Thermostable and thermoactive ␤-mannanase (1,4-␤-D-mannan mannanohydrolase [EC 3.2.1.78]), ␤-mannosidase (␤-D-mannopyranoside hydrolase [EC 3.2.1.25]), and ␣-galactosidase (␣-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 ␤-mannanase was an extracellular monomeric enzyme with a molecular mass of 65 kDa. The optimal temperature for activity was 90 to 92؇C, with half-lives (t 1/2) of 34 h at 85؇C, 13 h at 90؇C, and 35 min at 100؇C. The ␤-mannosidase and ␣-galactosidase were found primarily in cell extracts. The ␤-mannosidase was a homodimer consisting of approximately 100-kDa molecular mass subunits. The optimal temperature for activity was 87؇C, with t 1/2 of 18 h at 85؇C, 42 min at 90؇C, and 2 min at 98؇C. The ␣-galactosidase was a 61-kDa monomeric enzyme with a temperature optimum of 100 to 103؇C and t 1/2 of 9 h at 85؇C, 2 h at 90؇C, and 3 min at 100؇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.

An α‐galactosidase and a β‐mannanase produced by the hyperthermophilic bacterium, Thermotoga neapolitana 5068 (TN5068), separately and together, were evaluated for their ability to hydrolyze guar gum in relation to viscosity reduction of guar‐based hydraulic fracturing fluids used in oil and gas well stimulation. In such applications, premature guar gum hydrolysis at lower temperatures before the fracturing process is completed is undesirable, whereas thermostability and thermoactivity are advantageous. Hyperthermophilic enzymes presumably possess both characteristics. The purified α‐galactosidase was found to have a temperature optimum of 100–105°C with a half‐life of 130 minutes at 90°C and 3 min at 100°C, while the purified β‐mannanase was found to have a temperature optimum of 91°C and a half‐life of 13h at this temperature and 35 min at 100°C. These represent the most thermostable versions of these enzymes yet reported. At 25°C, TN5068 culture supernatants, containing the two enzyme activities, reduced viscosity of a 0.7% (wt) guar gum solution by a factor of 1.4 after a 1.5‐h incubation period and by a factor of 2.4 after 5 h. This is in contrast to a viscosity reduction of 100‐fold after 1.5 h and 375‐fold after 5 h for a commercial preparation of these enzymes from Aspergillus niger. In contrast, at 85°C, the TN5068 enzymes reduced viscosity by 30‐fold after 1.5 h and 100‐fold after 5 h compared to a 2.5‐fold reduction after 5 h for the control. The A. niger enzymes were less effective at 85°C (1.6‐fold reduction after 1.5 h and a 4.2‐fold reduction after 5 h), presumably due to their thermal lability at this temperature. Furthermore, it was determined that the purified β‐mannanase alone can substantially reduce viscosity of guar solutions, while the α‐galactosidase alone had limited viscosity reduction activity. However, the α‐galactosidase appeared to minimize residual particulate matter when used in conjunction with the β‐mannanase. This could be the result of extensive hydrolysis of the α‐1,6 linkages between mannose and galactose units in guar, allowing more extensive hydrolysis of the mannan chain by the β‐mannanase. The use of thermostable enzymatic breakers from hyperthermophiles in hydraulic fracturing could be used to improve well stimulation and oil and gas recovery. © 1996 John Wiley & Sons, Inc.