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From our past research, our research team has had difficulties predicting successful mutation sites for PETase. Prof. Ngo has suggested that we refer to the structure of the enzyme, more specifically, the active site of the enzyme. We could possibly refer to the binding energies and the positions of hydrogen bonds in the protein structure, as well as the catalytic site of the enzyme. We can look specifically into each amino acid, nucleotide, and see which is the key nucleotide that is responsible for the catalytic activity of the entire enzyme.
Moreover, he speculated that a PETase-MHETase chimera with a stoichiometric ratio of 1:1 will demonstrate a higher efficiency of PET digestion. This chimeric protein could ensure a MHETase molecule in close proximity for the digestion of products resulting from the degradation of PET by PETase, so that the products do not have to diffuse over long distances to reach the active site of MHETase.
In addition, the HPLC data our team obtained variations among different replicates of the same protein samples. In response to this, Prof. Ngo pointed out that the enzymes should be frozen in liquid nitrogen before stored at -80oC , and only a small aliquot should be thawed every time. The remaining portion after an experiment should be discarded. This method, known as flash freezing, can preserve enzyme activity effectively.
On top of that, we expressed our concern over the production cost and the storage of the enzymes. In response to this, Prof. Ngo suggested that we overexpress the chimeric enzyme in E. coli and directly treat the plastic without purifying/extracting the enzymes. This would be a much more cost-effective method of applying the enzymes to a large-scale recycling facility.
We have constructed a PETase-MHETase chimera, linking the two enzymes in a 1:1 ratio by a 12 amino-acid serine-glycine linker. In our experiments, we have expressed and tested the rate of PET degradation using the chimera. We also studied the degradation rate of PET using our engineered chimera using a protein cocktail of PETase and MHETase in a 1:1 and 1:2 ratio.
We have followed the procedures of flash freezing in our experiments before storing the enzymes with liquid nitrogen at -80°C. After doing so, we have observed that the HPLC data among different replicates of the same protein samples showed less variations.
In addition, we have contacted various PET-recycling companies for the implementation of our engineered enzymatic system. Prof. Ngo’s advice supports our project idea to overexpress chimeric enzymes in E. coli.
In our interview with Prof. Chan, he pointed out that in order to publicize our project, we can promote our research outcome, which is the design of the chimeric protein. This can help integrate our project with proposed implementation to achieve the ultimate goal of alleviating plastic pollution problem in real world.
Moreover, our team also had doubts as to whether linking the C-terminus of MHETase to the N-terminus of PETase would show a higher hydrolytic activity as compared to the connection between the N-terminus of MHETase and the C-terminus of PETase. Prof. Chan noted that enzymatic reactions are more of a result from random motion, so whether the other configurations can increase degrading activity or not can only be concluded from experimental results.
