Home Science & Technology The recently discovered enzyme may allow the disposal of agro-industrial waste

The recently discovered enzyme may allow the disposal of agro-industrial waste

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Brazilian researchers have identified, characterized and confirmed the functions of two new families of enzymes with biotech potential.

One method of reducing dependence on oil and other fossil fuels is to convert agro-industrial waste into molecules of importance to society, such as biofuels and biochemicals. Brazil is well positioned to lead this shift as one of the world’s major producers of plant biomass, but lignocellulosic raw materials (containing lignin, hemicellulose and cellulose) are difficult to deconstruct or (more technically) resistant to microbial and enzymatic degradation.

Brazilian scientists are looking to nature for clues on how to improve the depolymerization of these materials by increasing the availability of the sugars they contain. A research team from the Brazilian National Laboratory of Biodegradable Resources (LNBR), a division of the Brazilian Center for Energy and Materials Research (CNPEM), conducted an interdisciplinary study involving omics (genomics, proteomics, metabolomics, etc.) and sin. (so Paul’s condition), and discovered two new families of enzymes with biotechnological potential that are produced by microorganisms in the intestinal capybara. CNPEM is a private non-profit organization controlled by the Ministry of Science, Technology and Innovation (MCTI).

Both families of enzymes act on components of plant cell walls and can therefore be used to produce biofuels, biochemicals and biomaterials. One of them also has potential applications in the dairy industry because it promotes lactose degradation.

Intestinal microorganisms capybara

Microorganisms present in the digestive tract of animals may have unique molecular strategies for depolymerizing this biomass. Credit: Gabriela Felix Persinotti

“One of our research areas is exploring Brazil’s diversity in search of new microbial mechanisms that reduce the reluctance of lignocellulosic waste. We noted that the capybara is a highly adapted herbivore capable of producing energy from unruly plant waste, and that it has been little studied, ”said Mario Thiago Murakami, LNBR’s research director and the latest author of a study published in The nature of communication.

Capybara (Hydrochoerus hydrochaeris) is the world’s largest rodent that lives in the world, and very efficiently converts sugar contained in plants into energy, although in some circles it is disliked because it may contain a tick that carries Brazilian spotted fever, a rare but very deadly infectious disease. caused by the bacterium Rickettsia rickettsii.

“There is a lot of research on ruminants, especially cattle, but there is relatively little information on monocotyledonous herbivores. Unlike ruminants, capybaras digest grass and other plant substances in the cecum, the first part of the large intestine. In light of their highly efficient sugar conversion, and also because capybaras in the Piracicaba region [of São Paulo state] feeding on sugar cane, among other plants, we hypothesized that microorganisms present in the digestive tract of animals may have unique molecular strategies for depolymerization of this biomass, which is very important for the Brazilian industry, “said Gabriela Felixi Persicotin LNBR. and the author-correspondent of the article.

The study was supported by FAPESP through a thematic project and a doctoral fellowship awarded to Marianne Abragao Bueno de Morais.

New methodology

The interdisciplinary approach used in the study included multi-omics (genomics, transcriptomics, and metabolomics used to characterize the molecular aspects of capybara gut microbiota) and bioinformatics, as well as CNPEM particle accelerators to analyze detected enzymes at the atom. “I can’t recall any studies that would combine all of these techniques, including the use of synchrotron light [a source of extremely bright electromagnetic radiation that helps scientists observe the inner structures of materials]”- said Murakami. “In this study, our analysis went all the way from the microbial community to the atomic structure of some proteins.”

The researchers analyzed samples collected from the cecum and rectum of three female capybaras euthanized in Tatoo (Sao Paulo) in 2017 in accordance with local policies to control the capybara population. The animals were neither pregnant nor infected with R. rickettsii.

“Samples of the cecum and rectum were taken by abdominal surgery. The material was frozen in liquid nitrogen. DNA and RNA the samples were taken in the laboratory and given to large-scale sequencing using integrative omics, ”Persinotti said.

They began by sequencing marker genes, in this case 16S, present in all bacteria and archaea. “With this first sequencing, we were able to detect differences between caecal and rectal samples and identify the major microorganisms in them. The 16S gene gave us a superficial answer to which microorganisms are present and much more or less, but did not tell us which enzymes are produced by microorganisms and which enzymes are present in their genome, ”she explained. “To do this, we used another omic technique – metagenomics. We donated DNA from the entire microbial community in the gastrointestinal tract to the capybara for large-scale sequencing, obtaining more data. By deploying a variety of bioinformatics tools, we were able to not only identify the genomes present in each of the samples and the genes in each of the genomes, but also find out which genes were new and which microorganisms had never been described. In this way, we were able to predict the functions of genes that could help depolymerize biomass and convert sugar into energy. ”

The researchers also wanted to know which microorganisms were most active at the time the samples were collected – in other words, which genes were actually detected by the microorganisms. To this end, they used metatranscriptomics, for which the raw material is RNA. “Another method we used was metabolomics to confirm which metabolites are produced by microorganisms,” Persinotti said. “Combining all this information from omics, bioinformatics and actual and potential gene expression, we were able to decipher the role of gut microorganisms in achieving such highly efficient conversion of plant fibers and find out which genes were involved in the process.”

They then analyzed all of this data to identify genes that could play a key role in reducing plant fiber resistance, focusing mainly on hitherto unknown targets. “The selection strategy focused on new genomes with many genes involved in depolymerizing plant biomass,” Persinotti said. “We saw how these genes were organized in the genomes of microorganisms, and used this information to find out if there are nearby genes with unknown functions that may be involved in the cleavage of unruly plant fiber. This is important because it drives the search for new genes, but only if we were able to demonstrate these results experimentally at a later stage could we establish the creation of these new families of enzymes. ”

Having identified these candidates, they proceeded to a biochemical demonstration of their functions. “We synthesized genes in vitro and expressed them with bacteria to produce the appropriate proteins,” Persinotti said. “We conducted several enzymatic and biochemical analyzes to identify the functions of these proteins and where they act. We determined the atomic structures of proteins using synchrotron light and other methods. With this functional and structural information, we were able to conduct other experiments to find out which areas of proteins are crucial to their activities, and to analyze the molecular mechanisms underlying their functions. ”

According to Murakami, the double-check ensured that new families were indeed involved. “We chose a gene not very similar to the one we studied before, in a set of sequences that theoretically formed the universe of the newly discovered family. We synthesized the gene, purified it, characterized it biochemically and showed that the sequence has the same functional properties as the previous one, ”he explained. “In other words, we described the second member of the new family to be absolutely sure that these proteins really make up the new family.”

New enzymes and cocktails

According to Persinotti, one of the recently discovered families, GH173, has potential applications in the food sector, and another, CBM89, is linked to carbohydrate recognition and could help produce second-generation ethanol from sugar cane and straw.

Researchers are also developing enzyme cocktails with mushrooms that overproduce enzymes, and newly discovered enzymes can naturally be incorporated into these fungal platforms. “The discovery of new enzyme families can be integrated with technology transfer to support innovation,” Murakami said. “In our group, we are very interested in exploring this great Brazilian treasure of biodiversity, particularly to understand what we call dark genomic matter – part of these complex microbial communities with unknown potential. Our center has an excellent infrastructure for this purpose, and together with our partnerships with public universities it has made it possible to conduct such research in Brazil. Indeed, 99% of the work was done here, from conceptual design to execution, analysis and writing. Given the enormous richness of Brazil’s biodiversity, it was to be expected that we would have the conditions and opportunities for discoveries like these.

Reference: “The intestinal microbiome of the largest of the living rodents contains unprecedented enzymatic systems for the degradation of plant polysaccharides” Lucelia Cabral, Gabriela F. Persinotti, Douglas AA Paishao, Marcele P. Martins, Mariana AB Monais, Maria, Maria Dominguez, L. Sforka, Renan A. S. Pyrolla, Wesley S. Generoso, Clalton A. Santos, Lucas F. Masiel, Nicolas Terapon, Vincent Lombard, Bernard Anrisat and Mario T. Murakami, February 2, 2022, The nature of communication.
DOI: 10.1038 / s41467-022-28310-y

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