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.
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Authors | Eleonora 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 |
Journal | Microbiological research
(Microbiol Res)
Vol. 169
Issue 2-3
Pg. 213-20
( 2014)
ISSN: 1618-0623 [Electronic] Germany |
PMID | 23838121
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Copyright | Copyright © 2013 Elsevier GmbH. All rights reserved. |
Chemical References |
- Bacterial Proteins
- Xylosidases
- exo-1,4-beta-D-xylosidase
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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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