Extremophilic microorganisms are adapted to survive in ecological niches such as at high temperatures, extremes of pH, high salt concentrations and high pressure. These microorganisms produce unique biocatalysts that function under extreme conditions comparable to those prevailing in various industrial processes. Some of the enzymes from extremophiles have already been purified and their genes successfully cloned in mesophilic hosts. In this review we will briefly discuss the biotechnological significance of extreme thermophilic (optimal growth 70-80 degrees C) and hyperthermophilic (optimal growth 85-100 degrees C) archaea and bacteria. In particular, we will focus on selected extracellular-polymer-degrading enzymes, such as amylases, pullulanases, cyclodextrin glycosyltransferases, cellulases, xylanases, chitinases, proteinases and other enzymes such as esterases, glucose isomerases, alcohol dehydrogenases and DNA-modifying enzymes with potential use in food, chemical and pharmaceutical industries and in environmental biotechnology.

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A total of 26 proteolytic moderate halophiles were isolated and characterized. Most isolates were members of the genus Salinivibrio (16 strains), while others were identified as Bacillus (4 strains), Salinicoccus (2 strains), or members of the gamma-Proteobacteria (4 strains). Strain CP76 was selected as the best producer of an extracellular protease, designated CP1, and was used for further studies. Sequence analysis of the 16S rRNA gene in addition to phenotypic tests led to the placement of this organism in the genus Pseudoalteromonas. Maximal protease production was detected at the end of the exponential growth phase. This CP1 protease was purified and biochemically characterized, showing optimal activity at 55 degrees C, pH 8.5, and high tolerance to a wide range of NaCl concentrations (0 to 4 M NaCl). The most interesting features of this enzyme are its moderate thermoactivity, its activity at a range of pH values (6-10), and, especially, its salt tolerance (optimal activity at 7.5% total salts). The purified protease has a molecular mass of 38 kDa, and the N-terminal amino acid sequence determined showed similarity to metalloproteases previously described. The protease activity was strongly inhibited by EDTA, PMSF, and Pefabloc. No significant inhibition was detected with E-64, bestatin, chymostatin, or leupeptin. These results suggest that Pseudoalteromonas sp. strain CP76 produces an extracellular metalloprotease moderately thermotolerant and stable at high salt concentrations.

Two thermophilic and thermostable enzymes, isolated from Sulfolohus solfataricus, S-adenosylhomocysteine hydrolase and 5'-methylthioadenosine phosphorylase, were exposed to 10.4 GHz microwave radiation in order to discriminate between thermal and non-thermal microwave effects. The exposure causes a non-thermal, irreversible and time-dependent inactivation of both enzymes; the inactivation rate is related to the energy absorbed and is independent of the enzyme concentration. The influence of salts on enzyme inactivation has also been investigated. Conformational changes of 5-adenosylhomocysteine hydrolase, detected by fluorescence and circular dichroism techniques, suggest that microwaves induce protein structural rearrangements not related to temperature.

The gene encoding a thermoactive pullulanase from the hyperthermophilic anaerobic archaeon Desulfurococcus mucosus (apuA) was cloned in Escherichia coli and sequenced. apuA from D. mucosus showed 45.4% pairwise amino acid identity with the pullulanase from Thermococcus aggregans and contained the four regions conserved among all amylolytic enzymes. apuA encodes a protein of 686 amino acids with a 28-residue signal peptide and has a predicted mass of 74 kDa after signal cleavage. The apuA gene was then expressed in Bacillus subtilis and secreted into the culture fluid. This is one of the first reports on the successful expression and purification of an archaeal amylopullulanase in a Bacillus strain. The purified recombinant enzyme (rapuDm) is composed of two subunits, each having an estimated molecular mass of 66 kDa. Optimal activity was measured at 85°C within a broad pH range from 3.5 to 8.5, with an optimum at pH 5.0. Divalent cations have no influence on the stability or activity of the enzyme. RapuDm was stable at 80°C for 4 h and exhibited a half-life of 50 min at 85°C. By high-pressure liquid chromatography analysis it was observed that rapuDm hydrolyzed ␣-1,6 glycosidic linkages of pullulan, producing maltotriose, and also ␣-1,4 glycosidic linkages in starch, amylose, amylopectin, and cyclodextrins, with maltotriose and maltose as the main products. Since the thermoactive pullulanases known so far from Archaea are not active on cyclodextrins and are in fact inhibited by these cyclic oligosaccharides, the enzyme from D. mucosus should be considered an archaeal pullulanase type II with a wider substrate specificity.Pullulanases (pullulan-6-glucanohydrolase [EC 3.2.1.41]) are classified as type I or type II (amylopullulanase) depending on their ability to degrade ␣-1,4 glycosidic linkages in starch, amylopectin, and related oligosaccharides. Unlike type II, pullulanase type I is unable to attack ␣-1,4 glycosidic linkages. Both pullulanase type I and type II attack ␣-1,6 glycosidic linkages in pullulan, producing maltotriose. All pullulanases known to date are unable to degrade cyclodextrins. In contrast, pullulan hydrolase type I (neopullulanase) and type II (isopullulanase) are able to cleave ␣-1,4 glycosidic linkages in pullulan, releasing panose and isopanose, respectively, and are highly active on cyclodextrins (32). Enzymes that degrade cyclodextrins faster than starch are designated cyclodextrinases (EC 3.2.1.54; cyclomaltodextrinase) (30).In recent years, a large number of pullulanases have been isolated, particularly from thermophilic microorganisms (32). The research on pullulanases from thermophiles is interesting not only for understanding enzyme stability but also for discovering improved enzymes for application in the industrial starch hydrolysis process. The first step in the conversion of starch to glucose is liquefaction, which runs at temperatures of 95 to 105°C in the presence of a thermostable ␣-amylase. Due to the absence of thermoactive enzymes, saccharification follows at a temperature...