Biocatalytic recycling: UK researchers develop enzyme evolving platform for PET depolymerization

25 Aug 2022 --- Researchers from The Manchester Institute of Biotechnology (MIB) have developed an engineering platform capable of evolving enzymes for PET depolymerization with characteristics that would be necessary for an industrial-scale PET degrading biocatalyst.

Elizabeth Bell, a leading scientist in this research tells PackagingInsights that “biocatalytic recycling is an exciting complementary strategy to add to the plastics recycling toolkit. Previously one of the challenges to using biocatalytic recycling on a wider scale was the lack of suitable enzymes and an inability to engineer them to meet process demands quickly and efficiently.”

“The platform is flexible, allowing us to change the selection pressures put on the enzymes in order to tailor them to our needs. Using this workflow, we have shown that we can increase the speed, thermostability and longevity of polymer deconstructing enzymes.”

“In addition, [the platform can] engineer enzymes to degrade more challenging semi-crystalline PET material which other biocatalysts struggle to depolymerize, as demonstrated by the evolution of HotPETase,” Bell adds. 

“Biocatalytic recycling will prove especially effective for multi-material packaging as we demonstrate that our evolved enzyme HotPETase can selectively depolymerize the PET portion of a PET/PE composite film lid, leaving the PE portion to be recycled using alternative methods.”

A hot take on PETases
Despite the availability of mechanical recycling processes for PET, recycling rates are still low as a result of challenges in gathering and separating diverse post-consumer waste streams as well as deteriorating polymer characteristics after numerous processing cycles. 

Bell says the platform can develop plastic degrading enzymes with a range of desirable characteristics.Due to these difficulties, depolymerizing PET into its individual monomers has garnered attention as a way to circularize the PET life cycle. Chemical recycling processes can be used to accomplish this. Enzymatic depolymerization has lately come to light as a potentially alluring substitute.

HotPETase is an engineered version of IsPETase, designed to meet certain industrial recycling requirements. IsPETase is poorly active and poorly thermostable so it would not survive in an industrial process, in fact, it is completely inactive over 40°C.

Ideally, to depolymerize the PET efficiently, it needs to operate at the glass transition temperature of PET (60-70°C) as at these temperatures the polymer chains in the PET become more mobile and so more accessible to enzyme action.

HotPETase can depolymerize PET in reactions up to 70°C, it is also a lot faster than IsPETase and other PET degrading enzymes. It can also depolymerize semi-crystalline forms of PET, which are more challenging to break down enzymatically, more rapidly than IsPETase. 

Tricky temperatures
Enzymatic recycling is a promising new strategy for plastics recycling, but there are a number of hurdles to overcome before it can become a widely-used recycling process. One of these challenges the researchers faced is the ability to engineer plastic degrading enzymes to meet certain industrial requirements.

Bell tells PackagingInsights that they have now developed a platform that can speed up this process and develop plastic degrading enzymes with a range of desirable characteristics. 

“We demonstrated this by engineering an enzyme, HotPETase, that can operate at elevated temperatures, is able to deconstruct crystalline PET more rapidly than other reported PETases, and can selectively depolymerize the PET portion of a real-world packaging composite film lid that is challenging to recycle using other methods.”

She maintains that in the future, this platform should offer novel opportunities to create enzymes that can degrade different types of plastic. 

Process difficulties
Highlighting the challenges of the process, Bell says “the HotPETase enzyme was developed from IsPETase using a process called directed evolution. This is like natural evolution but on a laboratory scale.”Despite the availability of mechanical recycling processes for PET, recycling rates are still low.

“We chose specific amino acids that make up the enzyme that we thought were important for catalysis and mutated them to see whether any of the mutated variants had a better ability to degrade PET than the starting enzyme.”

This required the researchers to screen thousands of enzyme variants for plastic depolymerizing activity, which was something that had proved very difficult to do in the past, and hence required them to design a new high-throughput screening and analysis platform, the “first of its kind” for plastic depolymerizes.

The platform was then fully automated using robots so the scientists could screen around 2000 enzymes for PET depolymerizing activity in 24 hours. Once this platform was set up, they then changed the selection pressures on the enzyme, looking for mutants that could not only efficiently depolymerize PET but could do so at higher temperatures and act upon more crystalline substrates that are like those that might be found in plastic waste.

“Again, creating enzymes that meet multiple requirements is traditionally quite challenging, but using our new directed evolution workflow this became achievable. The resulting enzyme, HotPETase, can depolymerize semi-crystalline PET more rapidly than other PETases at elevated temperatures for longer time periods.”

The future of enzymatic recycling
For large-scale biocatalytic plastics recycling to become feasible, a whole range of enzymes will be needed that can operate under specific process conditions on different types of plastics.

“As these enzymes either do not occur in nature or are not suitable for industrial processes they will need to be engineered. The platform we have developed offers the opportunity to engineer polymer degrading enzymes with characteristics tailored toward the processes we want to use the enzymes in,” Bell says.

“We can then use our enzyme evolution platform again, to tailor the enzyme even further so that it meets all of the conditions necessary to be a great industrial biocatalyst.”

“In the future, the platform could also be used to engineer enzymes that act on different types of plastics in order to create a suite of enzymes for a more comprehensive biocatalytic recycling strategy,” she concludes.

By Mieke Meintjes