Abstract
In the last years, the production of ethanol fuel has started to change with the introduction of second-generation ethanol (2 G Ethanol) in the energy sector. However, in Brazil, the process of obtaining 2 G ethanol did not reach a basic standard to achieve relevant and economically viable results. Several studies have currently been addressed to solve these issues. A critical stage in the bioethanol production is the deployment of efficient and stable enzymes to catalyze the saccharification step into the process of biomass conversion. The present study comprises a screening for genes coding for plant biomass degradation enzymes, followed by cloning a selected gene, addressing its heterologous expression, and characterizing enzymatic activity towards cellulose derived substrates, with a view to second-generation ethanol production. A cDNA database of the Cotton Boll Weevil, Anthonomus grandis (Coleoptera: Curculionidae), an insect that feeds on cotton plant biomass, was used as a source of plant biomass degradation enzyme genes. A larva and adult midgut-specific β-1,4-Endoglucanase-coding gene (AgraGH45-1) was cloned and expressed in the yeast Pichia pastoris. Its amino acid sequence, including the two catalytic domains, shares high identity with other Coleoptera Glycosyl Hydrolases from family 45 (GH45). AgraGH45-1 activity was detected in a Carboxymethylcellulose (CMC) and Hydroxyethylcellulose (HEC) degradation assay and the optimal conditions for enzymatic activity was pH 5.0 at 50 °C. When compared to commercial cellulase from Aspergillus niger, Agra GH45-1 was 1.3-fold more efficient to degrade HEC substrate. Together, these results show that AgraGH45-1 is a valid candidate to be engineered and be tested for 2 G ethanol production.