Researchers have discovered a novel enzyme (a β-glucosidase) in microorganisms living in the Brazilian Amazon that could boost efficiency of the sugarcane bagasse saccharification process, which accounts for up to 50% of the global costs of cellulosic ethanol production.

The team isolated, characterized and reproduced the enzyme, proving it to be compatible with two essential stages of the production of second-generation (cellulosic) ethanol: fermentation and saccharification. If these two stages can be performed simultaneously, the sugar and ethanol industry could cut its costs substantially because a single reactor can be used for all processes involved, economizing on reagents.

The study involved researchers at Brazil’s National Energy & Materials Research Center (CNPEM), Petrobras, the University of São Paulo (USP), and the Federal University of São Carlos (UFSCar), also in São Paulo State, and was supported by the Sao Paulo Research Foundation - FAPESP. An article by the research team has been published in Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics.

Although they do not act directly on cellulose, β-glucosidases play a fundamental role in its degradation, since the activities of endoglucanase and exoglucanase are inhibited by cellobiose. In addition to the production of glucose by hydrolysis of cellobiose, they also mitigate the inhibitory effect on the enzymatic complex. However, glucose may act by inhibiting the activity of β-glucosidases; thus, there is an increasing demand for enzymes insensitive to inhibition by its product and with high thermal stability to improve the saccharification process of lignocellulosic materials. Currently, enzymes of the cellulolytic complex, such as β-glucosidases, are of great biotechnological interest, mainly due to their application in the enzymatic saccharification of lignocellulosic materials, such as sugarcane bagasse, for the production of biofuels.

In this study, a novel β-glucosidase from a freshwater metagenome of Lake Poraquê from the Amazon region was recombinantly expressed and biochemically and structurally characterized. … our data indicate that AmBgl-LP can be used as component of cocktails for biomass degradation, reinforcing the importance of metagenomic approach for both purposes.

—Toyama et al.

The enzymes required to convert complex sugars into simple sugars account for between 30% and 50% of the cost of cellulosic ethanol, said Mario Tyago Murakami, a researcher at CNPEM and one of the principal investigators for the project.

The enzymes’ conversion efficiency is currently 50%-65%. That means between 50% and 35% of the sugar available in the biomass is ‘lost’ during saccharification. Our study set out to find biocatalysts that can help enhance enzyme efficiency.

—Mario Murakami

In the arsenal of necessary enzymes acting synergistically, beta-glucosidases are crucially important for their role in the last step of the cellulose saccharification cascade.

We know the saccharification rate falls as the proportion of saccharification product rises, because the presence of the product inhibits enzyme action. This is a sort of general rule. In this specific case, the glucose produced inhibits the action of beta-glucosidase. This technological constraint has been exhaustively studied. To increase saccharification efficiency, the beta-glucosidase has to be highly glucose tolerant.

—Mario Murakami

Owing to genetic specificities that derive from differences in the evolutionary process, homologous enzymes can display varying degrees of resistance to inhibition by the product. The aim of this study was to find beta-glucosidases suited to the biomass that is available in Brazil; to this end, the researchers focused on natural processes occurring in the Amazon Forest and Cerrado (Brazilian savanna) biomes.

Flavio Henrique da Silva, Full Professor in UFSCar's Department of Genetics & Evolution and co-principal investigator for the project, was responsible for this bioprospecting enterprise. The most promising discovery occurred in Lake Poraquê in the upper Solimões, near Coari, Amazonas. Samples taken from the local non-cultivable microbial community contained genes encoding beta-glucosidases with the requisite industrial potential.

In his enzymological studies, Silva found that the beta-glucosidase encoded by microorganisms in Lake Poraquê could be an efficient catalyst in sugarcane bagasse saccharification and was highly glucose-tolerant. The next step was taken by members of Murakami’s team who specialize in mechanistic structural biology and who elucidated this enzyme’s functioning at the molecular and atomic level.

With regard to molecular structure, the oligomeric study evidenced a protein that differed from others in its category, with a unique quaternary architecture.

This study corroborated previous research by the group regarding the structural determinants for the enzyme's product tolerance, validating our mechanistic model. In addition, we found that this beta-glucosidase is effective under temperature and pH conditions compatible with the hydrolysis process.

—Mario Murakami

This information is highly relevant because it suggests the enzyme discovered by the group could be used in simultaneous saccharification and fermentation (SSF). Because it is effective at temperatures compatible with the growth of yeast, this beta-glucosidase enables the release of carbohydrates resulting from saccharification and their fermentation by yeast to occur at the same time. This strategy mitigates inhibition by the product because the sugars are consumed by the yeast as they are released, avoiding inhibition of the enzyme due to excessive glucose production.

The next step will be to study combining the enzyme with existing fungal enzyme cocktails to enhance efficiency by boosting saccharification.

Once the gene of interest has been extracted using gene libraries for non-cultivable microorganisms and possible rational modifications based on knowledge of the structure to increase thermostability, it’s transferred to other hosts by means of molecular biology techniques,. The host in question is Trichoderma, a filamentous fungal genus that already has an arsenal of carbohydrate-active enzymes. The addition of beta-glucosidase from the Amazon will boost its potential. The goal is to enhance the efficiency of an industrial microbial platform that already exists.

—Mario Murakami

The researchers plan to apply for a patent on the fungus engineered with the enzyme.

Resources

• Danyelle Toyama, Mariana Abrahão Bueno de Morais, Felipe Cardoso Ramos, Letícia Maria Zanphorlin, Celisa Caldana Costa Tonoli, Augusto Furio Balula, Fernando Pellon de Miranda, Vitor Medeiros Almeida, Sandro Roberto Marana, Roberto Ruller, Mario Tyago Murakami, Flavio Henrique-Silva (2018) “A novel β-glucosidase isolated from the microbial metagenome of Lake Poraquê (Amazon, Brazil),” Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Volume 1866, Issue 4, Pages 569-579 doi: