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The discovery of an international group of scientists, which includes two Brazilians, may help to minimize pollution caused by the material

An international group of scientists, in which two Brazilians from the University of Campinas (Unicamp) participate, managed to improve the performance of PETase, an enzyme capable of feeding on polyethylene terephthalate, PET plastic. After PETase was discovered in a new species of bacteria in 2016, the group of researchers worked to obtain the structure of the enzyme and understand how it works. In the process, by accident, they ended up creating a mutation of the enzyme that has an even greater affinity for PET - that is, a greater potential to degrade plastic.

The work has enormous potential for practical use, as it is estimated that between 4.8 and 12.7 million tons of plastic are released into the oceans every year - a number that is only likely to grow. Plastics, which accumulate even on the most remote beaches on the planet, are so used precisely because of their resistance to degradation, which is what threatens the environment the most. When discarded, a PET bottle, for example, can remain in the environment for 800 years - in addition to the growing and alarming problem of microplastics.

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With all this, it is easy to understand the great interest aroused by the discovery of an enzyme capable of digesting polyethylene terephthalate. This enzyme, called PETase, has now increased its ability to degrade plastic. The novelty was described in an article published in Proceedings of the National Academy of Sciences of the United States of America (PNAS).

Two researchers from the Institute of Chemistry at the State University of Campinas (IQ-Unicamp) participated in the research, in collaboration with researchers from the United Kingdom (University of Portsmouth) and the United States (National Renewable Energy Laboratory). They are the postdoctoral student Rodrigo Leandro Silveira and his supervisor, the head professor and dean of Research at Unicamp Munir Salomão Skaf.

“Mainly used in the manufacture of beverage bottles, polyethylene terephthalate is also widely used in the manufacture of clothes, rugs and other objects. In our research, we characterized the three-dimensional structure of the enzyme capable of digesting this plastic, we engineered it, increasing its degradation power, and we demonstrated that it is also active in polyethylene-2,5-furandicarboxylate (PEF), a substitute for PET manufactured by from renewable raw materials,” Silveira told Agência FAPESP.

Interest in PETase emerged in 2016, when a group of Japanese researchers, led by Shosuke Yoshida, identified a new species of bacteria, the Ideonella sakaiensis, able to use polyethylene terephthalate as a source of carbon and energy – in other words, able to feed on PET . It is, to date, the only known organism with this ability. It literally grows on PET.

"In addition to identifying the Ideonella sakaiensis, the Japanese discovered that it produced two enzymes that are secreted into the environment. One of the secreted enzymes was precisely PETase. Because it has a certain degree of crystallinity, PET is a polymer that is very difficult to degrade. We technically use the term 'recalcitrance' to name the property that certain tightly packed polymers have to resist degradation. PET is one of them. But PETase attacks it and breaks it down into small units – mono(2-hydroxyethyl) terephthalic acid (MHET). The MHET units are then converted into terephthalic acid [by a second enzyme] and absorbed and metabolized by the bacteria,” said Silveira.

All known living things use biomolecules to survive. All except Ideonella sakaiensis, which manages to use a synthetic molecule, manufactured by humans. This means that this bacterium is the result of a very recent evolutionary process that took place over the last few decades. It managed to adapt to a polymer that was developed in the early 1940s and only began to be used on an industrial scale in the 1970s. For that, PETase is the key piece.

“PETase does the hardest part, which is breaking the crystalline structure and depolymerizing PET into MHET. The work of the second enzyme, the one that transforms MHET into terephthalic acid, is already much simpler, since its substrate is formed by monomers to which the enzyme has easy access because they are dispersed in the reaction medium. Therefore, the studies focused on PETase”, explained Silveira.

The next step was to study PETase in detail and this was the contribution of the new research. “Our focus was to find out what gave PETase the ability to do something that other enzymes weren't able to do very efficiently. For that, the first step was to obtain the three-dimensional structure of this protein”, he said.

“Obtaining the three-dimensional structure means finding the x, y and z coordinates of each of the thousands of atoms that make up the macromolecule. Our British colleagues did this work using a well-known and widely used technique called X-ray diffraction,” he explained.

Modified enzyme binds better to polymer

Once the three-dimensional structure was obtained, the researchers began to compare PETase with related proteins. The closest thing is a cutinase from the bacterium Thermobifida fusca, which degrades cutin, a kind of natural varnish that coats plant leaves. Certain pathogenic microorganisms use cutinases to break the cutin barrier and appropriate the nutrients present in the leaves.

Image: PETase structure, in blue, with a PET chain (in yellow) attached to its active site, where it will be degraded. Press Release/Rodrigo Leandro Silveira.

“We found that, in the region of the enzyme where the chemical reactions take place, the so-called 'active site', PETase had some differences in relation to cutinase. It has a more open active site. Through computer simulations – and this was the part I contributed the most – we were able to study the molecular movements of the enzyme. While the crystallographic structure, obtained by X-ray diffraction, provides static information, the simulations make it possible to have dynamic information and discover the specific role of each amino acid in the PET degradation process”, explained the researcher from IQ-Unicamp.

The physics of molecule motion results from the electrostatic attractions and repulsions of the huge array of atoms and temperature. Computer simulations allowed a better understanding of how PETase binds and interacts with PET.

“We found that PETase and cutinase have two different amino acids in the active site. Using molecular biology procedures, we then produce mutations in PETase, with the aim of transforming it into cutinase”, said Silveira.

“If we were able to do this, we would show why PETase is PETase, that is, we would know which components give it such peculiar property of degrading PET. But to our surprise, by trying to suppress the peculiar activity of PETase, that is, by trying to transform PETase into cutinase, we produce an even more active PETase. We were looking to reduce the activity and, instead, we increased it”, he said.

This required further computational studies to understand why the mutant PETase was better than the original PETase. With the modeling and simulations, it was possible to see that the changes produced in PETase favor the coupling of the enzyme with the substrate.

The modified enzyme binds better to the polymer. This coupling depends on geometric factors, that is, the “key and lock” type fit between the two molecules. But also thermodynamic factors, that is, the interactions between the various components of the enzyme and the polymer. The elegant way to describe this is to say that modified PETase has “greater affinity” for the substrate.

In terms of a future practical application, of obtaining an ingredient capable of degrading tons of plastic waste, the study was a huge success. But the question of what makes PETase a PETase remains unanswered.

“Cutinase has amino acids a and b. PETase has amino acids x and y. We imagine that, by exchanging x and y for a and b, we would be able to transform PETase into cutinase. Instead, we produce an improved PETase. In other words, the two amino acids are not the explanation for the differential behavior of the two enzymes. It's another thing,” said Silveira.

ongoing evolution

Cutinase is an ancient enzyme, while PETase is a modern enzyme, resulting from the evolutionary pressure that made it possible to Ideonella sakaiensis adapt to an environment that contains only or mainly polyethylene terephthalate as a source of carbon and energy.

Among the many bacteria unable to use this polymer, some mutation generated a species that managed to do so. This bacterium began to reproduce and grow much more than the others because it had enough food. With that, she developed. At least that is the explanation provided by standard evolutionary theory.

“The fact that we got a better enzyme by making a small change strongly suggests that this evolution is not yet complete. There are still new evolutionary possibilities to be understood and explored, with a view to obtaining even more efficient enzymes. Improved PETase is not the end of the road. It's just the beginning,” said Silveira.

With a view to application, the next step is to move from laboratory to industrial scale. For this, other studies, related to reactor engineering, process optimization and cost reduction will be necessary.