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Cloning and Expression of the xynA1 Gene Encoding a Xylanase of the GH10 Group in Caulobacter crescentus

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Abstract

Caulobacter crescentus (NA1000 strain) are aquatic bacteria that can live in environments of low nutritional quality and present numerous genes that encode enzymes involved in plant cell wall deconstruction, including five genes for β-xylosidases (xynB1–xynB5) and three genes for xylanases (xynA1–xynA3). The overall activity of xylanases in the presence of different agro-industrial residues was evaluated, and it was found that the residues from the processing of corn were the most efficient in inducing bacterial xylanases. The xynA1 gene (CCNA_02894) encoding a predicted xylanase of group 10 of glyco-hydrolases (GH10) that was efficiently overexpressed in Escherichia coli LMG194 using 0.02 % arabinose, after cloning into the vector pJet1.2blunt and subcloning into the expression vector pBAD/gIII, provided a fusion protein that contained carboxy-terminal His-tags, named XynA1. The characterization of pure XynA1 showed an enzymatic activity of 18.26 U mL−1 and a specific activity of 2.22 U mg−1 in the presence of xylan from beechwood as a substrate. XynA1 activity was inhibited by EDTA and metal ions such as Cu2+ and Mg2+. By contrast, β-mercaptoethanol, dithiothreitol (DTT), and Ca2+ induced recombinant enzyme activity. Kinetic data for XynA1 revealed K M and V max values of 3.77 mg mL−1 and 10.20 μM min−1, respectively. Finally, the enzyme presented an optimum pH of 6 and an optimum temperature of 50 °C. In addition, 80 % of the activity of XynA1 was maintained at 50 °C for 4 h of incubation, suggesting a thermal stability for the biotechnological processes. This work is the first study concerning the cloning, overexpression, and enzymatic characterization of C. crescentus xylanase.

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Acknowledgments

L. Graciano, J.M. Corrêa, F.G.N. Vieira, and A. Bosetto were fellows of the Coordination of Improvement of Higher Education Personnel (CAPES). R.C.G. Simão was partially supported by Araucaria Foundation (process 630/2014).

Conflict of Interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Centro de Ciências Exatas e Tecnológicas, Universidade Estadual do Oeste do Paraná, Cascavel, PR, Brazil

    Luciana Graciano, Juliana Moço Corrêa, Fabíola Giovanna Nesello Vieira & Adilson Bosetto

  2. Centro de Ciências Biológicas e da Saúde, Universidade Estadual do Oeste do Paraná, Cascavel, PR, Brazil

    Eduardo Alexandre Loth

  3. Centro de Ciências Médicas e Farmacêuticas, Universidade Estadual do Oeste do Paraná, Rua Universitária, 2069, Cascavel, PR, 85814-110, Brazil

    Marina Kimiko Kadowaki, Rinaldo Ferreira Gandra & Rita de Cássia Garcia Simão

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  1. Luciana Graciano
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  2. Juliana Moço Corrêa
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  3. Fabíola Giovanna Nesello Vieira
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Correspondence to Rita de Cássia Garcia Simão.

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Graciano, L., Corrêa, J.M., Vieira, F.G.N. et al. Cloning and Expression of the xynA1 Gene Encoding a Xylanase of the GH10 Group in Caulobacter crescentus . Appl Biochem Biotechnol 175, 3915–3929 (2015). https://doi.org/10.1007/s12010-015-1560-z

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  • DOI: https://doi.org/10.1007/s12010-015-1560-z

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