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 mannan ohydrolase [EC 3.2.1.78]), beta-mannosidase (beta-D-mannopyranoside hy drolase [EC 3.2.1.25]), and alpha-galactosidase (alpha-D-galactoside g alactohydrolase [EC 3.2.1.22]) were purified to homogeneity from cell extracts and extracellular culture supernatants of the hyperthermophil ic eubacterium Thermotoga neapolitana 5068 grown on guar gum-based med ia, The beta-mannanase was an extracellular monomeric enzyme with a mo lecular mass of 65 kDa, The optimal temperature for activity was 90 to 92 degrees C, with half-lives (t(1/2)) 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-m annosidase was a homodimer consisting of approximately 100-kDa molecul ar mass subunits. The optimal temperature for activity was 87 degrees C, with t(1/2) 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 en zyme with a temperature optimum of 100 to 103 degrees C and t(1/2) 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 version s of these types yet reported and probably act synergistically to hydr olyze extracellular galactomannans to monosaccharides by T. neapolitan a for nutritional purposes, The significance of such substrates in geo thermal environments remains to be seen.