the normally used regeneration system. The whole cell approach tends to over reduce the wanted product to an alcohol, which compromises the efficiency of this method. The cell provides the two cofactors but also use them for their own metabolism. Because of that the enzymatic activity is lower. In the end we simulated the enzymatic activity of the CAR in the reaction where our system provides the ATP as a cofactor. With our system cytotoxic products and products in high concentrations can be synthesised. M.A.R.S. enables a comparable activity after 2 hours to that of the whole cell approach, without production of unwanted secondary metabolites. In the long run, significantly higher product conversion can be achieved.
The enzyme carboxylate reductase (CAR) needs NADPH and ATP as cofactors. It´s part of very important enzymatic processes and enzyme cascades to produce pharmaceutical precursors such as R-PAC.
As a first reaction of the specific cascade, on which we set a special focus on, the CAR synthetizes a benzaldehyde out of a carbon acid. In a series of experiments, we tested the normally used regeneration system, whole cells as cofactor regeneration approach and did some negative controls to prove the dependency on ATP of this specific enzyme, too. We could represent the activity of the enzyme under the different conditions in form of kinetic curves. As a result, we saw less main product conversion with the whole cell system that with the normally used regeneration system. The whole cell approach tends to over reduce the wanted product to an alcohol, which compromises the efficiency of this method. The cell provides the two cofactors but also use them for their own metabolism. Because of that the enzymatic activity is lower. In the end we simulated the enzymatic activity of the CAR in the reaction where our system provides the ATP as a cofactor. With our system cytotoxic products and products in high concentrations can be synthesised. M.A.R.S. enables a comparable activity after 2 hours to that of the whole cell approach, without production of unwanted secondary metabolites. In the long run, significantly higher product conversion can be achieved.
Words of gratitude
M.A.R.S. enables the industry to produce any specific relevant chemical, without the ineffectiveness of whole cell approaches or the unsustainable implications of purely chemical regeneration systems. We thank Prof. Dörte Rother and Douglas Weber for supporting us and giving us an opportunity to work on project M.A.R.S. at FZ Jülich.
