Research team engineers a better plastic-degrading enzyme

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A breakthrough in enzyme research led by the U.S. Department of Energy's National Renewable Energy Laboratory (NREL) and the United Kingdom's University of Portsmouth has led to an improved variant of an enzyme that can break down ubiquitous plastic bottles made of polyethylene terephthalate, or PET.

While working to solve the crystal structure of PETase–a recently discovered enzyme that digests PET–the team inadvertently engineered an enzyme to be even better at degrading the man-made substance. Although the improvement is modest, this unanticipated discovery suggests that there is much more room to further improve these enzymes, moving scientists closer to solving the problem of an ever-growing amount of discarded plastics that take centuries to biodegrade.

The paper, "Characterization and engineering of a plastic-degrading aromatic polyesterase," was published this week in the Proceedings of the National Academy of Sciences (PNAS). The lead authors from the research team–NREL's Gregg Beckham, University of Portsmouth's John McGeehan, and Lee Woodcock from the University of South Florida–were attempting to understand how PETase evolved from likely working on natural substances to digesting synthetic materials when the serendipitous discovery was made.

The urgency of this work is as striking as the images pulled from recent headlines: 8 million metric tons of plastic waste, including PET bottles, enter the oceans each year, creating huge man-made islands of garbage. Experts estimate that by 2050, there will be as much waste plastic in the ocean by mass as there are fish. It's a global environmental problem that poses a serious risk to wildlife, particularly in marine environments.

Now imagine something as simple as a microbe that can degrade those plastic bottles. The good news: these organisms exist. A bacterium, Ideonella sakaiensis 201-F6, was discovered in the soil of a Japanese PET bottle recycling plant more than a year ago. The bad news: it doesn't work fast enough to solve plastic recycling at the industrial scale.

To begin experiments, the research team wanted to find out exactly how effective PETase was at digesting PET. NREL Senior Scientist Bryon Donohoe and postdoctoral researcher Nic Rorrer tested PETase by taking samples of PET from the soda bottles in Beckham's office and ran an experiment with PETase. "After just 96 hours you can see clearly via electron microscopy that the PETase is degrading PET," said Donohoe. "And this test is using real examples of what is found in the oceans and landfills."

But what if the researchers could engineer the enzyme to work a hundred times or a thousand times better?

"We originally set out to determine how this enzyme evolved from breaking down cutin–the waxy substance on the surface of plants–with cutinase, to degrading synthetic PET with PETase," said Beckham. After all, PET, patented as a plastic in the 1940s, has not existed in nature for very long. "We hoped to determine its structure to aid in protein engineering, but we ended up going a step further and accidentally engineered an enzyme with improved performance at breaking down these plastics. What we've learned is that PETase is not yet fully optimized to degrade PET–and now that we've shown this, it's time to apply the tools of protein engineering and evolution to continue to improve it."

NREL and the University of Portsmouth collaborated closely with a multidisciplinary research team at the Diamond Light Source in the UK, a large synchrotron that uses intense beams of X-rays 10 billion times brighter than the sun to act as a microscope powerful enough to see individual atoms. Using their beamline I23, an ultra-high-resolution 3D model of the PETase enzyme was generated in exquisite detail.

With help from the computational modeling scientists at the University of South Florida and the University of Campinas in Brazil, the team discovered that PETase looks very similar to a cutinase, but it has some unusual surface features and a much more open active site. These differences indicated that PETase must have evolved in a PET-containing environment to enable the enzyme to degrade PET. To test that hypothesis, the researchers mutated the PETase active site to make it more like a cutinase.

And this is where the unexpected happened. "Surprisingly, we found that the PETase mutant outperforms the wild-type PETase in degrading PET," said Rorrer. "Understanding how PET binds in the PETase catalytic site using computational tools helped illuminate the reasons for this improved performance. Given these results, it's clear that significant potential remains for improving its activity further."

Another significant aspect of the research: the discovery that PETase can also degrade polyethylene furandicarboxylate, or PEF, a bio-based substitute for PET plastics. The enhanced oxygen barrier properties of PEF could lead to its widespread use in bottles, which could ultimately find their way into the environment, thus adding to the pollution problem. "We were thrilled to learn that PETase works even better on PEF than on PET," said Beckham. "It is literally drilling holes through the PEF sample. This shows that by using PETase, PEF is even more biodegradable than PET."

While the invention of highly durable plastics has had positive impacts for humankind's quality of life, it's that very durability that is causing the plastics pollution problem. The structure of PET is too crystalline to be easily broken down and while PET can be recycled, most of it is not. PET that is recycled often exhibits inferior material properties as well. In addition, PEF plastics, although bio-based, are not biodegradable, and would still end up as waste in landfills and in the seas.

The team's goal is to use their findings to continue to improve the new enzymes to break down these man-made plastics, but in a fraction of the time. "Few could have predicted that in the space of 50 years, single-use plastics such as drink bottles would be found washed up on beaches across the globe," said McGeehan. "We can all play a significant part in dealing with the plastic problem. But the scientific community who ultimately created these 'wonder-materials,' must now use all the technology at their disposal to develop real solutions."

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The work reported in PNAS was enabled by funding from NREL's Laboratory Directed Research and Development (LDRD) program, the University of Portsmouth, and the UK's Biotechnology and Biological Sciences Research Council.

NREL is the U.S. Department of Energy's primary national laboratory for renewable energy and energy efficiency research and development. NREL is operated for the Energy Department by The Alliance for Sustainable Energy, LLC.

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https://www.nrel.gov/news/press/2018/research-team-engineers-a-better-plastic-degrading-enzyme.html

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