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

Cellulosomes prepared by the cellulose affinity digestion method from Clost ridium thermocellum culture supernatant hydrolyses carob galactomannan duri ng incubation at 60 degrees and pH 6.5. A recombinant phage expressing mann ase activity was isolated from a library of C. thermocellum genomic DNA con structed in lambda ZAPII. The cloned fragment of DNA containing a putative mannanse gene (manA) was sequenced, revealing an ORF of 1767 nt, encoding i n protein (mannanase A; Man26A) of 589 aa with a molecular mass of 66816 Da . The putative catalytic domain (CD) of Man26A, identified by gene sectioni ng and sequence comparisons, displayed up to 32% identity with other mannan ases belonging to family 26. Immediately downstream of the CD and separated from it by a short proline/threonine linker was a duplicated 24-residue do ckerin motif, which is conserved in all C. thermocellum cellulosomal enzyme s described thus far and mediates their attachment to the cellulosome-integ rating protein (CipA). Man26A consisting of the CD alone (Man26A') was hype rexpressed in Escherichia coil BL21(DSS) and purified. The truncated enzyme hydrolysed soluble and insoluble mannan, displaying a temperature optimum of 65 degrees C and a pH optimum of 6.5, but exhibited no activity against other plant cell wall polysaccharides. Antiserum raised against Man26A' cro ssreacted with a polypeptide with a molecular mass of 70 000 Da that is par t of the C. thermocellum cellulosome. A second variant of Man26A containing the N-terminal segment of 130 residues and the CD (Man26A'') bound to ivor y-nut mannan and weakly to soluble Carob galactomannan and insoluble cellul ose. Man26A' consisting of the CD alone did not bind to these polysaccharid es. These results indicate that the N-terminal 130 residues of mature Man26 A may constitute a weak mannan-binding domain. Sequence comparisons reveale d a lack of identity between this region of Man26A and other polysaccharide -binding domains, but significant identity with a region conserved in the t hree family 26 mannanases from the anaerobic fungus Piromyces equi.