A highly thermostable endo-(1,4)-beta-mannanase from the marine bacterium Rhodothermus marinus

Rhodothermus marinus ATCC 43812, a thermophilic bacterium isolated from mar ine hot springs, possesses hydrolytic activities for depolymerising substra tes such as carob-galactomannan. Screening of expression libraries identifi ed mannanase-positive clones. Subsequently, the corresponding DNA sequences were determined, eventually identifying a coding sequence specifying a 997 amino acid residue protein of 113 kDa. Analyses revealed an N-terminal dom ain of unknown function and a C-terminal mannanase domain of 550 amino acid residues with homology to known mannanases of glycosidase family 26. Actio n pattern analysis categorised the R. marinus mannanase as an endo-acting e nzyme with a requirement for at least five sugar moieties for effective cat alytic activity. When expressed in Escherichia coli, purified gene product with catalytic activity was mainly found as two protein fragments of 45 kDa and 50 kDa. The full-length protein of 113 kDa was only detected in crude extracts of ii. marinus, while truncated protein-containing fractions of th e original source resulted in a major active protein of 60 kDa. Biochemical analysis of the mannanase revealed a temperature and pH optimum of 85 degr ees C and pH 5.4, respectively. Purified, E. coli-produced protein fragment s showed high heat stability, retaining more than 70% and 25% of the initia l activity after 1 h incubation at 70 degrees C and 90 degrees C, respectiv ely. In contrast, R. marinus-derived protein retained 87% activity after 1 h at 90 degrees C. The enzyme hydrolysed carob-galactomannan (locust bean g um) effectively and to a smaller extent guar gum, but not yeast mannan.