Sequencing and expression of a beta-mannanase gene from the extreme thermophile Dictyoglomus thermophilum Rt46B.1, and characteristics of the recombinant enzyme.
A beta-mannanase gene (manA) was isolated from the extremely thermophilic bacterium Dictyoglomus thermophilum Rt46B.1. ManA is a single-domain enzyme related to one group of beta-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 degrees C and 5.0. In the absence of substrate, the enzyme showed no loss of activity at 80 degrees C over 16 h, while at 90 degrees 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 beta-mannanase. Sequence comparisons with related beta-mannanases has allowed the design of consensus PCR primers for the identification and isolation of related genes.
Gibbs
M D
MD
Department of Biological Sciences, Macquarie University, North Ryde, Sydney, New South Wales 2109, Australia.
Reeves
R A
RA
Sunna
A
A
Bergquist
P L
PL
D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000201:enzymology / Q000235:genetics*; D016384:Consensus Sequence; D005798:Genes, Bacterial; D008361:Mannosidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D044902:beta-Mannosidase
D011994:Recombinant Proteins; D008361:Mannosidases; D044902:beta-Mannosidase