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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.
AuthorsEleonora Campos, 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
JournalMicrobiological research (Microbiol Res) Vol. 169 Issue 2-3 Pg. 213-20 ( 2014) ISSN: 1618-0623 [Electronic] Germany
PMID23838121 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
CopyrightCopyright © 2013 Elsevier GmbH. All rights reserved.
Chemical References
  • Bacterial Proteins
  • Xylosidases
  • exo-1,4-beta-D-xylosidase
Topics
  • Amino Acid Sequence
  • Bacterial Proteins (chemistry, genetics, isolation & purification, metabolism)
  • Cloning, Molecular
  • Enterobacter (enzymology, genetics, isolation & purification)
  • Kinetics
  • Molecular Sequence Data
  • Open Reading Frames
  • Soil Microbiology
  • Xylosidases (chemistry, genetics, isolation & purification, metabolism)

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