Abstract
The xynB1 gene (CCNA 01040) of Caulobacter crescentus that encodes a bifunctional enzyme containing the conserved β-Xylosidase and α-l-Arabinofuranosidase (β-Xyl I-α-l-Ara) domains was amplified by PCR and cloned into the vector pJet1.2Blunt. The xynB1 gene was subcloned into the vector pPROEX-hta that produces a histidine-fused translation product. The overexpression of recombinant β-Xyl I-α-l-Ara was induced with IPTG in BL21 (DE3) and the resulting intracellular protein was purified with pre-packaged nickel-Sepharose columns. The recombinant β-Xyl I-α-l-Ara exhibited a specific β-Xylosidase I activity of 1.25 U mg−1 to oNPX and a specific α-l-Arabinofuranosidase activity of 0.47 U mg−1 to pNPA. The predominant activity of the recombinant enzyme was its β-Xylosidase I activity, and the enzymatic characterization was focused on it. The β-Xylosidase I activity was high over the pH range 3–10, with maximal activity at pH 6. The enzyme activity was optimal at 45 °C, and a high degree of stability was verified over 240 min at this temperature. Moreover, β-Xylosidase activity was inhibited in the presence of the metals Zn2+ and Cu2+, and the enzyme exhibited KM and VMax values of 2.89 ± 0.13 mM and 1.4 ± 0.04 μM min−1 to oNPX, respectively. The modeled structure of β-xylosidase I showed that its active site is highly conserved compared with other structures of the GH43 family. The increase in the number of contact residues responsible for maintaining the dimeric structure indicates that this dimer is more stable than the tetramer form.
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Acknowledgments
L. Graciano and J. M. Corrêa are fellows of Fundação Araucária and Fundação Parque Tecnológico Itaipu (PTI C&T, FTPI-BR), respectively. This work was supported by “Fundação Araucária, Fundo Paraná/Secretaria da Ciência Tecnologia e Ensino Superior (SETI)” and “Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).”
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Graciano, L., Corrêa, J.M., Gandra, R.F. et al. The cloning, expression, purification, characterization and modeled structure of Caulobacter crescentus β-Xylosidase I. World J Microbiol Biotechnol 28, 2879–2888 (2012). https://doi.org/10.1007/s11274-012-1099-x
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DOI: https://doi.org/10.1007/s11274-012-1099-x