title: Purification and characterization of a GH43 β-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteria.
abstract: 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 2.92 mM and 1.32 seg(-1), respectively. Activity was also demonstrated on xylobiose (X2), with Km 17.8 mM and Kcat 380 s(-1). These results demonstrated that Xyl43 is a functional beta-xylosidase and it is the first evidence of this activity for Enterobacter sp.
authors.affiliation: Instituto de Biotecnología, CICVyA, Instituto Nacional de Tecnología Agropecuaria (INTA), Buenos Aires, Argentina. Electronic address: eleonoracampos@gmail.com.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Campos
authors.affiliation: Unidad Biocarburantes, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Madrid, Spain.
authors.forename: María José
authors.initials: MJ
authors.lastname: Negro Alvarez
authors.affiliation: Instituto de Biotecnología, CICVyA, Instituto Nacional de Tecnología Agropecuaria (INTA), Buenos Aires, Argentina.
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Sabarís di Lorenzo
authors.affiliation: Instituto de Biotecnología, CICVyA, Instituto Nacional de Tecnología Agropecuaria (INTA), Buenos Aires, Argentina.
authors.forename: Sergio
authors.initials: S
authors.lastname: Gonzalez
authors.affiliation: Instituto de Suelos, CIRN, Instituto Nacional de Tecnología Agropecuaria (INTA), Buenos Aires, Argentina.
authors.forename: Marcela
authors.initials: M
authors.lastname: Rorig
authors.affiliation: Instituto de Biotecnología, CICVyA, Instituto Nacional de Tecnología Agropecuaria (INTA), Buenos Aires, Argentina.
authors.forename: Paola
authors.initials: P
authors.lastname: Talia
authors.affiliation: Instituto de Suelos, CIRN, Instituto Nacional de Tecnología Agropecuaria (INTA), Buenos Aires, Argentina.
authors.forename: Daniel H
authors.initials: DH
authors.lastname: Grasso
authors.affiliation: Unidad Biocarburantes, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Madrid, Spain.
authors.forename: Felicia
authors.initials: F
authors.lastname: Sáez
authors.affiliation: Unidad Biocarburantes, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Madrid, Spain.
authors.forename: Paloma
authors.initials: P
authors.lastname: Manzanares Secades
authors.affiliation: Unidad Biocarburantes, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Madrid, Spain.
authors.forename: Mercedes
authors.initials: M
authors.lastname: Ballesteros Perdices
authors.affiliation: Instituto de Biotecnología, CICVyA, Instituto Nacional de Tecnología Agropecuaria (INTA), Buenos Aires, Argentina.
authors.forename: Angel A
authors.initials: AA
authors.lastname: Cataldi
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D004754:Enterobacter / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D007700:Kinetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D012988:Soil Microbiology; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase
keywords: Beta-xylosidase; Enterobacter; GH43; Lignocellulosic biofuels