A highly thermostable endo-(1,4)-beta-mannanase from the marine bacterium Rhodothermus marinus.
Rhodothermus marimus ATCC 43812, a thermophilic bacterium isolated from marine hot springs, possesses hydrolytic activities for depolymerising substrates such as carob-galactomannan. Screening of expression libraries identified 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 domain of unknown function and a C-terminal mannanase domain of 550 amino acid residues with homology to known mannanases of glycosidase family 26. Action pattern analysis categorised the R. marinus mannanase as an endo-acting enzyme with a requirement for at least five sugar moieties for effective catalytic 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 R. marinus, while truncated protein-containing fractions of the original source resulted in a major active protein of 60 kDa. Biochemical analysis of the mannanase revealed a temperature and pH optimum of 85 degrees C and pH 5.4, respectively. Purified, E. coil-produced protein fragments showed high heat stability, retaining more than 70% and 25% of the initial activity after 1 h incubation at 70 degrees C and 90 degrees C, respectively. In contrast, R. marinus-derived protein retained 87% activity after 1 h at 90 degrees C. The enzyme hydrolysed carob-galactomannan (locust bean gum) effectively and to a smaller extent guar gum, but not yeast mannan.
Politz
O
O
Department of Dermatology, Benjamin Franklin Medical Centre, The Free University of Berlin, Germany. politz@medizin.fu-berlin.de
Krah
M
M
Thomsen
K K
KK
Borriss
R
R
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005690:Galactose / Q000031:analogs & derivatives; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D008351:Mannans / Q000378:metabolism; D008361:Mannosidases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D012623:Seawater; D017422:Sequence Analysis, DNA; D014871:Water Microbiology; D044902:beta-Mannosidase
D008351:Mannans; D011994:Recombinant Proteins; C012990:galactomannan; D008361:Mannosidases; D044902:beta-Mannosidase; C041867:endo-1,4-beta-D-mannanase; D005690:Galactose