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Cloning, expression and characterization of C. crescentus xynA2 gene and application of Xylanase II in the deconstruction of plant biomass

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Abstract

Biotechnology offers innovative alternatives for industrial bioprocesses mainly because it uses enzymes that biodegrade the hemicellulose releasing fermentable sugars. Caulobacter crescentus (C. crescentus) has seven genes responsible for xylanolytic cleavage, 5 to β-xylosidases (EC 3.2.1.37) and 2 for endoxylanases, like xynA2 (CCNA_03137) that encodes Xylanase II (EC 3.2.1.8) of the glycohydrolases-GH10 group. The xynA2 gene was amplified by PCR, cloned into the pTrcHisA vector e efficiently overexpressed in E. coli providing a His-tag fusion protein. Recombinant xylanase (XynA2) was purified by affinity chromatography using a nickel sepharose column and exhibited a single 43 kDa band on SDS-PAGE gel. XynA2 showed an optimum alkaline pH (8) and stability at alkaline pH for 24 h. Although C. crescentus is mesophilic, XynA2 has optimum temperature of 60 °C and is thermo-resistance at 65 °C. XynA maintains 66% of the enzymatic activity at high temperatures (90 °C) without being denatured.The enzyme displayed a xylanolitic activity free of cellulase to xylan from beechwood and it was not inhibited in the presence of 50 μmol mL−1 of xylose. In addition, dithiothreitol (DTT) induced XynA2 activity, as it improved its kinetic parameters by lowering the KM (5.78 μmol mL−1) and increasing the KCat/KM ratio (1.63 U s−1). Finally, C. crescentus XynA2 efficiently hydrolyzed corn straw with high release of reducing sugars that can be applied in different branches of the industry.

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Funding

D. Jacomini, L. Bussler and J.M. Corrêa were fellows of CAPES, the Coordination of Improvement of Higher Education Personnel.

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Authors and Affiliations

  1. Laboratório de Bioquímica Molecular, Centro de Ciências Exatas e Tecnológicas, Universidade Estadual do Oeste do Paraná, Cascavel,, Paraná, 85814-110, Brazil

    Débora Jacomini, Larissa Bussler & Rita de Cássia Garcia Simão

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

    Juliana Moço Corrêa, Marina Kimiko Kadowaki, Alexandre Maller, José Luis da-Conceição Silva & Rita de Cássia Garcia Simão

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  1. Débora Jacomini
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Contributions

The manuscript corresponds to an original work of the authors and was not previously submitted to Molecular Biology Reports or elsewhere. This work was carried out in collaboration among all authors whose names appears on the submission. Authors DJ and LB performed the experiments and wrote the first draft of the manuscript. Author JMC wrote the protocol. Authors MKK and AM designed the study and performed the statistical analysis. Authors DJ and JLCS managed the literature searches. Author RCGS managed the critical analyses of the study and wrote the final version of the manuscript. All authors read and approved the final version of the manuscript.

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Correspondence to Rita de Cássia Garcia Simão.

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Jacomini, D., Bussler, L., Corrêa, J.M. et al. Cloning, expression and characterization of C. crescentus xynA2 gene and application of Xylanase II in the deconstruction of plant biomass. Mol Biol Rep 47, 4427–4438 (2020). https://doi.org/10.1007/s11033-020-05507-2

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