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Heterologous Expression of Aspergillus niger β-d-Xylosidase (XlnD): Characterization on Lignocellulosic Substrates

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Abstract

The gene encoding a glycosyl hydrolase family 3 xylan 1,4-beta-xylosidase, xlnD, was successfully cloned from Aspergillus niger strain ATCC 10864. The recombinant product was expressed in Aspergillus awamori, purified by column chromatography, and verified by matrix-assisted laser desorption ionization, tandem time of flight (MALDI-TOF/TOF) mass spectroscopy of tryptic digests. The T max was determined using differential scanning microcalorimetry (DSC) to be 78.2 °C; the K m and k cat were found to be 255 μM and 13.7 s−1, respectively, using pNP-β-d-xylopyranoside as substrate. End-product inhibition by d-xylose was also verified and shown to be competitive; the K i for this inhibition was estimated to be 3.3 mM. XlnD was shown to efficiently hydrolyze small xylo-oligomers to monomeric xylose, making it a critical hydrolytic activity in cases where xylose is to be recovered from biomass conversion processes. In addition, the presence of the XlnD was shown to synergistically enhance the ability of an endoxylanase, XynA from Thermomyces lanuginosus, to convert xylan present in selected pretreated lignocellulosic substrates. Furthermore, the addition of the XynA/XlnD complex was effective in enhancing the ability of a simplified cellulase complex to convert glucan present in the substrates.

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Acknowledgements

This work was funded by the DOE Office of the Biomass Program. We would also like to acknowledge the CAFI pretreatment group for providing some of the corn stover samples used in this study and Cornell Proteomics and Mass Spectrometry core facility for providing MS data regarding the identification of our purified enzyme.

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Authors and Affiliations

  1. National Renewable Energy Laboratory, Chemical and Biosciences Center, 1617 Cole Blvd., Golden, CO, 80401, USA

    Michael J. Selig, Eric P. Knoshaug, Stephen R. Decker, John O. Baker, Michael E. Himmel & William S. Adney

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  1. Michael J. Selig
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  2. Eric P. Knoshaug
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  3. Stephen R. Decker
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  4. John O. Baker
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  5. Michael E. Himmel
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  6. William S. Adney
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Correspondence to Michael J. Selig.

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Selig, M.J., Knoshaug, E.P., Decker, S.R. et al. Heterologous Expression of Aspergillus niger β-d-Xylosidase (XlnD): Characterization on Lignocellulosic Substrates. Appl Biochem Biotechnol 146, 57–68 (2008). https://doi.org/10.1007/s12010-007-8069-z

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  • DOI: https://doi.org/10.1007/s12010-007-8069-z

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