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

• Published in: Microbiological research 2014-02, Vol.169 (2-3), p.213-220
• Main Authors: Campos, Eleonora, Negro Alvarez, María José, Sabarís di Lorenzo, Gonzalo, Gonzalez, Sergio, Rorig, Marcela, Talia, Paola, Grasso, Daniel H., Sáez, Felicia, Manzanares Secades, Paloma, Ballesteros Perdices, Mercedes, Cataldi, Angel A.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Beta-xylosidase; Cloning, Molecular; Enterobacter; Enterobacter - enzymology; Enterobacter - genetics; Enterobacter - isolation & purification; GH43; Kinetics; Lignocellulosic biofuels; Molecular Sequence Data; Open Reading Frames; Soil Microbiology; Xylosidases - chemistry; Xylosidases - genetics; Xylosidases - isolation & purification; Xylosidases - metabolism
• ISSN: 0944-5013; 1618-0623
• DOI: 10.1016/j.micres.2013.06.004

The use of lignocellulosic biomass for second generation biofuels requires optimization of enzymatic breakdown of plant cell walls. In this work, cellulolytic bacteria were isolated from a native and two cultivated forest soil samples. Amplification of glycosyl hydrolases was attempted by using a low stringency-degenerate primer PCR strategy, 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 glycosyl hydrolases (GH) families of interest. Using this approach, a fragment containing an open reading frame (ORF) with 98% identity to a putative GH43 beta-xylosidase coding gene from Enterobacter cloacae was amplified and cloned. The full protein was expressed in Escherichia coli as N-terminal or C-terminal His-tagged fusions and purified under native conditions. Only N-terminal fusion protein, His-Xyl43, presented beta-xylosidase activity. On pNPX, optimal activity was achieved at pH 6 and 40°C and Km and Kcat values were 2.92mM and 1.32seg−1, respectively. Activity was also demonstrated on xylobiose (X2), with Km 17.8mM and Kcat 380s−1. These results demonstrated that Xyl43 is a functional beta-xylosidase and it is the first evidence of this activity for Enterobacter sp.