CHARACTERIZATION OF A MULTIDOMAIN CELLULASE FROM AN EXTREMELY THERMOPHILIC ANAEROBE STRAIN NA10

The nucleotide sequence of a beta-glucanase gene from an extremely the rmophilic anaerobe NA10 was determined. The open reading frame extende d over 3000 bp and encoded a polypeptide with a molecular mass of 113 kDa, The deduced amino acid sequence of this protein exhibited high ho mology to a bifunctional cellulase CeIB of Caldocellum saccharolyticum . Based on the homology to CeIB, the NA10 beta-glucanase appears to co mprise three domains: N-terminal, central, and C-terminal domains. Amo ng these, N- and C-terminal domains apper to be catalytic domains, and the central domain to be a cellulose binding domain. These domains we re joined with each other by proline and threonine rich segments (PT b ox). The GST-fused C-terminal domain showed CMCase and MUCase activiti es, but the activities of the GST-fused N-terminal domain were very we ak. Zymogram analysis revealed that recombinant Escherichia coil conta ining the beta-glucanase gene produced a protein with a molecular mass of approximately 113 kDa, which was in good agreement with that deduc ed from the DNA sequence. However, Western blot analysis indicated tha t the amount of this full length protein was very small. Several small er abundant proteins which exhibited CMCase activity were also detecte d. Northern blot analysis indicated that there appear to be putative i nternal transcriptional initiation sites. Generation of small molecula r mass species appear to be due to alternative transcription and trans lation from the initiation sites within the gene, or partial proteolys is.