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

Abstract

Genomic walking PCR was used to obtained a 4,567-bp nucleotide sequence from Caldibacillus cellulovorans. Analysis of this sequence revealed that there were three open reading frames, designated ORF1, ORF2, and ORF3. Incomplete ORF1 encoded a putative C-terminal cellulose-binding domain (CBD) homologous to members of CBD family IIIb, while putative ORF3 encoded a protein of 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 unknown function, an internal CBD (D2), a beta-mannanase catalytic domain (D3), and a C-terminal CBD (D4). All four domains were linked via proline-threonine-rich peptides. Both of the CBDs exhibited sequence similarity to family IIIb CBDs, while the mannanase catalytic domain exhibited homology to the family 5 glycosyl hydrolases. The purified recombinant enzyme ManAd3 expressed from the cloned catalytic domain (D3) exhibited optimum activity at 85 degrees C and pH 6.0 and was extremely thermostable at 70 degrees C. This enzyme exhibited high specificity with the substituted galactomannan locust bean gum, while more substituted galacto- and glucomannans were poorly hydrolyzed. Preliminary studies to determine the effect of the recombinant ManAd3 and a recombinant thermostable beta-xylanase on oxygen-delignified Pinus radiata kraft pulp revealed that there was an increase in the brightness of the bleached pulp.

FIG. 1

Diagrammatic representation and partial restriction map of ORF1, ORF3, and manA and the domains encoded by the genes. The positions of PCR primers dom1F and newcbdt1R used to amplify the manA fragment from the genomic DNA of C. cellulovorans for construction of pSUN22 are shown. The positions of PCR primers mannF and mannR used to amplify the manAd3 fragment from pSUN22 for construction of expression vector pSUN23 are also shown. The asterisk indicates the stop codon. D, domain; PT, proline-threonine linker peptide.

FIG. 2

Alignment of sequences homologous to the C. cellulovorans ManAd1 sequence. Conserved residues are highlighted. The sequences used were sequences of Streptomyces olivaceoviridis Chb1 (GenBank accession no. X78535) (37), Streptomyces reticuli Chb2 (GenBank accession no. Y14315) (25), Serratia marcescens ChiA (GenBank accession no. L38384) (15), Serratia marcescens CBP21 (GenBank accession no. AB015998) (41), Streptomyces coelicolor CBP (GenBank accession no. AL031155) (35), Streptomyces halstedii CBP (GenBank accession no. U51222) (16), Caldibacillus cellulovorans ManAd1 (GenBank accession no. AF163837) (this study), and Pseudoalteromonas sp. Chi2 (GenBank accession no. AF007895) (44).