Biochemical confirmation and characterization of the family-57-like alpha-amylase of Methanococcus jannaschii.

The gene encoding a family-57-like alpha-amylase in the hyperthermophilic archaeon Methanococcus jannaschii, has been cloned into Escherichia coli. Extremely thermoactive alpha-amylase was confirmed in partially purified enzyme solution of the recombinant culture. This enzyme activity had a temperature optimum of 120 degrees C and a pH optimum 5.0-8.0. The amylase activity is extremely stable against denaturants. Hydrolysis of large sugar polymers with alpha-1-6 and alpha-1-4 linkages yields products including glucose polymers of 1-7 units. Highest activity is exhibited on amylose. The catalyst exhibited a half-life of 50 h at 100 degrees C, among the highest reported thermostabilities of natural amylases.

Novel, thermostable family-13-like glycoside hydrolase from Methanococcus jannaschii.

A novel glycoside hydrolase from the hyperthermophilic archaeon Methanococcus jannaschii has been cloned into Escherichia coli. Extremely thermoactive and thermostable amylolytic activity was confirmed in partially purified enzyme solution. This enzyme exhibited a temperature optimum of 100 degrees C and a pH optimum pH 5.0-8.0. Hydrolysis of large 1,6-alpha- and 1,4-alpha-linked polysaccharides yielded glucose polymers of 1-7 units. Incubation with amylose displayed the highest activity. The catalyst was activated and stabilized by Ca2+ and exhibited extreme thermostability at 100 degrees C with a half-life of 78 h.

Bioenergetic Response of the Extreme Thermoacidophile Metallosphaera sedula to Thermal and Nutritional Stresses.

The bioenergetic response of the extremely thermoacidophilic archaeon Metallosphaera sedula to thermal and nutritional stresses was examined. Continuous cultures (pH 2.0, 70(deg)C, and dilution rate of 0.05 h(sup-1)) in which the levels of Casamino Acids and ferrous iron in growth media were reduced by a step change of 25 to 50% resulted in higher levels of several proteins, including a 62-kDa protein immunologically related to the molecular chaperone designated thermophilic factor 55 in Sulfolobus shibatae (J. D. Trent, J. Osipiuk, and T. Pinkau, J. Bacteriol. 172:1478-1484, 1990), on sodium dodecyl sulfate-polyacrylamide gels. The 62-kDa protein was also noted at elevated levels in cells that had been shifted from 70 to either 80 or 85(deg)C. The proton motive force ((Delta)p), transmembrane pH ((Delta)pH), and membrane potential ((Delta)(psi)) were determined for samples obtained from continuous cultures (pH 2.0, 70(deg)C, and dilution rate of 0.05 h(sup-1)) and incubated under nutritionally and/or thermally stressed and unstressed conditions. At 70(deg)C under optimal growth conditions, M. sedula was typically found to have a (Delta)p of approximately -190 to -200 mV, the result of an intracellular pH of 5.4 (extracellular pH, 2.0) and a (Delta)(psi) of +40 to +50 mV (positive inside). After cells had been shifted to either 80 or 85(deg)C, (Delta)(psi) decreased to nearly 0 mV and internal pH approached 4.0 within 4 h of the shift; respiratory activity, as evidenced by iron speciation in parallel temperature-shifted cultures on iron pyrite, had ceased by this point. If cultures shifted from 70 to 80(deg)C were shifted back to 70(deg)C after 4 h, cells were able to regain pyrite oxidation capacity and internal pH increased to nearly normal levels after 13 h. However, (Delta)(psi) remained close to 0 mV, possibly the result of enhanced ionic exchange with media upon thermal damage to cell membranes. Further, when M. sedula was subjected to an intermediate temperature shift from 73 to 79(deg)C, an increase in pyrite dissolution (ferric iron levels doubled) over that of the unshifted control at 73(deg)C was noted. The improvement in leaching was attributed to the synergistic effect of chemical and biological factors. As such, periodic exposure to higher temperatures, followed by a suitable recovery period, may provide a basis for improving bioleaching rates of acidophilic chemolithotrophs.