One of the main challenges of second-generation biofuel production is identifying enzymes 

produced by microorganisms for use in a cocktail of enzymes to catalyze biomass hydrolysis 

in which the enzymes act together to break down the carbohydrates in sugarcane trash and 

bagasse, for example, and convert them into simple sugars for fermentation.

A group of researchers at the University of Campinas (UNICAMP), working in partnership with 

colleagues at the Brazilian Biorenewables National Laboratory (LNBR) in Campinas, S?o 

Paulo State, Brazil, have discovered that Trichoderma harzianum, a fungus found in the 

Amazon, produces an enzyme with the potential to play a key role in enzyme cocktails.

The enzyme, which is called β-glucosidase and belongs to glycoside hydrolase family 1 (GH1), 

acts in the last stage of biomass degradation to produce free glucose for fermentation and 

conversion into ethanol. In the laboratory, however, the researchers observed that high levels 

of glucose inhibited the activity of β-glucosidase.

"We also found that the enzyme's optimal catalytic activity occurred at 40 °C. This represented 

another obstacle to use of the enzyme because in an industrial setting, the enzymatic 

hydrolysis of biomass is performed at higher temperatures, typically around 50 °C," said 

Clelton Aparecido dos Santos, a postdoctoral researcher at UNICAMP's Center for Molecular 

Biology and Genetic Engineering (CBMEG) with a scholarship from FAPESP.

Based on an analysis of the enzyme's structure combined with genomics and molecular 

biology techniques, the researchers were able to modify the structure to solve these problems 

and considerably enhance its biomass degradation efficiency.

The study resulted from a project with a regular research grant from FAPESP and a Thematic 

Project also supported by FAPESP. The findings are published in the journal Scientific Reports.

"The modified protein we developed proved far more efficient than the unmodified enzyme and 

can be used to supplement the enzyme cocktails sold today to break down biomass and 

produce second-generation biofuels," Santos told.

To arrive at the modified protein, the researchers initially compared the crystal structure of the 

original molecule with structures of other wild-type β-glucosidases in the GH1 and GH3 

glycoside hydrolase families. The results of the analysis showed that glucose-tolerant GH1 

glucosidases had a deeper and narrower substrate channel than other β-glucosidases and that 

this channel restricted glucose access to the enzyme's active site.