A novel thermostable multidomain 1,4-beta-xylanase from 'Caldibacillus cellulovorans' and effect of its xylan-binding domain on enzyme activity.

The nucleotide sequence of the complete xynA gene, encoding a novel multidomain xylanase XynA of 'Caldibacillus cellulovorans', was determined by genomic-walking PCR. The putative XynA comprises an N-terminal domain (D1), recently identified as a xylan-binding domain (XBD), homologous to non-catalytic thermostabilizing domains from other xylanases. D1 is followed by a xylanase catalytic domain (D2) homologous to family 10 glycosyl hydrolases. Downstream of this domain two cellulose-binding domains (CBD), D3 and D4, were found linked via proline-threonine (PT)-rich peptides. Both CBDs showed sequence similarity to family IIIb CBDs. Upstream of xynA an incomplete open reading frame was identified, encoding a putative C-terminal CBD homologous to family IIIb CBDs. Two expression plasmids encoding the N-terminal XBD plus the catalytic domain (XynAd1/2) and the xylanase catalytic domain alone (XynAd2) were constructed and the biochemical properties of the recombinant enzymes compared. The absence of the XBD resulted in a decrease in thermostability of the catalytic domain from 70 degrees C (XynAd1/2) to 60 degrees C (XynAd2). Substrate-specificity experiments and analysis of the main products released from xylan hydrolysis indicate that both recombinant enzymes act as endo-1, 4-beta-xylanases, but differ in their ability to cleave small xylooligosaccharides.