Purification and characterization of extremely thermostable beta-mannanase, beta-mannosidase, and alpha-galactosidase from the hyperthermophilic eubacterium Thermotoga neapolitana 5068.
Thermostable and thermoactive beta-mannanase (1,4-beta-D-mannan mannanohydrolase [EC 3.2.1.78]), beta-mannosidase (beta-D-mannopyranoside hydrolase [EC 3.2.1.25]) and alpha-galactosidase (alpha-D-galactoside galactohydrolase [EC 3.2.1.22]) were purified to homogeneity from cell extracts and extracellular culture supernatants of the hyperthermophilic eubacterium Thermotoga neapolitana 5068 grown on guar gum-based media. The beta-mannanase was an extracellular monomeric enzyme with a molecular mass of 65 kDa. The optimal temperature for activity was 90 to 92 degrees C, with half-lives (t1/2) of 34 h at 85 degrees C, 13 h at 90 degrees C, and 35 min at 100 degrees C. The beta-mannosidase and alpha-galactosidase were found primarily in cell extracts. The beta-mannosidase was a homodimer consisting of approximately 100-kDa molecular mass subunits. The optimal temperature for activity was 87 degrees C, with t1/2 of 18 h at 85 degrees C, 42 min at 90 degrees C, and 2 min at 98 degrees C. The alpha-galactosidase was a 61-kDa monomeric enzyme with a temperature optimum of 100 to 103 degrees C and t1/2 of 9 h at 85 degrees C, 2 h at 90 degrees C, and 3 min at 100 degrees C. These enzymes represent the most thermostable and thermoactive versions of these types yet reported and probably act synergistically to hydrolyze extracellular galactomannans to monosaccharides by T. neapolitana for nutritional purposes. The significance of such substrates in geothermal environments remains to be seen.
Duffaud
G D
GD
Department of Chemical Engineering, North Carolina State University, Raleigh 27695-7905, USA.
McCutchen
C M
CM
Leduc
P
P
Parker
K N
KN
Kelly
R M
RM
D002240:Carbohydrate Sequence; D004795:Enzyme Stability; D005690:Galactose / Q000031:analogs & derivatives; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D006207:Half-Life; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008351:Mannans / Q000096:biosynthesis / Q000737:chemistry; D008361:Mannosidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity; D000519:alpha-Galactosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D044902:beta-Mannosidase
D008351:Mannans; C012990:galactomannan; D008361:Mannosidases; D000519:alpha-Galactosidase; D044902:beta-Mannosidase; D005690:Galactose