Applied Microbiology and Biotechnology

, Volume 45, Issue 1–2, pp 86–93

Cloning, sequencing and overexpression in Escherichia coli of a xylanase gene, xynA from the thermophilic bacterium Rt8B.4 genus Caldicellulosiruptor

  • P. P. Dwivedi
  • M. D. Gibbs
  • D. J. Saul
  • P. L. Bergquist
Original Paper Applied Genetics and Regulations

DOI: 10.1007/s002530050653

Cite this article as:
Dwivedi, P.P., Gibbs, M.D., Saul, D.J. et al. Appl Microbiol Biotechnol (1996) 45: 86. doi:10.1007/s002530050653

Abstract

A genomic library of the extremely thermophilic eubacterial strain Rt8B.4 was constructed in λZapII and screened for the expression of xylanase activity. One recombinant bacteriophage showed xylanase, xylosidase and arabinosidase activity. Sequence analysis and homology comparisons showed that this plasmid derivative, pNZ2011, was composed of 6.7 kb thermophilic DNA and contained what appeared to be an operon-like structure involving genes associated with xylose metabolism. The xylanase gene, xynA was shown to code for a multi-domain protein. Xylanase activity was shown to be associated with the carboxy-terminal domain (domain 2) by deletion analysis and also by selezctive polymerase chain reaction (PCR) amplification and expression of the individual domains. Denaturing polyacrylamide gel analysis of the protein encoded by the PCR product showed three main overexpressed proteins to be present in cell extracts, presumably caused by proteolytic degradation in the Escherichia coli host. The xylanase activity from domain 2 is associated with a 36-kDa protein, which is stable at 70°C for at least 12 h at pH 7. The small size of this active enzymatic domain and its temperature stability suggest that it may be of value in the enzyme-enhanced bleaching of kraft pulp.

Copyright information

© Springer-Verlag 1996

Authors and Affiliations

  • P. P. Dwivedi
    • 1
  • M. D. Gibbs
    • 1
  • D. J. Saul
    • 1
  • P. L. Bergquist
    • 2
  1. 1.Centre for Gene Technology, Molecular Genetics and Microbiology, School of Biological SciencesUniversity of AucklandAucklandNew Zealand
  2. 2.School of Biological SciencesMacquarie UniversityNew South WalesAustralia

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