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-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.
Sunna
A
A
Department of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia.
Gibbs
M D
MD
Chin
C W
CW
Nelson
P J
PJ
Bergquist
P L
PL
D000595:Amino Acid Sequence; D001420:Bacteria, Aerobic / Q000201:enzymology* / Q000235:genetics; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005690:Galactose / Q000031:analogs & derivatives; D006863:Hydrogen-Ion Concentration; D008351:Mannans / Q000378:metabolism*; D008361:Mannosidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature; D014934:Wood; D044902:beta-Mannosidase
D008351:Mannans; D011994:Recombinant Proteins; C012990:galactomannan; D008361:Mannosidases; D044902:beta-Mannosidase; D005690:Galactose