Molecular cloning, sequencing, and expression of a novel multidomain mannanase gene from Thermoanaerobacterium polysaccharolyticum

The manA gene of Thermoanaerobacterium polysaccharolyticum was cloned in Es cherichia coli. The open reading frame of manA is composed of 3,291 bases a nd codes for a preprotein of 1,097 amino acids with an estimated molecular mass of 119,627 Da, The start codon is preceded by a strong putative riboso me binding site (TAAGGCGGTG) and a putative -35 (TTCGC) and -10 (TAAAAT) pr omoter sequence, The ManA of T. polysaccharolyticum is a modular protein, S equence comparison and biochemical analyses demonstrate the presence of an N-terminal leader peptide, and three other domains in the following order: a putative mannanase-cellulase catalytic domain, cellulose binding domains 1 (CBD1) and CBD2, and a surface-layer-like protein region (SLH-1, SLH-2, a nd SLH-3). The CBD domains show no sequence homology to any cellulose bindi ng domain yet reported, hence suggesting a novel CBD, The duplicated CBDs, which lack a disulfide bridge, exhibit 69% identity, and their deletion res ulted in both failure to bind to cellulose and an apparent loss of carboxym ethyl cellulase and mannanase activities, At the C-terminal region of the g ene are three repeats of 59, 67, and 56 amino acids which are homologous to conserved sequences found in the S-layer-associated regions within the xyl anases and cellulases of thermophilic members of the Bacillus-Clostridium c luster. The ManA of T. polysaccharolyticum, besides being an extremely acti ve enzyme, is the only mannanase gene cloned which shows this domain struct ure.