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The modified enzyme may increase the production of second-generation ethanol

Publicado em 17 junho 2019

Using the protein produced by the fungus inhabiting the Amazon, Brazilian scientists have developed a molecule capable of increasing the release of glucose from biomass for fermentation

One of the main challenges associated with the production of second generation biofuels is the identification of enzymes produced by microorganisms for use in an enzyme cocktail to catalyze the hydrolysis of biomass, in which enzymes work together, for example to break down carbohydrates in the garbage and pancreas cane and convert them into sugars easy to ferment.

A group of researchers from the University of Campinas (UNICAMP), working with colleagues from the Brazilian Biorenewables National Laboratory (LNBR) in Campinas, Sao Paulo, Brazil, discovered that Trichoderma harzianum, a fungus found in the Amazon, produces an enzyme that can play a key role in enzyme cocktails.

The enzyme, which is called ß-glucosidase and belongs to the family of glycoside hydrolases 1 (GH1), works in the last stage of biomass degradation, producing free glucose for fermentation and conversion to ethanol. However, in the laboratory, scientists have observed that high glucose levels inhibit ß-glucosidase activity.

'We also found that the optimal catalytic activity of the enzyme occurred at 40 ° C. This was another hurdle in using the enzyme because under industrial conditions enzymatic biomass hydrolysis is carried out at higher temperatures, typically around 50 ° C,' said Clelton Aparecido dos Santos, researcher postdoctoral at the Center for Molecular Biology and Genetic Engineering UNICAMP (CBMEG) from the FAPESP scholarship.

Based on the analysis of the enzyme structure in combination with genomics and molecular biology techniques, researchers were able to modify the structure to address these problems and significantly increase the efficiency of biomass degradation.

The study resulted from a project with a regular research grant FAPESP and Thematic Project supported by FAPESP. The findings were published in the journal Scientific reports.

"The modified protein we developed turned out to be much more efficient than the unmodified enzyme and could be used to complement the enzyme cocktails sold today to break down biomass and produce second generation biofuels," Santos said.

To obtain the modified protein, the researchers initially compared the crystal structure of the original molecule with those of other wild-type ß-glucosidases in the GH1 and GH3 glycosidase hydrolases families. The results of the analysis showed that glucose tolerant GH1 glucosidases had a deeper and narrower substrate channel than other ß-glucosidases and that this channel restricted glucose access to the active site of the enzyme.

Smaller glucose tolerant ß-glucosidases had a shallower but wider input channel at the active site, allowing more of the glucose produced by these enzymes to enter the final stage of biomass degradation. Stopped glucose blocks the protein channel and reduces its catalytic activity.

Based on this observation, the researchers used a molecular biology technique known as site-directed mutagenesis to replace the two amino acids that can act as "guards" when entering the active site of the enzyme, admitting glucose or blocking it. Analysis of their experiments showed that the modification narrowed the channel to the active site.

"The active site of the mutant enzyme has shrunk to the size of glucose-tolerant ß-glucosidase GH1," Santos said.

Increased efficiency

Researchers conducted a series of experiments to measure better protein yield in biomass degradation, especially sugar cane pomace, agro-industrial waste with high potential for profitable use in Brazil. During a research internships abroad with a scholarship from the research foundation São Paulo – FAPESP, Santos worked with a research group led by Paul Dupree, a professor at the University of Cambridge in Great Britain, on the analysis of the enzyme-releasing glucose yield when it is a different source of plant biomass they were transformed.

The analysis showed that the catalytic efficiency of the modified enzyme was 300% higher than that of the wild type enzyme in terms of glucose release. What's more, it was more tolerant to glucose, so more glucose was released from all the tested plant biomass resources. The mutation also increased the thermal stability of the enzyme during fermentation.

"The mutation of two amino acids in the active site made the enzyme superegressive. It is ready for industrial use, "said Anete Pereira de Souza, professor of UNICAMP and principal project researcher. "One of the advantages of the enzyme is that it is produced in vitro, not from a modified fungus or other organism, so it can be mass produced at a relatively low cost."

Research shows how genetics can contribute to the development of 2G ethanol

More information:

Clelton A. Santos et al., Modified ß-glucosidase GH1 shows increased glucose tolerance and increased sugar release from lignocellulosic materials, Scientific reports (2019). DOI: 10.1038 / s41598-019-41300-3

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The modified enzyme may increase the production of second-generation ethanol (2019, 17 June)
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