CLONING OF THE XYNB GENE FROM DICTYOGLOMUS-THERMOPHILUM RT46B.1 AND ACTION OF THE GENE-PRODUCT ON KRAFT PULP

A two-step PCR protocol was used to identify and sequence a family 11 xylanase gene from Dictyoglomus thermophilum Rt46B.1. Family II xylana se 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 famil y 11 xylanase genes. On the basis of the sequences of a representative sample of the GXCFs a single family 11 xylanase gene (xynB) was ident ified. The entire gene sequence was obtained in the second step by usi ng genomic walking PCR to amplify Rt46B.1 genomic DNA fragments upstre am and downstream of the xynB GXCF region. The putative XynB peptide ( M-r, 39,800) encoded by the Rt46B.1 xynB open reading frame was a mult idomain 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 23-amino-acid linker pe ptide. Seven xylanases which differed at their N and C termini were pr oduced from different xynB expression plasmids. All seven xylanases ex hibited optimum activity at pH 6.5. However, the temperature optima of the XynB xylanases varied from 70 to 85 degrees C. Pretreatment of Pi nus radiata and eucalypt kraft-oxygen pulps with XynB resulted in mode rate xylan solubilization and a substantial improvement in the bleacha bility of these pulps.