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 multido main xylanase XynA of 'Caldibacillus cellulovorans', was determined by geno mic-walking PCR. The putative XynA comprises an N-terminal domain (D1), rec ently identified las a xylan-binding domain (XBD), homologous to noncatalyt ic thermostabilizing domains from other xylanases. D1 is followed by a xyla nase catalytic domain (D2) homologous to family 10 glycosyl hydrolases. Dow nstream of this domain two cellulose-binding domains (CBD), D3 and D4, were found linked via proline-threonine (PT)-rich peptides. Both CBDs showed se quence 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 p lus the catalytic domain (XynAd1/2) and the xylanase catalytic domain alone (XynAd2) were constructed and the biochemical properties of the recombinan t enzymes compared. The absence of the XBD resulted in a decrease in thermo stability oil: the catalytic domain from 70 degreesC (XynAd1/2) to 60 degre esC (XynAd2). Substrate-specificity experiments and analysis of the main pr oducts released from xylan hydrolysis indicate that both recombinant enzyme s act as endo-1,4-beta -xylanases, but differ in their ability to cleave sm all xylooligosaccharides.