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.

No abstract

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.

The enzymatic hydrolysis of mannan-based hemicelluloses is technologically important for applications ranging from pulp and paper processing to food processing to gas and oil well stimulation. In many cases, thermostability and activity at elevated temperatures can be advantageous. To this end, the genes encoding beta-mannosidase (man2) and beta-mannanase (man5) from the hyperthermophilic bacteria Thermotoga neapolitana 5068 and Thermotoga maritima were isolated, cloned, and expressed in Escherichia coli. The amino acid sequences for the mannosidases from these organisms were 77% identical and corresponded to proteins with an M(r) of approximately 92 kDa. The translated nucleotide sequences for the beta-mannanase genes (man5) encoded polypeptides with an M(r) of 76 kDa that exhibited 84% amino acid sequence identity. The recombinant versions of Man2 and Man5 had similar respective biochemical and biophysical properties, which were also comparable to those determined for the native versions of these enzymes in T. neapolitana. The optimal temperature and pH for the recombinant Man2 and Man5 from both organisms were approximately 90 degrees C and 7.0, respectively. The presence of Man2 and Man5 in these two Thermotoga species indicates that galactomannan is a potential growth substrate. This was supported by the fact that beta-mannanase and beta-mannosidase activities were significantly stimulated when T. neapolitana was grown on guar or carob galactomannan. Maximum cell densities increased by at least tenfold when either guar or carob galactomannan was added to the growth medium. For T. neapolitana grown on guar at 83 degrees C, Man5 was secreted into the culture media, whereas Man2 was intracellular. These localizations were consistent with the presence and lack of signal peptides for Man5 and Man2, respectively. The identification of the galactomannan-degrading enzymes in these Thermotoga species adds to the list of biotechnologically important hemicellulases produced by members of this hyperthermophilic genera.