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Appl Environ Microbiol, May 1998, p. 1759-1765, Vol. 64, No. 5
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Cloning of the xynB Gene from Dictyoglomus thermophilum Rt46B.1 and Action of the Gene Product on Kraft Pulp

Daniel D. Morris,1,2 Moreland D. Gibbs,2 Charles W. J. Chin,3 Mei-Hsien Koh,3 Ken K. Y. Wong,4 Robert W. Allison,4 Peter J. Nelson,3 and Peter L. Bergquist1,2,*

Centre for Gene Technology, School of Biological Sciences, University of Auckland, Auckland,1 and PAPRO New Zealand, FRI Ltd., Rotorua,4 New Zealand; and School of Biological Sciences, Macquarie University, Sydney, New South Wales 2109,2 and CSIRO Forestry and Forest Products, Ian Wark Laboratories, Clayton, Victoria 3168,3 Australia

Received 30 December 1997/Accepted 23 January 1998

A two-step PCR protocol was used to identify and sequence a family 11 xylanase gene from Dictyoglomus thermophilum Rt46B.1. Family 11 xylanase consensus fragments (GXCFs) were amplified from Rt46B.1 genomic DNA by using different sets of consensus PCR primers that exhibited broad specificity for conserved motifs within fungal and/or bacterial family 11 xylanase genes. On the basis of the sequences of a representative sample of the GXCFs a single family 11 xylanase gene (xynB) was identified. The entire gene sequence was obtained in the second step by using genomic walking PCR to amplify Rt46B.1 genomic DNA fragments upstream and downstream of the xynB GXCF region. The putative XynB peptide (Mr, 39,800) encoded by the Rt46B.1 xynB open reading frame was a multidomain enzyme comprising an N-terminal catalytic domain (Mr, 22,000) and a possible C-terminal substrate-binding domain (Mr, 13,000) that were separated by a short serine-glycine-rich 23-amino-acid linker peptide. Seven xylanases which differed at their N and C termini were produced from different xynB expression plasmids. All seven xylanases exhibited optimum activity at pH 6.5. However, the temperature optima of the XynB xylanases varied from 70 to 85°C. Pretreatment of Pinus radiata and eucalypt kraft-oxygen pulps with XynB resulted in moderate xylan solubilization and a substantial improvement in the bleachability of these pulps.

* Corresponding author. Mailing address: Research Office, Macquarie University, Sydney, New South Wales 2109, Australia. Phone: 61 2 9850 8614. Fax: 61 2 9850 8799. E-mail: peter.bergquist{at}mq.edu.au.

Appl Environ Microbiol, May 1998, p. 1759-1765, Vol. 64, No. 5
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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