Resumo

The use of lignocellulosic biomass for second generation biofuels requires optimization of the biochemical conversion of cellulose to its fermentable components. The objective of this work was the identification of cellulase and hemicellulase encoding genes from bacteria isolated from native forest soils. Presence of cellulolytic microorganisms was confirmed in a soil sample obtained from a native forest area in the sub-tropical region of Iguazu, in Argentina, by most probable number assay (MPN). Cellulolyitic degrading bacteria were isolated from tubes with filter paper degradation signs by streak out in defined media with carboximethylcellulose (CMC) as sole carbon source. Colonies with cellulolytic and xylanolytic activity were identified by a degradation halo on carboxi-methylcellulose or xylan plates, visualized by Congo red staining. Amplification of glycosyl hydrolases coding genes was attempted using a degenerate primer approach. Primers were designed by search of conserved domains of GH families of interest. Total DNA pooled from positive colonies was used as template. A fragment of 1.5 kb comprising a full gene with 98% identity with a gene encoding a putative GH43 family beta-xylosidase from Enterobacter cloacae was amplified from a pool of cellulolytic bacteria DNA using the degenerate primer approach. The fragment was fully sequenced and specific primers were designed in order to amplify, clone and express the GH43 gene as a fusion protein to a 6His tag. The recombinant His-tagged full protein, named ECXyl43, was expressed and purified in its native form. It showed a high beta-xylosidase activity on pNPX substrate, when compared to a commercial hemicellulase mixture from Aspergillus niger. As a conclusion, this study showed that native forest soils are an attractive source for the bioprospection of enzymes for cellulose and hemicellulose deconstruction. ECXyl43 is currently being evaluated as additive for commercial enzymes used in lignocellulosic biomass degradation for the generation of bioethanol.