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An -galactosidase gene from Pyrococcus furiosus was identified, cloned and functionally expressed in Escherichia coli. It is the first -galactosidase from a hyperthermophilic archaeon described to date. The gene encodes a unique amino acid sequence compared to other -galactosidases. Highest homology was found with -amylases classified in family 57 of glycoside hydrolases. The 364 amino acid protein had a calculated mass of 41.6 kDa. The recombinant -galactosidase specifically catalyzed the hydrolysis of para-nitrophenyl--galactopyranoside, and to some extent that of melibiose and raffinose. The enzyme proved to be an extremely thermo-active and thermostable -galactosidase with a temperature optimum of 115°C and a half-life time of 15 hours at 100°C. The pH optimum is between 5.0 and 5.5. Sequence analysis showed four conserved carboxylic residues. Site-directed mutagenesis was applied to identify the potential catalytic residues. Glu117Ala showed decreased enzyme activity, which could be rescued by the addition of azide or formate. It is concluded that glutamate 117 is the catalytic nucleophile, whereas the acid/base catalyst remains to be identified.

In recent years, many efforts have been made to isolate enzymes from extremophilic organisms in the hope to unravel the structural basis for hyperstability and to obtain hyperstable biocatalysts. Here we show how a moderately stable enzyme (a thermolysin-like protease from Bacillus stearothermophilus, TLP-ste) can be made hyperstable by a limited number of mutations. The mutational strategy included replacing residues in TLP-ste by residues found at equivalent positions in naturally occurring, more thermostable variants, as well as rationally designed mutations. Thus, an extremely stable 8-fold mutant enzyme was obtained that was able to function at 100 degrees C and in the presence of denaturing agents. This 8-fold mutant contained a relatively large number of mutations,whose stabilizing effect is generally considered to result from a reduction of the entropy of the unfolded state ("rigidifying" mutations such as Gly --> Ala, Ala --> Pro, and the introduction of a disulfide bridge). Remarkably, whereas hyperstable enzymes isolated from natural sources often have reduced activity at low temperatures, the 8-fold mutant displayed wild-type-like activity at 37 degrees C.

In recent years, many efforts have been made to isolate enzymes from extremophilic organisms in the hope to unravel the structural basis for hyperstability and to obtain hyperstable biocatalysts. Here we show how a moderately stable enzyme (a thermolysin-like protease from Bacillus stearothermophilus, TLP-ste) can be made hyperstable by a limited number of mutations. The mutational strategy included replacing residues in TLP-ste by residues found at equivalent positions in naturally occurring, more thermostable variants, as well as rationally designed mutations. Thus, an extremely stable 8-fold mutant enzyme was obtained that was able to function at 100 degrees C and in the presence of denaturing agents. This 8-fold mutant contained a relatively large number of mutations,whose stabilizing effect is generally considered to result from a reduction of the entropy of the unfolded state ("rigidifying" mutations such as Gly --> Ala, Ala --> Pro, and the introduction of a disulfide bridge). Remarkably, whereas hyperstable enzymes isolated from natural sources often have reduced activity at low temperatures, the 8-fold mutant displayed wild-type-like activity at 37 degrees C.

Desulfovibrio gigas NCIMB 9332 cells grown in ethanol-containing medium with 0.1 mu M tungstate contained a benzylviologen-linked aldehyde oxidoreductase, The enzyme was purified to electrophoretic homogeneity and found to be a homodimer with a subunit M(r) of 62,000, It contained 0.68 a 0.08 W, 4.8 Fe, and 3.2 +/- 0.2 labile S per subunit, After acid iodine oxidation of the purified enzyme, a fluorescence spectrum typical for form A of molybdopterin was obtained, Acetahdehyde, propionaldehyde, and benzaldehyde were excellent substrates, with apparent K-m values of 12.5, 10.8, and 20 mu M, respectively, The natural electron acceptor is not yet known; benzylviologen was used as an artificial electron acceptor (apparent K-m, 0.55 mM), The enzyme was activated by potassium ions and strongly inhibited by cyanide, arsenite, and iodoacetate, In the as-isolated enzyme, electron paramagnetic resonance studies readily detected W(V) as a complex signal with g values in the range of 1.84 to 1.97, The dithionite-reduced enzyme exhibited a broad signal at low temperature,vith g = 2.04 and 1.92; this is indicative of a [4Fe-4S](1+) cluster interacting with a second paramagnet, possibly the S = 1 system of W(TV), Until now W-containing aldehyde oxidoreductases had only been found in two Clostridium strains and two hyperthermophilic archaea, The D. gigas enzyme is the first example of such an enzyme in a gram-negative bacterium.

Desulfovibrio gigas NCIMB 9332 cells grown in ethanol-containing medium with 0.1 mu M tungstate contained a benzylviologen-linked aldehyde oxidoreductase, The enzyme was purified to electrophoretic homogeneity and found to be a homodimer with a subunit M(r) of 62,000, It contained 0.68 a 0.08 W, 4.8 Fe, and 3.2 +/- 0.2 labile S per subunit, After acid iodine oxidation of the purified enzyme, a fluorescence spectrum typical for form A of molybdopterin was obtained, Acetahdehyde, propionaldehyde, and benzaldehyde were excellent substrates, with apparent K-m values of 12.5, 10.8, and 20 mu M, respectively, The natural electron acceptor is not yet known; benzylviologen was used as an artificial electron acceptor (apparent K-m, 0.55 mM), The enzyme was activated by potassium ions and strongly inhibited by cyanide, arsenite, and iodoacetate, In the as-isolated enzyme, electron paramagnetic resonance studies readily detected W(V) as a complex signal with g values in the range of 1.84 to 1.97, The dithionite-reduced enzyme exhibited a broad signal at low temperature,vith g = 2.04 and 1.92; this is indicative of a [4Fe-4S](1+) cluster interacting with a second paramagnet, possibly the S = 1 system of W(TV), Until now W-containing aldehyde oxidoreductases had only been found in two Clostridium strains and two hyperthermophilic archaea, The D. gigas enzyme is the first example of such an enzyme in a gram-negative bacterium.

Substrate and product inhibition of hydrogen production during sucrose fermentation by the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus was studied. The inhibition kinetics were analyzed with a noncompetitive, nonlinear inhibition model. Hydrogen was the most severe inhibitor when allowed to accumulate in the culture. Concentrations of 5-10 mM H2 in the gas phase ( partial hydrogen pressure (pH2) of (1-2) · 104 Pa) initiated a metabolic shift to lactate formation. The extent of inhibition by hydrogen was dependent on the density of the culture. The highest tolerance for hydrogen was found at low volumetric hydrogen production rates, as occurred in cultures with low cell densities. Under those conditions the critical hydrogen concentration in the gas phase was 27.7 mM H2 ( pH2 of 5.6 · 104 Pa); above this value hydrogen production ceased completely. With an efficient removal of hydrogen sucrose fermentation was mainly inhibited by sodium acetate. The critical concentrations of sucrose and acetate, at which growth and hydrogen production was completely inhibited (at neutral pH and 70°C), were 292 and 365 mM, respectively. Inorganic salts, such as sodium chloride, mimicked the effect of sodium acetate, implying that ionic strength was responsible for inhibition. Undissociated acetate did not contribute to inhibition of cultures at neutral or slightly acidic pH. Exposure of exponentially growing cultures to concentrations of sodium acetate or sodium chloride higher than ca. 175 mM caused cell lysis, probably due to activation of autolysins. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 255-262, 2003. (Less)

In recent years, many efforts have been made to isolate enzymes from extremophilic organisms in the hope to unravel the structural basis for hyperstability and to obtain hyperstable biocatalysts. Here we show how a moderately stable enzyme (a thermolysin-like protease from Bacillus stearothermophilus, TLP-ste) can be made hyperstable by a limited number of mutations. The mutational strategy included replacing residues in TLP-ste by residues found at equivalent positions in naturally occurring, more thermostable variants, as well as rationally designed mutations. Thus, an extremely stable 8-fold mutant enzyme was obtained that was able to function at 100 degrees C and in the presence of denaturing agents. This 8-fold mutant contained a relatively large number of mutations,whose stabilizing effect is generally considered to result from a reduction of the entropy of the unfolded state ("rigidifying" mutations such as Gly --> Ala, Ala --> Pro, and the introduction of a disulfide bridge). Remarkably, whereas hyperstable enzymes isolated from natural sources often have reduced activity at low temperatures, the 8-fold mutant displayed wild-type-like activity at 37 degrees C.

Desulfovibrio gigas NCIMB 9332 cells grown in ethanol-containing medium with 0.1 mu M tungstate contained a benzylviologen-linked aldehyde oxidoreductase, The enzyme was purified to electrophoretic homogeneity and found to be a homodimer with a subunit M(r) of 62,000, It contained 0.68 a 0.08 W, 4.8 Fe, and 3.2 +/- 0.2 labile S per subunit, After acid iodine oxidation of the purified enzyme, a fluorescence spectrum typical for form A of molybdopterin was obtained, Acetahdehyde, propionaldehyde, and benzaldehyde were excellent substrates, with apparent K-m values of 12.5, 10.8, and 20 mu M, respectively, The natural electron acceptor is not yet known; benzylviologen was used as an artificial electron acceptor (apparent K-m, 0.55 mM), The enzyme was activated by potassium ions and strongly inhibited by cyanide, arsenite, and iodoacetate, In the as-isolated enzyme, electron paramagnetic resonance studies readily detected W(V) as a complex signal with g values in the range of 1.84 to 1.97, The dithionite-reduced enzyme exhibited a broad signal at low temperature,vith g = 2.04 and 1.92; this is indicative of a [4Fe-4S](1+) cluster interacting with a second paramagnet, possibly the S = 1 system of W(TV), Until now W-containing aldehyde oxidoreductases had only been found in two Clostridium strains and two hyperthermophilic archaea, The D. gigas enzyme is the first example of such an enzyme in a gram-negative bacterium.