Development of a highly active engineered PETase enzyme for polyester degradation
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Abstract
Polyethylene terephthalate ( PET ) accounts for ≈6% of global plastic production, contributing considerably to the global solid‐waste stream and environmental plastic pollution. Since the discovery of PET ‐depolymerizing enzymes, enzymatic PET recycling has been regarded as a promising method for plastic disposal, particularly in the context of a circular economy strategy. However, because the PET ‐degrading enzymes developed so far suffer from relatively limited thermostability and low catalytic efficiency, as well as degradation product inhibition, their large‐scale industrial applications are still largely hampered. To overcome these limitations, we engineered the current PET ‐hydrolyzing enzyme gold standard [the ICCG variant of leaf‐branch compost cutinase ( LCC ‐ ICCG )] using in silico protein design methods to develop a PET ‐hydrolyzing enzyme that features enhanced thermal stability and PET depolymerization activity. Our mutant, LCC ‐ ICCG ‐ C09 , features a 3.5 °C increase in melting temperature relative to the LCC ‐ ICCG enzyme. Under optimal reaction conditions (68 °C), the engineered enzyme hydrolyzes amorphous PET material into terephthalic acid ( TPA ) with a two‐fold higher efficiency compared to LCC ‐ ICCG . Owing to its enhanced properties, LCC ‐ ICCG ‐ C09 may be a promising candidate for future applications in industrial PET recycling processes.
Article activity feed
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Protein expression and purification
I am curious if there were differences in expression or yield between the different proteins. I could see that being important if the thought is to produce a bunch of this protein and use it to degrade PET.
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The position and orientation of the catalytic triad (D210, H242, and S265) overlaps perfectly with the catalytic triad in the parent enzymes
This is interesting! Do the protein design methods you used specifically try to preserve the catalytically active parts of the protein or was this something that you assessed when picking proteins? If not, might that be useful to consider when selecting proteins to test? For example, maybe the proteins with negligible activity had a lot of differences in the triad. That seems like something you could catch before purifying and testing activity.
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Conclusions
I'd love to see some discussion around your working hypothesis as it seems that you maybe disproved it? It might even be cool to have a bivariate plot with thermal stability on one axis and enzymatic activity on the other to see if there is a correlation. Additionally, because you tried 3 different methods to generate proteins and then evaluated them the same way, it might be useful to talk about which worked best, why that might be, pros/cons of the three methods, etc.
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data not shown
Again, it would be interesting to see this data, even if you just put it in the supplement!
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data not shown
This sounds like such a cool result, it would be great to be able to see the data.
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For the P06 and P08 variants, we did not set out to determine their melting temperature because of their negligible enzymatic activity
It might still be useful to determine the Tm for these proteins to help with testing the working hypothesis that you discussed in the intro that proteins with higher stability have higher enzymatic activity.
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