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Modified enzyme brings value to lignin monomers

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a EUGO active site positions selected for mutagenesis (blue spheres) and changes observed at all positions according to multiple sequence alignments (top). FAD cofactor and 4-n-Propylguaiacol (4PG) ligands are represented by gray and light blue balls, respectively. Residues selected by the Monte Carlo algorithm for all positions in the Rosetta Coupled Moves experiment are shown (bottom). b Ligand scores from Rosetta Coupled Moves (top) and binding energies from Autodock VINA (bottom) are displayed in a box plot showing the median of the data as the center line and the boxes represent the quartiles. Sections cover 25-75% of the distribution, and hints represent the smallest and largest outliers. credit: Nature Communications (2022). DOI: 10.1038/s41467-022-34912-3″ width=”800″ height=”397″/>

Computer analysis to improve activity. a EUGO active site positions selected for mutagenesis (blue spheres) and changes observed at all positions according to multiple sequence alignments (top). FAD cofactor and 4-n-Propylguaiacol (4PG) ligands are represented by gray and light blue balls, respectively. Residues selected by the Monte Carlo algorithm for all positions in the Rosetta Coupled Moves experiment are shown (bottom). b Ligand scores from Rosetta Coupled Moves (top) and binding energies from Autodock VINA (bottom) are displayed in box plots showing the median of the data as the center line, boxes from 25 to 75 for all selected variants. represents the interquartile section that covers the % of the distribution, and hints represent the smallest and largest outliers. credit: Nature Communications (2022). DOI: 10.1038/s41467-022-34912-3

The chemical industry faces the challenge of replacing fossil-based building blocks with environmentally friendly alternatives. Biomass is an interesting source of carbon-based molecules. Also, about 25% of all plant biomass is in the form of lignin, a biopolymer that so far can only be used as a solid fuel and is therefore underutilized.

A team of scientists, including ‘enzyme engineer’ Marco Fraaije at the University of Groningen, has developed an enzyme that can use lignin monomers. chemical synthesisThis enzyme is Nature Communications paper.

Plant biomass is an attractive source of carbon-based molecules. However, about a quarter of this material is lignin, a biopolymer that is difficult to break down into useful chemical building blocks. As a result, lignin is mainly used as a green fuel in energy production. That’s why scientists around the world are looking for ways to make better use of lignin.

fragrance

Professor Marco Fraaye, head of the Molecular Enzymes Group at the University of Groningen, is collaborating on a European research project to increase the value of biomass and is looking at lignin. “We knew that a group at the University of Leuven in Belgium had developed a chemical process to degrade this polymer, but unfortunately the resulting monomers are not very useful for chemical synthesis.”

This is why Fraaije and his colleagues within the SMARTBOX (Selective Modification of ARomatics by Biocatalytic Oxidation) project investigated how to modify this monomer.

“Chemical depolymerization of lignin can produce many different molecules, one of which seemed very useful,” says Fraaije. This aromatic monomer is called propylguaiacol and is almost identical to the compound used to make fragrances. is an aromatic molecule consisting of ring structure A short tail of three carbon atoms. “I wanted to insert double bond Since it is on the tail, it will be easier to use as a building block. And I knew an enzyme that might do the trick. ”

alcohol

However, tests have shown that this particular enzyme does not work well and produces the wrong product. was “Working with our partners in Barcelona, ​​we used computational tools to predict the modifications necessary to make the enzyme more stable, more selective, and to speed up the conversion of monomers into useful building blocks. Did.”

The first two steps were performed relatively quickly. “Thanks to you, computation is now possible. It’s been very reliable. Before, you just made a bunch of mutants and hoped that one of them would show an improvement.” Due to the increased potency, the enzyme remains active for several days.

This reduces the costs associated with replacing used enzyme batches. Also, enzymes change carbon tails to alkenes or alcohols. The latter is less useful, and by excluding water from the enzyme’s active site, the reaction may be forced towards the formation of the alkene.

upscaling

So far so good, but the stable selective enzyme has slowed down dramatically. “The structure of the enzyme was then determined by colleagues in Pavia,” he says Fraaije. “And it turned out that the substrate was covalently bound to the active site.” Covalent bonds are very strong chemical bonds, so releasing the substrate took a considerable amount of time. “Such covalent bonds between enzymes and substrates are very rare, but I encountered them during my own Ph.D. research.”

When this issue was resolved by another engineering round, enzyme worked brilliantly. “We’ve already done experiments that produce 1 gram of the final product,” says Fraaije. This molecule can be used in fragrances, but also serves as a starting compound for a variety of other compounds such as vanillin, polymers, fine chemicals, and epoxy resins.

Another project partner, the bio-based European pilot plant in Ghent (Belgium), will be responsible for expanding the production of modified propylguaiacol. Marco Fraaije’s work on the SMARTBOX project has ended. But he will no doubt find other enzymes to work with.

For more information:
Yiming Guo et al, Structural and computational aided engineering of an oxidase that produces isoeugenol from lignin-derived compounds, Nature Communications (2022). DOI: 10.1038/s41467-022-34912-3

Quote: Modified Enzymes Bring Value to Lignin Monomers (29 Nov 2022) Retrieved 30 Nov 2022 from https://phys.org/news/2022-11-enzyme-lignin-monomers.html

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