Improvement of parts
The usage of PET have brought a global problem that is accumulation of the discarded PET in the environment. And abandoned PET waste never truly leave the environment but are present as micro- and nanoplastics that are ingested by marine organisms and continue to accumulate in their bodies. Therefore, PET waste harms soil, marine and human, especially highly crystallized PET due to its durability. Despite some attempts improved the degradation activity have been done, PETase was still low in activity. It is quite clear that more studies need to be done to further improve the performance of PETase.
PET pollution is a serious problem in today's world, which has caused an indelible impact on our environment. This pollution problem is mainly caused by the high crystallization of PET which is difficult to degrade. In 2016, PETase was discovered, but the degradation efficiency of it is not high, which is due to its Tm value was so low that it could not function at high temperatures. Therefore, we try to improve the thermal stability of PETase to solve this problem.
We carried out a rational design to improve the thermal stability of PETase by introducing disulfide bond, hydrogen bond, salt bridge, proline, enhancing hydrophobic force, etc. Eventually, we built up a huge database of mutations including a single point library with more than 4000 mutants and a multi-point library with more than 500 combined mutations. We ended up with about 508 parts, most of these parts show significant improvement in thermal stability.
Here we’d like to list some parts with impressive effect, whose Tm values even reach 80 ℃, far exceeding PET's glassy temperature of 75 ℃. They are BBa_K3468004, BBa_K3468022, BBa_K3468023, BBa_K3468024, BBa_K3468025, BBa_K3468026, BBa_K3468027, BBa_K3468028, etc.
To be used in other PET degradation projects
In addition, they can use our modified enzymes in other PET degradation projects or apply our enzymes directly to other metabolic pathways.
To be used in next year's experiment
In the coming year, our iGEM team may carry out experimental verification based on our mutation database this year, and screen out the mutants with the most improved activity for PETase to transform our research into practical.
Establish the conversion model between DDG and Tm
We collected data on variants of PETase and Lipase A from published literature and found that there is a good linear relationship between Tm values predicted by FoldX and Tm values experimental measured. So we speculate that there is also a linear relationship between the relative changes in folding free energy due to point mutations and the 3D structures of the corresponding mutant enzymes (ΔΔG) and ΔTm. We analyzed the correlation coefficient between the ΔTm values of the mutant and the calculated ΔΔG values of the mutants . There was a negative correlation between the ΔTm values and ΔΔG values with relatively high accuracy. (Click thisfor details)
Be used for other projects about improving the thermal stability of enzyme
Generally, when it comes to the results of most software predicting the mutant of enzyme, the parameter measuring thermal stability is DDG.However, DDG can not directly reflect the thermal stability of protein.Therefore, it is particularly important of our model that it can convert DDG into Tm value so that we can predict the thermal stability of enzymes indirectly through software. Later people can also predict the Tm of enzyme by DDG.
Contribution on ideas
Improve enzyme activity indirectly by enhancing the thermal stability of the enzyme
In addition, we propose to improve the thermal stability of PETase to improve the activity of it. Compared with improving enzyme activity by modifying active sites, there are more sites to choose from by improving enzyme thermal stability. There will be more room for manipulation by improving the thermal stability and enzyme activity. Other teams can refer to this idea when reforming enzyme activity. Also, in other projects related to protein activity such as drug design, its activity can also be regulated by changing its thermal stability.
Contribution on tools
Summarize online tools and software for protein modification
Through reading literature and other channels, we summarized some online tools and software for predicting protein thermal stability based on different algorithms. When designing protein mutations, future teams can save a lot of time for reading literature and searching websites by referring to our summary, which can greatly improve their efficiency. Click thisfor details.
|Consensus Finder||http://kazlab.umn.edu/||Give advice on mutant|
|FireProt||https://loschmidt.chemi.muni.cz/fireprotweb/?action=help&||Give advice on mutant|
|I-mutant 3.0||http://gpcr.biocomp.unibo.it/cgi/predictors/I-Mutant3.0/I-Mutant3.0.cgi||Predict stability|