Purification and characterization of a GH43 -xylosidase from Enterobacter sp. identified and cloned from forest soil bacteria

CAMPOS ELEONORA; MARÍA JOSÉ NEGRO ALVAREZ; GONZALO SABARÍS DI LORENZO; SERGIO GONZALEZ; MARCELA RORIG; PAOLA TALIA; DANIEL H. GRASSO; FELICIA SÁEZ; PALOMA MANZANARES SECADES; MERCEDES BALLESTEROS PERDICES; ANGEL A. CATALDI

MICROBIOLOGICAL RESEARCH

ELSEVIER GMBH

Año: 2014 vol. 169 p. 213 - 220

0944-5013

The use of lignocellulosic biomass for second generation biofuels requires optimization of the biochemical conversion of plant cell wall to their fermentable components. The objective of this work was the isolation of cellulolytic bacteria from forest soils as a basis for identifying novel hydrolytic encoding genes. Soil samples were obtained from a native forest area and two cultivated forests (Pinus taeda and Eucalyptus grandis), in the sub-tropical region of Misiones, Argentina. The presence of cellulolytic microorganisms was determined by most probable number assay (MPN), using filter paper as sole carbon source and, from those samples with higher filter paper degrading activity, aerobic bacteria that could degrade cellulose and/or xylan were isolated. Amplification of glycosyl hydrolases was attempted by using a low stringency PCR strategy, with a mix of degenerate and specific primers and using total soil DNA and bulk DNA pooled from positive colonies as template. A set of primers was designed based on Acidothermus cellulolyticus genome, by search of conserved domains of glycosil hydrolases (GH) families of interest. Using this approach, we have amplified a fragment containing an open reading frame (ORF) with 98% identity to a putative GH43 beta-xylosidase coding gene from Enterobacter cloacae. This gene was cloned and the full protein was expressed in E. coli as N-terminal or C-terminal His- tagged fusions and purified under native conditions. Purified recombinant proteins were tested for beta-xylosidase activity, resulting in a high activity rate of the N-terminal fusion protein, His-Xyl43.