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

Anwar Sunna, Moreland D. Gibbs, Charles W J Chin, Peter J. Nelson, Peter L. Bergquist

Research output: Contribution to journalArticleResearchpeer-review

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 β-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°C and pH 6.0 and was extremely thermostable at 70°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 β-xylanase on oxygen-delignified Pinus radiata kraft pulp revealed that there was an increase in the brightness of the bleached pulp.

LanguageEnglish
Pages664-670
Number of pages7
JournalApplied and Environmental Microbiology
Volume66
Issue number2
DOIs
Publication statusPublished - Feb 2000

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Caldibacillus cellulovorans
beta-mannosidase
kraft pulp
Cellulose
cellulose
active sites
enzyme
Catalytic Domain
gene
Enzymes
enzymes
Genes
glucomannans
genes
beta-Mannosidase
Endo-1,4-beta Xylanases
genome walking
locust bean gum
Pinus
protein

Cite this

Sunna, Anwar ; Gibbs, Moreland D. ; Chin, Charles W J ; Nelson, Peter J. ; Bergquist, Peter L. / A gene encoding a novel multidomain β-1,4-mannanase from Caldibacillus cellulovorans and action of the recombinant enzyme on kraft pulp. In: Applied and Environmental Microbiology. 2000 ; Vol. 66, No. 2. pp. 664-670.
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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 β-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°C and pH 6.0 and was extremely thermostable at 70°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 β-xylanase on oxygen-delignified Pinus radiata kraft pulp revealed that there was an increase in the brightness of the bleached pulp.",
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A gene encoding a novel multidomain β-1,4-mannanase from Caldibacillus cellulovorans and action of the recombinant enzyme on kraft pulp. / Sunna, Anwar; Gibbs, Moreland D.; Chin, Charles W J; Nelson, Peter J.; Bergquist, Peter L.

In: Applied and Environmental Microbiology, Vol. 66, No. 2, 02.2000, p. 664-670.

Research output: Contribution to journalArticleResearchpeer-review

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