From November 13 to 19, the third Global Plastics Treaty Meeting was held in Nairobi (Kenya), which tried, albeit in vain, to find an agreement to hold "countries and companies accountable for their action, or inaction, on plastic pollution and its impact on our health, environment and economy". World leaders have set themselves a date to further discuss the issue in April 2024 in Ottawa, Canada, but in the meantime, plastics and microplastics continue to produce pollution, primarily in waters around the globe. Among the possible solutions, beyond reduction, is to find a method that destroys these plastics, something in which researchers from the Faculty of Chemical Sciences, who have designed, in collaboration with the Barcelona Supercomputing Center and the Institute of Catalysis of the CSIC, protein nanopores for the capture and degradation of microplastics.
Professors Sara García Linares and Élvaro Martínez del Pozo, from the Department of Biochemistry and Molecular Biology of the Faculty of Chemical Sciences, are two of the authors of the work, which has been published, after several exhaustive reviews, in Nature Catalysis. Both are also members of the "Structure-Function in Proteins" research group, which Martínez del Pozo himself directs, and whose decades of research have led to this promising result.
Sara García Linares explains that the Barcelona group, which specializes in bioinformatics, has created software that combines prediction algorithms and artificial intelligence. With this software they can "take a protein with a known structure, give it a substrate, and from there see what changes need to be made in that protein so that the substrate fits and can be modified".
At a certain point, Víctor Guallar, a member of the Barcelona Supercomputing Center, wanted to develop a pore-forming protein, and in his search he came across some actinoporins that UCM has been researching for decades, which are a family of pore-forming toxins found in sea anemones. According to Martínez del Pozo, "almost all poisons have pore-forming proteins, because the membrane is a good target to break a cell".
The idea, from Barcelona, was to "change this actinoporin to convert it into an esterase, an enzyme that hydrolyzes esters", which, simply explained, is a chemical process that is very relevant in the degradation of organic compounds in nature, but also in the synthesis of various chemical products.
Guallar’s team gave the Complutense the list of changes to be made, and Martínez del Pozo’s group designed these modified proteins, using basic genetic engineering techniques. Once two mutant versions were obtained, they were passed on to Manuel Ferrer’s group at CSIC to test the activity of these proteins.
In doing so, it was seen that not only did it degrade esters, but also that those it degraded had a very specific structure, very similar to that of PET, the plastic used massively in the manufacture of bottles and containers. This led to experiments with micro and nanoparticles of this plastic, to see if it also degraded them, with positive results.
According to García Linares, moreover, "the two mutants complement each other very well, because in one two amino acids have been changed and in the other two different amino acids, and although both mutants perform the same function, the degradation products are different. One goes to the end of degradation, to the minimum component of PET, and would be great for cleaning, for complete degradation, while the other stays a few steps earlier, which would allow PET to be recycled".
The protein
The Complutense has been studying this protein since the mid-1990s, but according to the two UCM professors, thanks to its structure, "it is a system that still has a lot of room for improvement, with new or multiplied activities. This protein is part of the toxic arsenal of a sea anemone that, if you go to the coast of northern Spain, you see it as soon as the tide goes out, you don’t even need to dive. This anemone, if its tentacles are touched with a microscopic harpoon, injects the poison, which is a cocktail of proteins, and one of them is the one used in this study, and "it is toxic because it makes holes in the cell membranes, which causes cell death".
The sting of this anemone is similar to that of a jellyfish, but for a human it is less powerful, because our skin is too tough for it. Martínez del Pozo affirms that his group, and also this project, tries to "convert toxins into benefits, in this case, making the protein of a poison, which can even kill you if injected in sufficient quantity, become a beneficial product". García Linares adds that, "the particle obtained is made of fats and protein, so it is completely safe and biodegradable. It is a toxic protein, but for it to take effect, the anemone, with its microscopic harpoon, would have to inject the protein, but on the outside it does absolutely nothing, and you could even eat it because in the acid PH of the stomach it would degrade without any consequences".
The initial interest in these proteins, according to Martínez del Pozo, was of a biophysical nature, because "proteins in general are either water-soluble or are inserted in a membrane. And this protein is perfectly soluble in water, but, in addition, without any kind of sequence changes or external modification, simply by interaction with a membrane it is able to insert itself into it. There are very few proteins that are in that intermediate gray zone".
For decades, the UCM has studied this molecule extensively at the molecular level and it is known that, if all proteins are made up of several amino acids, this particular one has 175, with regions structured in different ways and with different functions, some of which are important for insertion into the membrane, for sticking to it.... These functions have been known by changing parts of the protein during "many years of detailed study, so much so that we have been able to use all this information and now say that if it is changed in a particular way it will degrade an ester".
In vitro experiment
The UCM professors explain that "so far the experiments have been carried out in a tube, in an aqueous preparation, where a preparation of plastic nanoparticles is dropped, and it is seen how the particle, which is shaped like a field hockey puck with the pore inserted, decomposes them". What has not been tested is to do it with large amounts of water, which would be a next step, because "the innovative thing would be to be able to scale it up with contaminated water, pour a specific amount of the particle, leave it for a while and see what happens".
A great advantage of the system, which has attracted the attention of the magazine, is that "there are already other proteins that degrade this type of plastic, but they require the preparation to be at 70º, while this one degrades at room temperature, which is much better, since it is not necessary to heat and emit more COx#2#xx# to try to clean the plastic".
Another advantage is that "it is very simple to make, no innovative techniques or exotic materials are used to manufacture the system, so any interested company could scale it up without any problems. Bacteria are used to make the protein, as is used, for example, to make insulin".
Future possibilities
Once the initial discovery of PET degradation has been made, the Complutenses report that the work will continue with the search for new functionalities. The mutants already obtained degrade esters and now they want to make one that, for example, "degrades proteins, because there are many contaminants of a protein nature, or nylon, which is a major pollutant in the textile industry, which would be very interesting to eliminate".
García Linares clarifies that at present the problem of plastics is enormous on any scale, although a ton of large plastic in the sea, with a lot of effort and time, can be removed with nets. The problem, he adds, "is that the plastic, while it is in the sea, degrades, forming such tiny particles that there is no way to remove them. Therefore, the idea is to make filters that have this pore incorporated, and when filtering the water, the nanoparticles are cleaned".
Another idea for future work, and since the pore created by this protein is quite small, would be "to look for proteins that make larger pores, to be able to enlarge the size of particles that can be degraded, or to reduce the time it takes to degrade larger particles."
It would also be possible to "mix different activities in the same pore, modify them to degrade esters and other proteins or similar bonds, but all in the same structure, so that the filter is multifunctional, to remove different types of contaminants, including pharmacological ones".
In addition to the authors mentioned above, the other two authors from Complutense are Rafael Amigot-Sánchez, "who was doing his master’s degree at the UCM, but because he did not have the funding to support it, he had to go to the Carlos III Institute", and the doctoral student Diego Heras-Márquez.
