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 b acterium 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 expre ssion product, ManA, purified to homogeneity. The recombinant ManA is a mon omeric enzyme with a molecular mass of 40 kDa and an optimal temperature an d 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 d egrees C the enzyme had a half-life of 5.4 min. Hydrolysis of the galactoma nnan locust bean gum (LBG) by purified ManA released mainly mannose, mannob iose, and mannotriose, confirming that ManA is an endo-acting beta-mannanas e. Sequence comparisons with related beta-mannanases has allowed the design of consensus PCR primers for the identification and isolation of related g enes.