LNBR/CNPEMAn illustration depicting the three-dimensional structure of CelOCE, the enzyme that accelerates the conversion of cellulose into glucoseLNBR/CNPEM
Where might be the best place to find specialized microorganisms equipped to dismantle sugarcane’s tough cell walls, break down cellulose—a long-chain carbohydrate that stiffens plant structures but resists fermentation—and convert it into glucose, a sugar that ferments easily?
The answer: at a sugar and ethanol refinery where, for years, the ground has been blanketed by piles of sugarcane bagasse. It was in just such an environment, at sugar mills in rural São Paulo State, that researchers from Brazil’s National Laboratory of Biorenewables (LNBR) uncovered a tiny enzyme capable of accelerating the breakdown of cellulose into glucose molecules. The LNBR is part of the Brazilian Center for Research in Energy and Materials (CNPEM), based in Campinas.
Dubbed CelOCE—short for Cellulose Oxidative Cleaving Enzyme—this 110-amino acid enzyme is produced by a previously unknown bacterium, a member of an uncharacterized bacterial phylum (UBP4) found in sugarcane biomass waste and aquatic settings. Drawing on soil samples long buried beneath piles of cane residue, researchers extracted the bacterium’s DNA and identified the gene sequence encoding the enzyme.
“We’ve shown that bacteria in nature can exploit powerful redox chemistry to break down cellulose,” says LNBR scientific director Mario Murakami, who led the research team that reported the CelOCE discovery this February in Nature. “This enzyme has significant potential to boost yields at biomass-based biorefineries, including ethanol plants.”
Most of the study’s authors, whose work was partially funded by FAPESP, are based at CNPEM. The research team also included collaborators from the University of São Paulo’s Ribeirão Preto campus, Aix-Marseille University in France, and the Technical University of Denmark (DTU).
Eduardo Cesar / Revista Pesquisa FAPESPThe newly discovered enzyme was isolated from bacteria found in sugarcane bagasse at ethanol millsEduardo Cesar / Revista Pesquisa FAPESP
To simulate industrial-scale production, the Campinas-based researchers tested the enzyme in smaller bioreactors, with capacities of 65 liters (l) and 300 l. The results were striking: incorporating CelOCE boosted the performance of the commercial enzyme cocktail currently used to produce second-generation ethanol by 21%. This type of biofuel is produced by partially breaking down the rigid cell walls of plant residues—in the case of sugarcane, its leftover bagasse and tops. Sugars locked within these structures, like cellulose, are converted into glucose, which can then ferment to produce ethanol. This process is expensive and far less efficient than the conventional method of making ethanol from sugarcane juice, where sugars are already in a readily fermentable form. “So far, engineers have been able to improve the enzyme cocktail’s efficiency by only about 10% using LPMOs—enzymes discovered more than two decades ago that aid in cellulose breakdown,” explains Murakami. “The incremental efficiency gain delivered by our enzyme is roughly double what LPMOs alone can achieve.”
Murakami’s team has successfully mastered the entire process for producing the enzyme, and a patent application has already been filed. The researchers cloned the bacterial gene that naturally produces CelOCE and, using the CRISPR-Cas9 gene-editing tool, inserted it into the fungus Trichoderma reesei. This enables the fungus to simultaneously produce CelOCE along with the standard enzymes already used in commercial cellulose-degrading enzyme blends.
“One of the most exciting discoveries came when we used the Sirius synchrotron facility to determine the enzyme’s crystal structure,” recalls Clelton Aparecido dos Santos, a molecular biologist at LNBR and the study’s lead author. “We found that it has an unusual configuration, with a compact active site and a distinctive mode of interacting with cellulose.” The largest scientific instrument in Brazil, Sirius is a state-of-the-art synchrotron light source housed at CNPEM.
“It’s an extraordinary discovery,” remarks Igor Polikarpov of the Institute of Physics of São Carlos (IFSC) at the University of São Paulo, who was not involved in the research but specializes in structural biology, molecular biophysics, and their applications in bioenergy and biotechnology. “CelOCE is certain to be valuable in the enzymatic hydrolysis of cellulose-rich biomass and holds promise for second-generation ethanol production.”
Marcos Buckeridge, a plant physiologist at the Institute of Biosciences at the University of São Paulo, who studies bioenergy systems, agrees. “They made excellent use of the most advanced research techniques available, and as a result, provided a very thorough characterization of the enzyme,” says Buckeridge, who was also not involved in the CelOCE study. LNBR is currently in talks with two companies about the potential licensing of the biocatalyst for use in second-generation ethanol production.
The story above was published with the title “From soil to biofuel” in issue in issue 349 of march/2025.
Project
Enzymatic mechanisms of the microbiome in aquatic herbivores for complex carbohydrate depolymerization and metabolism (nº 21/04891-3); Grant Mechanism Thematic Project; Principal Investigator Mario Murakami (CNPEM); Investment R$4,097,625.98.
Scientific article
SANTOS, C. A. et al. A metagenomic “dark matter” enzyme catalyses oxidative cellulose conversion. Nature. Feb. 12, 2025.
