ABSTRACT Two genes, xynB and xynC, coding for xylanases were isolated from Thermotoga maritima FjSS3B.1 by a genomic-walking–PCR technique. Sequencing of the genes showed that they encode multidomain family 10 xylanases. Only XynB exhibited activity against xylan substrates. The temperature optimum (87°C) and pH optimum (pH 6.5) of XynB are different from the previously reported xylanase, XynA (also a family 10 enzyme), from this organism. The catalytic domain expressed without other domains has a lower temperature optimum, is less thermostable, and has optimal activity at pH 6.5. Despite having a high level of sequence similarity toxynB, xynC appears to be nonfunctional since its encoded protein did not show significant activity on xylan substrates.

Genes for xylanases from two different glycosyl hydrolase families from the bacterium Dictyoglomus thermophilum Rt46B.1 have been isolated and overexpressed in bacterial and fungal hosts. XynB has shown greater promise than XynA as a bleach-boosting agent for both pine and eucalypt kraft pulps in ECF and TCF sequences. Because of the limits of natural biodiversity, further development of XynB for increased pH range is being explored by enzyme evolution techniques which combine in vitro mutagenesis and recombination. Simulations using in silico population genetics are also being implemented to explore the available sequence space.

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 thexynB GXCF region. The putative XynB peptide (Mr, 39,800) encoded by the Rt46B.1xynB 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 differentxynB 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.

Dictyoglomus spp Rt46.B1 produces two thermophilic xylanases: a family 10 xylanase (XynA) and a family II xylanase (XynB). The Rt46.B1 XynB family 11 xylanase was able to enhance the bleachability of pine kraft oxygen pulp to ECF bleaching (DED) and eucalypt kraft oxygen pulp to both ECF bleaching (D[EO]DED) and TCF bleaching (Q[PO]), however, the Rt46.B1 XynA family 10 xylanase had little or no effect on final brightness of pine and eucalypt kraft oxygen pulps following ECF bleaching, even under very high enzyme loading (> 50XU/g pulp). XynA and XynB showed differences in their activities on the fibre-bound kraft pulp xylans, consistent with these differences in the efficacy of the XynA and XynB pretreatment on the final pulp brightness. Measured on oat spelts xylan, the apparent specific activity of XynB was at least 40-fold higher than that of XynA. At enzyme dosages of 10XU/g pulp, XynA released mostly xylose and xylobiose following extended incubation with pine kraft pulp, whereas XynB released xylobiose and xylotriose in addition to a range of higher molecular weight hydrolysis products. Additionally, XynA and XynB produced more or less identical amounts of reducing sugars from pine and eucalypt kraft oxygen pulps. However, XynB released almost twice the number of chromophoric materials from the pulps (as measured by absorbance of the hydrolysate at 280nm). Finally, the binding patterns of XynA and XynB to the kraft pulps were different in terms of the degree of pulp binding by XynA and XynB with respect to solution pH. 1 XU of XynA bound tightly to 0.1g pulp at low pH (pH6.0, 12.5mM sodium acetate buffer), but remained mostly unbound at high pH (pH10.0, 12.5mM CAPS buffer). In contrast, XynB remained mostly bound to the pulp at all pH values tested (6.0, 8.0 and 10.0). The presence of salt appeared to enhance the binding of both XynA and XynB to kraft pulp.

Xylanases can be grouped into two unrelated families, namely family F and family G. We report here the cloning of a family G xylanase gene (xynB) from the Dictyoglomus thermophilum strain Rt46B.1 and the characterisation of the expressed gene product (229B). Novel consensus-PCR and genomic-walking PCR techniques were used to isolate the xynB gene from Dictyoglomus thermophilum genomic DNA. Various 229B xylanases produced from xynB expression constructions had pH optima of 6.5, and temperature optimum ranges of between 70 and 85 degrees C. The 229B xylanase was active on kraft pulp as shown by the release of reducing sugars.

The celA, manA, and celB genes from Caldocellulosiruptor saccharolyticus compose a cellulase-hemicellulase gene cluster and are arranged on a 12-kb C. saccharolyticus genomic fragment of the recombinant lambda bacteriophage NZP lambda 2. The beginning of a fourth open reading frame (celC) which was homologous to the C. saccharolyticus manA and celA genes was located at the 3' end of the 12-kb NZP lambda 2 genomic fragment. Genome-walking PCR was used to isolate DNA fragments downstream of the C. saccharolyticus celB gene, and the entire nucleotide sequence of celC was obtained. From the preliminary nucleotide sequence, celC appeared to encode yet another multidomain bifunctional enzyme (CelC) consisting of an N-terminal endo-1,4-beta-D-glucanase domain (75% similar to CelA domain 1), two central cellulose-binding domains, and a C-terminal endo-1,4-beta-D-mannanase domain (98% similar to ManA domain 1). However, upon completion of the celC sequencing, two -1 frameshifts were identified in the region encoding the putative CelC mannanase domain. The isolated CelC mannanase domain exhibited no beta-mannanase activity, which supported this observation. Recombinant PCR was used to correct the celC frameshifts by inserting the appropriate nucleotides into the gene. The repaired celC fragment containing the base insertions (manB) expressed strong beta-mannanase activity on soluble mannan substrates and showed significant activity on kraft pulp as judged by the release of reducing sugars.