Sequencing and expression of a β-mannanase gene from the extreme thermophile Dictyoglomus thermophilum Rt46B.1, and characteristics of the recombinant enzyme

Moreland D. Gibbs, Rosalind A. Reeves, Anwar Sunna, Peter L. Bergquist

Research output: Contribution to journalArticleResearchpeer-review

Abstract

A β-mannanase gene (manA) was isolated from the extremely thermophilic bacterium Dictyoglomus thermophilum Rt46B.I. ManA is a single-domain enzyme related to one group of β-mannanases (glycosyl hydrolase family 26). The manA gene was expressed in the heat-inducible vector pJLA602 and the expression product, ManA, purified to homogeneity. The recombinant ManA is a monomeric enzyme with a molecular mass of 40 kDa and an optimal temperature and pH for activity of 80°C and 5.0. In the absence of substrate, the enzyme showed no loss of activity at 80°C over 16 h, while at 90°C the enzyme had a half-life of 5.4 min. Hydrolysis of the galactomannan locust bean gum (LBG) by purified ManA released mainly mannose, mannobiose, and mannotriose, confirming that ManA is an endo-acting β-mannanase. Sequence comparisons with related β-mannanases has allowed the design of consensus PCR primers for the identification and isolation of related genes.

LanguageEnglish
Pages351-357
Number of pages7
JournalCurrent Microbiology
Volume39
Issue number6
DOIs
Publication statusPublished - Dec 1999

Fingerprint

beta-Mannosidase
Enzymes
Genes
Hydrolases
Mannose
Half-Life
Hydrolysis
Hot Temperature
Bacteria
Polymerase Chain Reaction
Temperature

Keywords

  • mannose
  • hydrolase
  • recombinant enzyme
  • galactomannan
  • glycosyl hydrolase

Cite this

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title = "Sequencing and expression of a β-mannanase gene from the extreme thermophile Dictyoglomus thermophilum Rt46B.1, and characteristics of the recombinant enzyme",
abstract = "A β-mannanase gene (manA) was isolated from the extremely thermophilic bacterium Dictyoglomus thermophilum Rt46B.I. ManA is a single-domain enzyme related to one group of β-mannanases (glycosyl hydrolase family 26). The manA gene was expressed in the heat-inducible vector pJLA602 and the expression product, ManA, purified to homogeneity. The recombinant ManA is a monomeric enzyme with a molecular mass of 40 kDa and an optimal temperature and pH for activity of 80°C and 5.0. In the absence of substrate, the enzyme showed no loss of activity at 80°C over 16 h, while at 90°C the enzyme had a half-life of 5.4 min. Hydrolysis of the galactomannan locust bean gum (LBG) by purified ManA released mainly mannose, mannobiose, and mannotriose, confirming that ManA is an endo-acting β-mannanase. Sequence comparisons with related β-mannanases has allowed the design of consensus PCR primers for the identification and isolation of related genes.",
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Sequencing and expression of a β-mannanase gene from the extreme thermophile Dictyoglomus thermophilum Rt46B.1, and characteristics of the recombinant enzyme. / Gibbs, Moreland D.; Reeves, Rosalind A.; Sunna, Anwar; Bergquist, Peter L.

In: Current Microbiology, Vol. 39, No. 6, 12.1999, p. 351-357.

Research output: Contribution to journalArticleResearchpeer-review

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