Certain filamentous fungi have the ability to decompose very stubborn materials such as wood. Other pathogenic fungi pierce complex materials such as the surface of very resistant plant tissues, or even the particularly strong shells of insects. How do they manage to tackle such robust materials? “These fungi secrete enzymes that degrade matter. Among these enzymes, we find LPMOs (lytic polysaccharide monooxygenases).
These are very special molecules which use copper in their active sites and allow very powerful oxidative chemistry, essential for breaking chemical bonds such as those that we encounter in wood cellulose,” explains Bastien Bissaro, enzymologist and researcher at the INRAE “Biodiversity and Fungal Biotechnology” (BBF) laboratory, located within the Calanques National Park of Aix-Marseille University (AMU). He continues: “Their strength also comes from the fact that their active sites are located on the surface, which is rare for an enzyme.”
With an active site on the surface, the enzyme can attack very large molecules such as wood cellulose, which is a polymer. During a first study a> published in 2021, researchers from the Marseille unit had already assessed the potential of a thousand mushrooms to degrade recalcitrant materials from a collection of more than 3000 strains. They tested these organisms on more or less recalcitrant materials such as wood, artificial dyes or components with a structure approaching that of plastic. Several of the fungi identified have the famous LPMOs.
Transforming enzymes
By analyzing the results, the researchers said to themselves that they could transpose these properties degradation to other types of materials such as artificial polymers such as plastics. “In nature, LPMOs do not attack plastic, for them to do so, we need to give them a helping hand. For this, the substrate, that is to say here the plastic, must be recognized by the enzyme and then the latter triggers catalysis,” explains the scientist.
The Marseille team working on the first stage. LPMOs are molecules composed of two modules: one which allows catalysis, and the other which binds to the substrate. “We want to modify this docking module so that it clings to the plastic. For this we are looking for proteins produced by fungi with hydrophobic properties (like plastic). Such proteins are notably found on the surface of mushroom spores, making them “waterproof” and thus facilitating their dispersion in the air.
They are also secreted by certain fungi to allow them to adhere to hydrophobic surfaces (like plant leaves),” explains Bastien Bissaro. The researchers changed the amino acid sequence that codes for the protein in LPMO. The DNA thus recomposed is then transformed into a yeast which will produce a “chimeric” LPMO: it contains the original catalysis module and the modified binding module.
They subsequently tested the properties of these new molecules by mixing them with different types of plastic polymers such as polyesters (polyhydroxyalkanoates [PHA]polylactic acid [PLA] and polyethylene terephthalate [PET]), but also polyolefins (polystyrene [PS]polyethylene [PE] and polypropylene [PP]), under various reaction conditions, such as changing pH, for example.
“We have identified certain chimeras capable of binding with other polymers. The starting LPMO (unmodified) bound to PHA, biodegradable polyester, at 10%. The chimeras have reached 80% binding, however, they are no longer able to bind to cellulose,” specifies the researcher. The team then focused on the most effective chimera to see if it had the ability to degrade the polymer.
The first results show a reduction in the size of the PHA molecules as well as the formation holes on the surface of the plastic. “Our enzymes have activity, but they are still very far from recycling alone,” adds Bastien Bissaro. For this to be possible, the catalytic module of the LPMO would now have to be evolved. Especially since the chimeras developed by the laboratory act on the surface. To ensure complete degradation, they must be combined with other types of enzymes in an enzymatic cocktail capable of attacking in depth.
Degrade to recycle?
But when we talk about degradation of plastics, what exactly does this correspond to?
In certain cases, for example with PLA which is obtained from corn starch, it would be possible to imagine decomposition in nature. “PLA could be metabolized by microorganisms in compost, because it is not a toxic polymer. It would then disappear completely and become biomass. On the other hand, certain plastics, such as PET, PP or PS, from petrochemicals, can never be degraded in compost,” explains Bastien Bissaro.
A polymer can be represented as a necklace of identical pearls where each pearl symbolizes a monomer. The latter can then be reassembled to form a new polymer using specific chemical conditions. This can work for plastics such as PET which have “break points” between the beads, namely ester chemical functions, which are easy to break back to the monomer.
“This is not the case for polyolefins (PE, PP and PS) which have a very solid chemical structure, with no obvious “breaking point”. To separate them, oxygen atoms are introduced, but the product obtained in the end is not the monomer. We cannot therefore talk about recycling “, explains the researcher.
The products thus formed could be used differently, since they have different properties. But research is still in its infancy in this area and technical-economic constraints (such as added value and the volume of substances generated) play a preponderant role. “We must also think about developing polymers whose recyclability will be programmed from the design, and which will replace our current plastics. But in the meantime, the end of life of polymers that are not easily recyclable must be considered. So, we might as well try to degrade them and enhance them,” concludes Bastien Bissaro.
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