Cloning of the xynB gene from Dictyoglomus thermophilum Rt46b.1 and action of the gene product on kraft pulp

Daniel D. Morris, Moreland D. Gibbs, Charles W. J. Chin, Mei-Hsien Koh, Ken K. Y. Wong, Robert W. Allison, Peter J. Nelson, Peter L. Bergquist*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

105 Citations (Scopus)

Abstract

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 (M(r), 39,800) encoded by the Rt46B.1 xynB open reading hame was a multidomain enzyme comprising an N-terminal catalytic domain (M(r), 22,000) and a possible C-terminal substrate-binding domain (M(r), 13,000) that were separated by a short serine-glycine-rich 7,3-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.

Original languageEnglish
Pages (from-to)1759-1765
Number of pages7
JournalApplied and Environmental Microbiology
Volume64
Issue number5
Publication statusPublished - May 1998

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