A gene encoding a novel multidomain beta-1,4-mannanase from Caldibacillus cellulovorans and action of the recombinant enzyme on kraft pulp

Genomic walking PCR was used to obtained a 4,567-pp nucleotide sequence fro m Caldibacillus cellulovorans. Analysis of this sequence revealed that ther e were three open reading frames, designated ORF1, ORF2, and ORF3. Incomple te ORF1 encoded a putative C-terminal cellulose-binding domain (CBD) homolo gous to members of CBD family IIIb, while putative ORF3 encoded a protein o f unknown function. The putative ManA protein encoded by complete manA ORF2 was an enzyme with a novel multidomain structure and was composed of four domains in the following order: a putative N-terminal domain (D1) of unknow n function, an internal CBD (D2), a beta-mannanase catalytic domain (D3), a nd a C-terminal CBD (D4). All four domains were linked via proline-threonin e-rich peptides. Both of the CBDs exhibited sequence similarity to family I IIb CBDs, white the mannanase catalytic domain exhibited homology to the fa mily 5 glycosyl hydrolases. The purified recombinant enzyme ManAd3 expresse d from the cloned catalytic domain (D3) exhibited optimum activity at 85 de grees C and pH 6.0 and was extremely thermostable at 70 degrees C. This enz yme exhibited high specificity with the substituted galactomannan locust be an gum, while more substituted galacto- and glucomannans were poorly hydrol yzed. Preliminary studies to determine the effect of the recombinant ManAd3 and a recombinant thermostable beta-xylanase on oxygen-delignified Pinus r adiata kraft pulp revealed that there was an increase in the brightness of the bleached pulp.