Analysis of a Clostridium josui cellulase gene cluster containing the man5A gene and characterization of recombinant Man5A.

A cellulase gene cluster of Clostridium josui was sequenced, and was found to encode 11 proteins responsible for cellulosome (cellulolytic complex) formation, viz., cipA, cel48A, cel8A, cel9A, cel9B, orfX, cel9C, cel9D, man5A, cel9E, and cel5B, in order from the upstream side. All the predicted enzymes had a dockerin module, suggesting that these proteins are members of the C. josui cellulosome. Among these genes, the man5A gene encoding β-mannanase was expressed in Escherichia coli and the recombinant enzyme (rMan5A) was characterized. rMan5A showed strong activity toward carob galactomannan and low activity toward guar gum, suggesting that it prefers non-galactosylated mannan to galactomannan. This enzyme hydrolyzed ivory nut mannan to produce mainly mannotriose and larger mannooligosaccharides, and was not active toward mannotriose. An antiserum raised against the recombinant enzyme detected Man5A in the culture supernatants of C. josui, which was grown on either ball-milled cellulose or glucose as a carbon source.