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A family 26 mannanase produced by Clostridium thermocellum as a component of the cellulosome contains a domain which is conserved in mannanases from anaerobic fungi

Laboratory of Molecular Enzymology, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK¹
Department of Animal Science, University of Western Australia, Nedlands, WA 6009, Australia²
Department of Biological and Nutritional Sciences, The University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, UK³

Author for correspondence: Harry J. Gilbert. Tel: +44 191 2226609. Fax: +44 191 2228684. e-mail: h.j.gilbert{at}newcastle.ac.uk

Cellulosomes prepared by the cellulose affinity digestion method from Clostridium thermocellum culture supernatant hydrolysed carob galactomannan during incubation at 60 °C and pH 6·5. A recombinant phage expressing mannanase activity was isolated from a library of C. thermocellum genomic DNA constructed in ZAPII. The cloned fragment of DNA containing a putative mannanase gene (manA) was sequenced, revealing an ORF of 1767 nt, encoding a protein (mannanase A; Man26A) of 589 aa with a molecular mass of 66816 Da. The putative catalytic domain (CD) of Man26A, identified by gene sectioning and sequence comparisons, displayed up to 32% identity with other mannanases belonging to family 26. Immediately downstream of the CD and separated from it by a short proline/threonine linker was a duplicated 24-residue dockerin motif, which is conserved in all C. thermocellum cellulosomal enzymes described thus far and mediates their attachment to the cellulosome-integrating protein (CipA). Man26A consisting of the CD alone (Man26A') was hyperexpressed in Escherichia coli BL21(DE3) and purified. The truncated enzyme hydrolysed soluble and insoluble mannan, displaying a temperature optimum of 65 °C and a pH optimum of 6·5, but exhibited no activity against other plant cell wall polysaccharides. Antiserum raised against Man26A' cross-reacted with a polypeptide with a molecular mass of 70000 Da that is part of the C. thermocellum cellulosome. A second variant of Man26A containing the N-terminal segment of 130 residues and the CD (Man26A'') bound to ivory-nut mannan and weakly to soluble Carob galactomannan and insoluble cellulose. Man26A' consisting of the CD alone did not bind to these polysaccharides. These results indicate that the N-terminal 130 residues of mature Man26A may constitute a weak mannan-binding domain. Sequence comparisons revealed a lack of identity between this region of Man26A and other polysaccharide-binding domains, but significant identity with a region conserved in the three family 26 mannanases from the anaerobic fungus Piromyces equi.

Keywords: mannanase, Clostridium thermocellum, cellulosome, family 26

Abbreviations: CBD, cellulose-binding domain; CD, catalytic domain

The GenBank/EMBL accession number for the sequence reported in this paper is AJ242666.

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