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A novel thermostable multidomain 1,4-ß-xylanase from ‘Caldibacillus cellulovorans’ and effect of its xylan-binding domain on enzyme activity

Department of Biological Sciences, Macquarie University, North Ryde, Sydney, NSW 2109, Australia¹
Department of Molecular Medicine, University of Auckland Medical School, Private Bag 92019, Auckland, New Zealand²

Author for correspondence: Peter L. Bergquist. Tel: +61 2 9850 8614. Fax: +61 2 9850 8799. e-mail: peter.bergquist{at}mq.edu.au

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 °C (XynAd1/2) to 60 °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-ß-xylanases, but differ in their ability to cleave small xylooligosaccharides.

Keywords: thermophilic bacteria, thermostabilizing domain, xynA, hemicellulose degradation

Abbreviations: CBD, cellulose-binding domain; PT-linker, proline-threonine linker; TSD, thermostabilizing domain; XBD, xylan-binding domain; XU, xylanase unit

The GenBank accession number for the sequence reported in this paper is AF200304.

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