Search

Deception Island, an active stratovolcano located in the South Shetland Islands, Antarctica, provides excellent conditions for the thermophilic bacteria growth because of high ground temperatures in specific areas, such as Fumarole Bay where the temperatures are above the mesophilic range. Denaturing Gradient Gel Electrophoresis (DGGE) was used with the 16S ribosomal gene to analyse cultures of thermophilic bacteria from a soil sample taken from Fumarole Bay. Nine bands were sequenced and analysed from DGGE and they indicated the presence of bacteria from the generaGeobacillus,Bacillus,Brevibacillus,Thermusand uncultured sulphate reducing bacteria. Some of which have been reported in other Antarctic geothermal sites.Geobacillus,BacillusandBrevibacillusgenera were successfully cultivated in an enriched medium. A pure culture of one thermophilicGeobacillusbacterium was obtained closely related toGeobacillus jurassicus.

The rifting zone of Bransfield Strait, Antarctica, is tectonically and geologically unique. It is a back-arc basin that was opened by extensional forces associated to roll-back subduction after cessation of spreading activity of the Phoenix Ridge, and the transtension of the westward ending of Scotia-Antarctica Plate boundary. The Bransfield Rift/ Ridge is still active generating volcanism or magma rise to force hydrothermal activity. During the ANT-XXV/4 cruise onboard R/V "Polarstern", standard CTD and beam transmission measurements were done to determine temperature anomaly and turbidity. Water sampling was performed to determine δ 3 He and to find thermophilic microorganisms to examine the Orca seamount hydrothermal activity. A temperature anomaly of ~0.08 °C, a pick of turbidity, and high value of δ 3 He (>10%) were found inside Orca seamount. Results are consistent with a hydrothermal flux coming from the seamount. The report of the first observation of thermophilic and hyperthermophilic microorganisms in cold deep Antarctic waters is part of this study. Inside Orca seamount these microorganisms were found at three different depth levels close to the bottom. We suggest that the fluid migration from the volcano resulted from recent magmatic activity and provided the required elemental nutrients for microbial growth. Besides some thermophiles were found outside the seamount in a small quantity close to the seafloor. These would probably be related to subsidiary structures of the Orca seamount, or were transported by currents from other active volcanic sites as Deception Island. The finding of these thermophilic and hyperthermophilic microorganisms raise questions about the dispersal and their resistance in these extreme environments.; ¿Existe un flujo hidrotermal activo desde el monte submarino Orca en el estrecho Bransfield, Antártica?. La zona de rifting del estrecho Bransfield, Antártica, es tectónica y geológicamente única. Es una cuenca de trasarco originada por fuerzas de extensión asociadas a roll-back después del cese de la subducción y expansión de la dorsal Fénix, y la transtensión relacionada con la actividad del límite oeste de la Placa Scotia-Antártica. El rift/ dorsal del estrecho de Bransfield, actualmente activo, provoca volcanismo o ascenso de magma al que se asocia actividad hidrotermal. Durante el crucero ANT-XXV/4 del R/V "Polarstern", se realizaron mediciones estándar de CTD y transmisión de luz (haz) para determinar anomalías de temperatura y turbidez, y también muestreo de agua para establecer su valor de δ 3 He y presencia de microorganismos termofílicos y así evaluar la potencial actividad hidrotermal en el monte submarino Orca. Una anomalía de temperatura de ~0.08 °C, un pico de turbidez y un alto valor de δ 3 He (>10%) se encontraron dentro del cráter de Orca. Los resultados son consistentes con un flujo hidrotermal proveniente del monte submarino. También se encontraron, por primera vez, microorganismos termófilos e hipertermófilos en aguas centers and intra-plate volcanoes (e.g., German et al., 2000). Every mentioned site has its own associated tectonics, geodynamics and geological characteristics that produce distinctive geophysical and geochemical signatures, which are reflected in the variability in composition, size or volume, spatial distribution, frequency and transience of the deep-sea hydrothermal vents (Lupton et al., 1998; Baker et al., 2001; Hey et al., 2004; Hannington et al., 2005). The rifting zone of the Bransfield Strait, located between the South Shetland Islands and the Antarctic Peninsula (Fig. 1) is tectonically and geologically unique (Lawyer et al., 1996). Subduction and extension processes occur simultaneously and there is currently no clarity in their geological evolution frías profundas de la Antártica. Dentro del cráter del monte submarino Orca, estos microorganismos se localizaron en tres niveles de profundidad diferentes cerca del fondo. Nosotros sugerimos que la circulación de fluidos hidrotermales, producto de la actividad magmática reciente, proporcionaron los nutrientes y el ambiente propicio para el crecimiento de estos microorganismos. Además, se encontraron fuera del volcán, cerca del lecho marino, microorganismos termófilos en una pequeña cantidad. Estos estarían relacionados, probablemente, con estructuras subsidiarias del monte submarino Orca o fueron transportadas por corrientes desde otros centros volcánicos activos como la isla Decepción. El descubrimiento de estos microorganismos termófilos e hipertermófilos genera nuevas preguntas sobre su dispersión y resistencia en estos ambientes extremos.

The Nanoarchaeota, proposed as the fourth sub-division of the Archaea in 2002, are known from a single isolate, Nanoarchaeum equitans, which exists in a symbiotic association with the hyperthermophilic Crenarchaeote, Ignicoccus. N. equitans fails to amplify with standard archaeal 16S PCR primers and can only be amplified using specifically designed primers. We have designed a new set of universal archaeal primers that amplify the 16S rRNA gene of all four archaeal sub-divisions, and present two new sets of Nanoarchaeota-specific primers based on all known nanoarchaeal 16S rRNA gene sequences. These primers can be used to detect N. equitans and have generated nanoarchaeal amplicons from community DNA extracted from Chinese, New Zealand, Chilean and Tibetan hydrothermal sites. Sequence analysis indicates that these environments harbour novel nanoarchaeal phylotypes, which, however, do not cluster into clear phylogeographical clades. Mesophilic hypersaline environments from Inner Mongolia and South Africa were analysed using the nanoarchaeal-specific primers and found to contain a number of nanoarchaeal phylotypes. These results suggest that nanoarchaeotes are not strictly hyperthermophilic organisms, are not restricted to hyperthermophilic hosts and may be found in a large range of environmental conditions.

Enzymes produced by extremophiles offer an attractive solution to industrial biocatalysis since unlike most industrial enzymes today, extremozymes are more stable and robust, therefore making them ideal candidates for industrial applications. For industry, confirming the functionality of a biocatalyst under the conditions of a particular process makes the difference between obtaining or not the required product. Although the molecular approaches allow access to genes of interest in a fast way, to obtain large amounts of functional proteins, it is not possible by the sole use of genomics. Additionally, these approaches cannot discern properties such as: activity, pH and temperature stability, and inhibitors, only possible to measure through a functional approach. New technologies will need to integrate the functional approach into the large-scale biotechnology by implementing the technological capacity to analyse some intrinsic features to search for various enzymatic activities at the same time. This chapter will analyse the most recent and relevant advances for the discovery and development of extremophilic enzymes with a special emphasis in the implementation of functional screening strategies as well as rapid and efficient discovery tools. Furthermore, a group of selected biotransformations will be analysed in the context of the applicability of extremozymes in an industrial context.

The development of enzymes for industrial applications relies heavily on the use of microorganisms. The intrinsic properties of microbial enzymes, e.g., consistency, reproducibility, and high yields along with many others, have pushed their introduction into a wide range of products and industrial processes. Extremophilic microorganisms represent an underutilized and innovative source of novel enzymes. These microorganisms have developed unique mechanisms and molecular means to cope with extreme temperatures, acidic and basic pH, high salinity, high radiation, low water activity, and high metal concentrations among other environmental conditions. Extremophile-derived enzymes, or extremozymes, are able to catalyze chemical reactions under harsh conditions, like those found in industrial processes, which were previously not thought to be conducive for enzymatic activity. Due to their optimal activity and stability under extreme conditions, extremozymes offer new catalytic alternatives for current industrial applications. These extremozymes also represent the cornerstone for the development of environmentally friendly, efficient, and sustainable industrial technologies. Many advances in industrial biocatalysis have been achieved in recent years; however, the potential of biocatalysis through the use of extremozymes is far from being fully realized. In this article, the adaptations and significance of psychrophilic, thermophilic, and hyperthermophilic enzymes, and their applications in selected industrial markets will be reviewed. Also, the current challenges in the development and mass production of extremozymes as well as future prospects and trends for their biotechnological application will be discussed.

The biosynthesis of nanoparticles by microorganisms, on the contrary to chemical synthesis, is an environmentally-friendly process which has low energy requirements. In this investigation, we used the microorganism Geobacillus wiegelii, strain GWE1, an aerobic thermophile belonging to genus Geobacillus, isolated from a drying oven. This microorganism has the ability to reduce selenite evidenced by the change of color from colorless to red in the culture. Elemental analysis and composition of the particles were verified using transmission electron microscopy and energy-dispersive X-ray analysis. The nanoparticles have a defined spherical shape and a selenium elemental state. Previous experiments showed that the presence of the whole microorganism for the reduction of selenite was not necessary. The results strongly suggested that an intracellular NADPH/NADH-dependent reductase mediates selenium nanoparticles synthesis under aerobic conditions. The enzyme was purified and identified by mass spectroscopy MALDI-TOF TOF technique. The enzyme is a 1-pyrroline-5-carboxylate dehydrogenase. Histograms of nanoparticles sizes were obtained. Size distribution ranged from 40-160 nm, where 70% of nanoparticles have less than 100 nm in size. Spectroscopic analysis showed that the nanoparticles are composed of elemental selenium. To analyse the effect of pH in size and morphology of nanoparticles, the synthesis of them was carried out at different pHs (4.0, 5.0, 6.0, 7.0, 8.0). For thermostability studies samples were incubated at different temperatures (60, 80 and 100 ºC) for 1 h and 3 h. The size of all nanoparticles was less than 100 nm at pH 4.0; over 50% of nanoparticles have less than 100 nm at pH 5.0; at pH 6.0 and 8.0 over 90% of nanoparticles have less than 100 nm in size. At neutral pH (7.0) nanoparticles reach a size around 120 nm and only 20% of them were less than 100 nm. When looking at temperature effect, nanoparticles did not show a significant difference in size when they were incubated between 0 and 3 h at 60 ºC. Meanwhile at 80 °C the nanoparticles suspension lost its homogeneity. A change in size was observed from 0 h of incubation at 80ºC, observing a size range between 40-160 nm, with 20% of them over 100 nm. Meanwhile after 3 h of incubation at size range changed to 60-180 nm with 50% of them over 100 nm. At 100 °C the nanoparticles aggregate forming nanorod structures. In conclusion, these results indicate that is possible to modulate size and shape of biologically synthesized nanoparticles by modulating pH and temperature., {"references":["C. Ip, \"Selenium and ER stress response: Implication and exploitation\nfor cancer therapy\". Proceedings of the International Conference on\nSelenium in Biology and Medicine, July 2006, pp. 25-30, University\nWisconsin, Madison, pp: 63-63.","X. Gao, J. Zhang, L. Zhang, \"Hollow sphere selenium nanoparticles.\nTheir in vitro anti-hydroxyl radical effect\". Adv. Mater, vol. 14, no. 4,\npp. 290- 293, Feb. 2002.","H. Wang, J. Zhang, H. Yu, \"Elemental selenium at nano size possesses\nlower toxicity without compromising the fundamental effect on\nselenoenzymes: comparison with selenomethionine in mice\", Free\nRadic. Biol. Med., vol. 42, no. 10, pp. 1524-1533, May 2007.","D. Peng, J. Zhang, Q. Liu, E. Taylor, \"Size effect of elemental selenium\nnanoparticles (Nano-Se) at supranutritional levels on selenium\naccumulation and glutathione S-transferase activity\", J. Inorg. Biochem.,\nvol. 101, no. 10, pp. 1457-1463, Oct. 2007.","R. Oremland, M. Herbel, J. Blum, S. Langley, T. Beveridge, P. Jayan,\nT. Sutto, A. Ellis, S. Curran, \"Structural and spectral features of\nselenium nanospheres produced by Se-respiring bacteria\", Appl.\nEnviron. Microbiol., vol., 70, no. 1, pp. 52-60, Jan. 2004.","P. Tejo, N. Sharma, R. Prakash, K. Raina, J. Fellowes, C. Pearce, J.\nLloyd, R. Pattrick, \"Aerobic microbial manufacture of nanoscale\nselenium: exploiting nature's bio-nanomineralization potential\",\nBiotechnol. Lett., vol. 31, no. 12, pp. 1857-1862, Aug. 2009.","V. Yadav, N. Sharma, R. Prakash, K. Raina, L. Bharadwaj, P. Tejo,\n\"Generation of Selenium containing Nano-structures by soil bacterium,\nPseudomonas aeruginosa\", Biotechnol., vol. 7, no. 2, pp. 299-304,\n2008.","A. Abdelouas, W. Gong, W. Lutze, J. Shelnutt, R. Franco, I. Moura,\n\"Using cytochrome c3 to make selenium nanowires\" Chem. Mater., vol.\n12, no. 6, pp. 1510-1512, May 2000.","Y. Gorby, T. Beveridge, R. Blakemore, \"Characterization of the\nbacterial magnetosome membrane\". J. Bacteriol., vol. 170, no. 2, pp.\n834-841, Feb. 1988.\n[10] W. Leinfelder, K. Forchhammer, F. Zinoni, G. Sawers, M. Mandrand-\nBerthelot, A. Böck, \"Escherichia coli genes whose products are\ninvolved in selenium metabolism\". J. Bacteriol., vol. 170, no. 2, pp.\n540-546, Feb. 1988.\n[11] M. Tanaka, Y. Okamura, A. Arakaki, T. Tanaka, H. Takeyama, T.\nMatsunaga, \"Origin of magnetosome membrane: proteomic analysis of\nmagnetosome membrane and comparison with cytoplasmic\nmembrane\", Proteomics, vol. 6, no. 19, pp. 5234-5247, Oct. 2006.\n[12] A. Arakaki, J. Webb, T. Matsunaga, \"A novel protein tightly bound to\nbacterial magnetic particles in magnetospirillum magneticum strain\nAMB-1\" J. Biol. Chem. Vol. 278, no. 10, pp. 8745-8750, Mar. 2003.\n[13] G. Kaur, M. Iqbal, M. Bakshi, \"Biomineralization of fine selenium\ncrystalline rods and amorphous spheres\". J. Phys. Chem. C vol.\n113, no. 31, pp. 13670-13676, July 2009.\n[14] J. Dobias, E. Suvorova, R. Bernier-Latmani, \"Role of proteins in\ncontrolling selenium nanoparticle size\", Nanotechnology, vol. 22, no.\n19, pp. 1-9, May 2011.\n[15] S. He, Z. Guo, Y. Zhang, S. Zhang, J. Wang, N. Gu, \"Biosynthesis of\ngold nanoparticles using the bacteria Rhodopseudomonas\ncapsulata\". Mater Lett., vol. 61, no. 18, pp. 3984-3987, July 2007.\n[16] D. Correa-Llantén, J. Larraín-Linton, P. Muñoz, M. Castro., F.\nBoehmwald, J. Blamey, \"Characterization of the thermophilic bacterium\nGeobacillus sp. Strain GWE1 isolated from a sterilization oven\", Korean\nJ. Microbiol. Biotechnol., vol. 41, no. 3, pp. 278-283, Sept. 2013.\n[17] M. Amenábar, J. Blamey, \"Purification and characterization of a\nthermostable glutamate dehydrogenase from a thermophilic bacterium\nisolated from a sterilization drying oven\", B.M.B. Rep., vol., 45, no. 2,\npp. 91-95, Feb. 2012.\n[18] P. Fesharaki, P. Nazari, M. Shakibaie, S. Rezaie, M. Banoee, M.\nAbdollahi, A. Shahverdi, \"Biosynthesis of selenium nanoparticles\nusing KlebsielIa pneumoniae and their recovery by a simple\nsterilization process\", Braz. J. Microbiol., vol. 41, no. 2, pp. 461–466,\nJune 2010."]}

Lipases catalyze the hydrolysis and synthesis of triglycerides and their reactions are widely used in industry. The use of ionic liquids has been explored in order to improve their catalytic properties. However, the effect of these compounds on kinetic parameters of lipases has been poorly understood. A study of the kinetic parameters of Lip1, the most thermostable lipase from the supernatant of the strain ID17, a thermophilic bacterium isolated from Deception Island, Antarctica, and a member of the genus Geobacillus is presented. Kinetic parameters of Lip1 were modulated by the use of ionic liquids BmimPF6 and BmimBF4. The maximum reaction rate of Lip1 was improved in the presence of both salts. The highest effect was observed when BmimPF6 was added in the reaction mix, resulting in a higher hydrolytic activity and in a modulation of the catalytic efficiency of the enzyme. However, the catalytic efficiency did not change in the presence of BmimBF4. The increase of the reaction rates of Lip1 promoted by these ionic liquids could be related to possible changes in the Lip1 structure. This effect was measured by quenching of tryptophan fluorescence of the enzyme, when it was incubated with each liquid salt. In conclusion, the hydrolytic activity of Lip1 is modulated by the ionic liquids BmimBF4 and BmimPF6, improving the reaction rate and the catalytic efficiency of this enzyme when BmimPF6 was used. This effect is probably due to changes in the structure of Lip1 induced by the presence of these ionic liquids, stimulating its catalytic activity.

The hyperthermophilic archaeon Pyrococcus woesei , is a strict anaerobe heterotroph that grows up to 110°C. Its ferredoxin has now been purified to homogeneity under anaerobic conditions. P. woesei ferredoxin has a Mr value of 7,000 by gel electrophoresis. The Mr value was determined to be 14 000 and 6 500 by gel filtration in 0.2 M NaCl and 0.5 M NaCl, respectively, suggesting that the purified protein exists as a dimer at lower ionic strength. Air-oxidized P. woesei ferredoxin had an UV-visible absorption spectra typical of 4Fe-type ferredoxins with an A 390 /A 280 ratio of 0.63. Its reduction potential was −360 mV at room temperature as determined by direct electrochemistry. P. woesei ferredoxin showed to be extremely thermostable, being unaffected by a 12 h incubation at 95°C. Interestingly, its N-terminus sequence, which represents approximately 1/3 of the total sequence, was determined to be more closely related to the sequences of extremely thermophilic bacterial ferredoxins (≥ 76%) than it was to the sequence of a hyperthermophilic ferredoxin (56%) isolated from an archaeon from the same genus, P. furiosus .

Growth medium components and cultivation conditions for the extremely thermophilic Archaea Thermococcus celer and Pyrococcus woesei were optimized. A culture media based in marine water was formulated. Both Archaea demonstrated to be strictly anaerobic with optimal growth temperature of 85 degrees and 95 degrees C, respectively. Sodium sulfide, but not cysteine, was used as a sulfur and reductive capacity source. It was observed that hydrogen sulfide could be replaced by 30 microM titanium (III) nitrile acetate. The addition of elemental S(o) enhanced growth of both microorganisms, with T. celer far more sensitive than P. woesei to the absence of S(o). P. woesei utilized maltose as a carbon source, while T. celer was able to use only peptides from yeast extract, peptone and tryptone as its carbon source. Optimum carbon source concentrations were 1.25 g/L for T. celer and 5 g/L for P. woesei. Although both Archaea required peptides as a nitrogen source, the addition of ammonia chloride to a nitrogen-limited media did not stimulate growth, which suggests that neither Archaea appear to metabolize ammonia. The growth of P. woesei, but not T. celer, was stimulated considerably in the presence of iron. Co, Ni, Zn, Mo. Mn and Mg were essential trace elements needed for optimal growth of both bacteria.

The relation between gizzard erosion-black vomit (GE-BV) and gastric secretion is not completely understood. A pharmacological approach to reduce the presence of GE-BV in chicks due to fish meal in diets is also unknown. In this study the use of omeprazole, a H+/K+ ATPase inhibitor, and fish meals of different biotoxicological characteristics, showed that: 1) Omeprazole decreased total gastric acid content, GE scores and severe GE (SGE) cases, in a dose-dependent manner. This reduction was significant at levels higher than 20 mg omeprazole/Kg body weight (BW)/day (p < 0.01). The addition of 50 mg omeprazole/kg BW/day almost completely prevented the incidence of SGE cases and reduced in 50% GE score in chicks (p < 0.01). 2) A significant reduction in specific mortality, near 90%, was also seen with all toxic fish meals when omeprazole (50 mg/Kg BW/day) was added to experimental diets in comparison to control groups. However, no mortality was observed when omeprazole was added to diets containing non-toxic fish meals. 3) In chicks fed with toxic fish meals, addition of different amounts of omeprazole to diets changed the relative weight of proventriculus (p < 0.01) and gizzard (p < 0.05). Maximum effect was obtained with omeprazole concentration higher than 50 mg/Kg BW/day. 4) Omeprazole did not change feed intake in chicks fed with toxic fish meal. However, in some fish meal a reduction on weight gain was observed with the addition of omeprazole.

GWE1 strain is an example of anthropogenic thermophilic bacterium, recently isolated from dark crusty material from sterilization ovens by Correa-Llanten et al. (Kor. J. Microb. Biotechnol. 2013. 41(3):278-283). Thermostability is likely to arise from the adaptation of macromolecules such as proteins, lipids and nucleic acids. Complex lipid arrangement and/or type in the cell membrane are known to affect thermostability of microorganisms and efforts were made to understand the chemical nature of the polar lipids of membrane. In this work, we extracted total lipids from GWE1 cell membrane, separated them by TLC into various fractions and characterize the lipid structures of certain fractions with analytical tools such as (1)H, (13)C, (31)P and 2D NMR spectroscopy, ATR-FTIR spectroscopy and MS(n) spectrometry. We were able to identify glycerophosphoethanolamine, glycerophosphate, glycerophosphocholine, glycerophosphoglycerol and cardiolipin lipid classes and an unknown glycerophospholipid class with novel MS/MS spectra pattern. We have also noticed the presence of saturated iso-branched fatty acids with NMR spectra in individual lipid classes.

Antarctica, far from being an exclusively cold continent, has many geothermal sites with volcanic activity. Different islands belonging to this continent harbour different geothermal sites as thermal springs, fumaroles, hot soils and hydrothermal vents, providing ideal environments for the growth of thermophilic and hyperthermophilic microorganisms. Hyperthermophiles are an important source of enzymes and bioactive compounds with biotechnological applications.

The hyperthermophilic bacterium Thermotoga maritima and the hyperthermophilic archaeon Pyrococcus furiosus grow optimally at 80 and 100 degrees C, respectively, by the fermentation of carbohydrates to organic acids, CO2, and H2. Pyruvate is a major source of reductant for H2 production during fermentation, and pyruvate ferredoxin oxidoreductase (POR), a 4Fe-type ferredoxin, and hydrogenase have been previously purified from both species. P. furiosus utilizes a copper-iron-containing POR and a nickel-iron-containing hydrogenase, whereas the POR of T. maritima lacks copper and its hydrogenase lacks nickel. For all four enzymes and for the two ferredoxins, we have determined their reduction potentials (E degrees') and, where possible, thermodynamic parameters associated with electron transfer (delta S degrees and delta H degrees), using differential pulse voltammetry at temperatures ranging from 25 to 95 degrees C. At ambient temperature, the E degrees' values for all six proteins were comparable and spanned less than 50 mV, but their temperature dependence varied dramatically, even between analogous proteins, such that in the physiological-relevant temperature range the E degrees' values became widely separated. In most cases, transition points were observed in E degrees'/temperature profiles, and these generally corresponded with significant increases in catalytic activity, but occurred at lower temperatures in T. maritima than in P. furiosus. The two ferredoxins (and also P. furiosus rubredoxin) had much more negative entropy terms than were calculated for POR and hydrogenase, and these values were also more negative than those previously reported for mesophilic redox proteins. The reduction potentials measured at high temperatures and likely efficiencies of electron transfer between the various proteins were consistent with in vitro activity measurements.(ABSTRACT TRUNCATED AT 250 WORDS)

Four lipases were purified from ID17, a thermophilic bacterium belonging to Geobacillus genus isolated from Deception Island, Antarctica. Lipase activity was detected by opacity test and p-nitrophenyl laurate methods. Lipase production was better in a medium containing tryptone as the carbon and nitrogen source, without non-ionic detergents and pH 7.5. Proteins were ultrafiltered from supernatant and separated using anion exchange and size exclusion chromatography resulting in four distinct fractions with lipase activity (called Lip1–4). Purified lipases showed an optimal pH at 9.0, 9.5, 10.0 and 8.0 and temperature at 65, 70, 75 and 80 °C for Lip1–4, respectively. Lip1 and Lip2 showed higher activity using p-nitrophenol decanoate as substrate, whereas Lip3 and Lip4 prefer p-nitrophenol laurate. Based on their molecular weight Lip1 and Lip2 are trimeric and pentameric proteins, respectively, whereas Lip3 and Lip4 are monomeric proteins. Lip1 was exceptionally thermostable maintaining 70 % of its activity after incubating it at 70 °C for 8 h. Based on their characteristics, the four lipases obtained from ID17 are good candidates to understand the mechanisms of lipase stability and to be used in different types of industrial applications.

A thermostable superoxide dismutase from a thermophilic bacterium, called Geobacillus wiegeli (GWE1), isolated from the interior of a sterilization drying oven, was purified by anion-exchange and molecular size-exclusion liquid chromatography. On the basis of SDS-PAGE, the purified enzyme was found to be homogeneous and showed an estimated subunit molecular mass of 23.9 kDa. The holoenzyme is a homotetramer of 97.3 kDa. Superoxide dismutase exhibited maximal activity at pH 8.5 and at temperature around 60 oC. The enzyme was thermostable maintaining 50% of its activity even after 4.5 hours incubation at 60 oC and more than 70% of its activity after 30 min at 80 oC. When the microorganism was irradiated with UVA, an increase in the specific activity of superoxide dismutase was observed which was correlated with decreasing levels of anion superoxide, indicating the direct involvement of this enzyme in the capture of reactive oxygen species. This study reports the effects of UV radiation on a superoxide dismutase from a thermophilic bacterium isolated from an anthropogenic environment.

In Antarctica microorganisms are exposed to several conditions that trigger the generation of reactive oxygen species, such as high UV radiation. Under these conditions they must have an important antioxidant defense system in order to prevent oxidative damage. One of these defenses are pigments which are part of the non-enzymatic antioxidant mechanisms. In this work we focused on the antioxidant capacity of pigments from an Antarctic microorganism belonging to Pedobacter genus. This microorganism produces different types of pigments which belong to the carotenoids group. The antioxidant capacity of a mix of pigments was analyzed by three different methods: 1,1-diphenyl-2-picrylhydrazyl, ROS detection and oxygen electrode. The results obtained from these approaches indicate that the mix of pigments has a strong antioxidant capacity. The oxidative damage induced by UVB exposure to liposomes was also analyzed. Intercalated pigments within the liposomes improved its resistance to lipid peroxidation. Based on the analysis carried out along this research we conclude that the antioxidant properties of the mix of pigments protect this bacterium against oxidative damage. These properties make this mix of pigments a powerful antioxidant mixture with potential biotechnological applications.

A ferredoxin has been purified from one of the most ancient and most thermophilic bacteria known, Thermotoga maritima, which grows up to 90 degrees C. The reduced protein (M(r) approx. 6300) contains a single S = 1/2 [4Fe-4S]1+ cluster with complete cysteinyl ligation, and was unaffected after incubation at 95 degrees C for 12 h. It functioned as an electron carrier for T. maritima pyruvate oxidoreductase. Remarkably, the properties and amino acid sequence of this hyperthermophilic bacterial protein are much more similar to those of ferredoxins from hyperthermophilic archaea, rather than ferredoxins from mesophilic and moderately thermophilic bacteria.

The hyperthermophilic bacterium, Thermotoga maritima, is a strict anaerobe that grows up to 90 degrees C by carbohydrate fermentation. We report here on its pyruvate ferredoxin oxidoreductase (POR), the enzyme that catalyzes the oxidation of pyruvate to acetyl-CoA, the terminal oxidation step in the conversion of glucose to acetate. T. maritima POR was purified to electrophoretic homogeneity under strictly anaerobic conditions. It has a molecular weight of 113,000 and comprises four dissimilar subunits with M(r) values of approximately 43,000, 34,000, 23,000, and 13,000. It contains thiamine pyrophosphate (TPP) and at least two ferredoxin-type [4Fe-4S] clusters per molecule, as determined by iron analysis and EPR spectroscopy. CoASH was absolutely required for pyruvate oxidation activity, while the addition of TPP was stimulatory. The apparent Km values at 80 degrees C for pyruvate, CoASH, and TPP were 14.5, 0.34, and 0.043 mM, respectively, and the corresponding apparent Vm values ranged from 154 to 170 mumol of pyruvate oxidized/min/mg (units/mg). The apparent Km and Vm values for T. maritima ferredoxin, the proposed physiological electron carrier for POR, were 26 microM and 280 units/mg, respectively. POR did not use 2-oxoglutarate, phenyl pyruvate, or indolyl pyruvate as substrates. The enzyme was extremely thermostable: the temperature optimum for pyruvate oxidation was above 90 degrees C, and the time for a 50% loss of activity (t50%) at 80 degrees C (under anaerobic conditions) was 15 h. The enzyme was also very sensitive to inactivation by oxygen, with a t50% in air at 25 degrees C of 70 min.(ABSTRACT TRUNCATED AT 250 WORDS)

[1] The Chilean Altiplano is the westernmost part of a large volcanic-sedimentary plateau in the central Andes. High solar irradiance and rapid increase of temperature have contributed to make it a hot spot of global climatic change. In this study, we describe microbial diversity in the summit lake of the Simba volcano (5,870 m) and the evaporitic basins of Salar de Aguas Calientes (4,200 m) and Laguna Lejia (4,325 m) using both culture and culture-independent methods. The results obtained were analyzed together with available information from related environments to describe the traits of the microbial community driven by main environmental factors. Isolated cultures exhibit high resistance to all three types of UV radiation, further supporting the adaptation of microorganisms to the high altitude environment. The microbial community structures at Salar de Aguas Calientes and Laguna Lejia are similar to those from other saline systems and cold environments where Bacteroidetes is the major bacterial group. The abundance of sequences related to alphaproteobacteria and methanogenic populations likely reflects the importance of aerobic anoxigenic phothosynthesis and the cycling of one-carbon compounds in the high altitude lake ecosystems. Geochemistry and microbial communities at Simba as well as those reported in the Licancabur summit lake provide evidence for sulfur-rich environments but under different conditions. Those differences between neighboring mountain lake ecosystems highlight the effect of volcanic activity on microbial communities. The hypothetical ecosystem model described in this work provides a clue to follow the microbial community responses to geophysical environment coupled with rapid climate change.

As never before, biological and medical sciences in Latin America have been caught unprepared with the explosive development of genome biology with its concomitant use of high-throughput technologies, large scale database management, sophisticated instrumentation and collaborative research. In the past, new biological technologies, such as those developed during the genetic engineering revolution, were readily assimilated and successfully used by laboratories in the Region. A typical, small sized university laboratory with a set of shared instruments, managed to produce results that were acceptable in good or regular impact journals. Nowadays, a

Thermococcus celer cells contain a single hydrogenase located in the cytoplasm, which has been purified to apparent homogeneity using three chromatographic steps: Q-Sepharose, DEAE-Fast Flow, and Sephacryl S-200. In vitro assays demonstrated that this enzyme was able to catalyze the oxidation as well as the evolution of H2. T. celer hydrogenase had an apparent MW of 155,000+/-30,000 by gel filtration. When analyzed by SDS polyacrylamide gel electrophoresis a single band of 41,000+/-2,000 was detected. Hydrogenase activity was also detected in situ in a SDS polyacrylamide gel followed by an activity staining procedure revealing a single band corresponding to a protein of apparent Mr 84,000+/-3,000. Measurements of iron and acid-labile sulfide in different preparations of T. celer hydrogenase gave values ranging from 24 to 30 g-atoms Fe/mole of protein and 24 to 36 g-atoms of acid-labile sulfide per mole of protein. Nickel is present in 1.9-2.3 atoms per mole of protein. Copper, tungsten, and molybdenum were detected in amounts lower than 0.5 g-atoms per mole of protein. T. celer hydrogenase was inactive at ambient temperature, exhibited a dramatic increase in activity above 70 degrees C, and had an optimal activity above 90 degrees C. This enzyme showed no loss of activity after incubation at 80 degrees C for 28 h, but lost 50% of its initial activity after incubation at 96 degrees C for 20 h. Hydrogenase exhibited a half-life of approximately 25 min in air. However, after treating the air-exposed sample with sodium dithionite, more than 95% of the original activity was recovered. Copper sulfate, magnesium chloride and nitrite were also inactivators of this enzyme.

The reduction potentials of the metalloproteins pyruvate ferredoxin oxidoreductase (POR), ferredoxin, and hydrogenase isolated from hyperthermophilic Thermococcus celer (Topt = 88 degrees C) were determined as a function of temperature from 10 to 85 degrees C. Square-wave voltammetry experiments were carried out on 15 microL samples directly at an unmodified "edge-polished" pyrolytic graphite electrode using MgCl2 as an electrode promoter. POR exhibited two voltammetric waves with peaks at -280 and -403 mV at room temperature, indicating multiple redox centers, and a single wave at -420 mV at 85 degrees C. These waves displayed different temperature-dependent peak positions and peak heights, indicating that these redox centers have different thermodynamic and kinetic properties. Ferredoxin displayed a single linear temperature-dependent voltammetric wave at -280 mV at room temperature and -327 mV at 85 degrees C. Hydrogenase displayed a single biphasic temperature-dependent voltammetric wave at -197 mV at room temperature and -211 mV at 85 degrees C. Thermodynamic parameters associated with electron transfer, namely standard enthalpies and entropies for the redox centers in the various proteins, are reported.

High enolase activity, as measured by the conversion of 2-phosphoglycerate to phosphoenolpyruvate, was found in the cytoplasm of Pyrococcus furiosus (an anaerobic, hyperthermophilic archaeon that grows optimally at 100 degrees C). In this organism, the enzyme probably functions in a sugar fermentation pathway. The enzyme was purified to homogeneity. It had a temperature optimum of90 degrees C and a pH optimum of 8.1. The enzyme was extremely thermostable with a time for 50% inactivation at 100 degrees C of 40 min. In contrast, an enolase from yeast was totally inactivated in 1 min at 88 degrees C. Both the P. furiosus and yeast enzymes required a metal ion for activity, but whereas the yeast enzyme has an absolute requirement for Mg2+, the P. furiosus enolase was equally active in the presence of Mn2+. Both enzymes were competitively inhibited by citrate. P. furiosus enolase, as for mesophilic enolases, probably has a homodimeric structure with subunit M(r) greater than 45,000. A highly conserved sequence of eight amino acids in the N-terminal region was found in enolases from P. furiosus and a wide range of other organisms including bacteria, yeast, birds, and mammals. Substantial differences in the thermal properties of the hyperthermophilic enzyme compared with that from less extreme thermophiles and mesophiles might be due to a substantially enhanced composition of hydrophobic amino acids.

Pyruvate ferredoxin oxidoreductase (POR) has been previously purified from two hyperthermophiles, the archaeon Pyrococcus furiosus (Pf, Topt = 100 degrees C) and the bacterium Thermotoga maritima (Tm, Topt = 80 degrees C). Each catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2 near the optimal growth temperature of the organism and are virtually inactive at 25 degrees C. Both PORs contain a thiamine pyrophosphate (TPP) cofactor and at least two [4Fe-4S] ferredoxin-type clusters. We have now shown, using EPR spectroscopy and metal analyses, that PfPOR also contains an unusual copper center that is not present in Tm POR. In addition, distinct catalytic intermediates were generated in both enzymes by the addition, separately and in combination, of the substrates pyruvate and CoASH, and these were examined by EPR spectroscopy. The addition of pyruvate to oxidized Pf POR produced an isotropic signal centered at g = 2.01, which was measurably broader in the presence of pyruvate-2(13)C. This signal, which was assigned to a (hydroxyethyl)thiamine pyrophosphate radical intermediate, was not observed in Tm POR under the same experimental conditions. Incubation of the oxidized enzymes with CoASH resulted in the partial reduction of the copper site in Pf POR and the partial reduction of a novel iron-sulfur center in Tm POR, which was not seen in the dithionite-reduced enzyme. The addition of both pyruvate and CoASH to the PORs in their oxidized states resulted in the reduction of the same iron-sulfur centers that are reduced by sodium dithionite.(ABSTRACT TRUNCATED AT 250 WORDS)

Pyrococcus furiosus grows optimally at 100°C by carbohydrate fermentation. It is thought to contain a novel tungsten-dependent, NAD(P)-independent glycolytic pathway in which one of the oxidation steps is catalyzed by a tungsten-containing aldehyde ferredoxin oxidoreductase. The enzyme that catalyzes the terminal oxidation step, pyruvate ferredoxin oxidoreductase (POR), has now been purified. POR has a molecular mass of 100 kDa and is comprised of three subunits (45, 31 and 24 kDa). It lacks tungsten but contains thiamine pyrophosphate (TPP) and two ferredoxin-type [4Fe-4S] clusters per molecule which, by EPR spectroscopy, can be differentiated by their relaxation properties. The enzyme requires CoASH but not TPP for pyruvate oxidation activity and will not use 2-oxoglutarate, phenyl pyruvate or indole pyruvate as substrates. POR is virtually inactive at 25°C and shows a temperature optimum for pyruvate oxidation above 90°C. The apparent K m values for pyruvate, CoASH and P. furiosus ferredoxin at 80°C are 460, 100 and 70 μM, respectively. Carbon monoxide was a potent inhibitor of pyruvate oxidation (apparent K i = 7 μ M ). The half-life of activity ( t 50% ) in air at 25°C was 15 min and the t 50% value at 80μC (under anaerobic Conditions) was 23 min. Based on molecular comparisons with PORs from mesophilic organisms, it is proposed that P. furiosus POR may represent an ancestral form of a pyruvate-oxidizing enzyme.

The gene for ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, sequenced, and expressed in Escherichia coli. The coding region confirmed the determined amino acid sequence. Putative archaeon-type transcriptional regulatory elements were identified. The fdxA gene appears to be an independent transcriptional unit. Recombinant ferredoxin was indistinguishable from the protein purified from P. furiosus in its thermal stability and in the potentiometric and spectroscopic properties of its [4Fe-4S] cluster.

Background The use of microorganisms in the synthesis of nanoparticles emerges as an eco-friendly and exciting approach, for production of nanoparticles due to its low energy requirement, environmental compatibility, reduced costs of manufacture, scalability, and nanoparticle stabilization compared with the chemical synthesis. Results The production of gold nanoparticles by the thermophilic bacterium Geobacillus sp. strain ID17 is reported in this study. Cells exposed to Au3+ turned from colourless into an intense purple colour. This change of colour indicates the accumulation of intracellular gold nanoparticles. Elemental analysis of particles composition was verified using TEM and EDX analysis. The intracellular localization and particles size were verified by TEM showing two different types of particles of predominant quasi-hexagonal shape with size ranging from 5–50 nm. The mayority of them were between 10‒20 nm in size. FT-IR was utilized to characterize the chemical surface of gold nanoparticles. This assay supports the idea of a protein type of compound on the surface of biosynthesized gold nanoparticles. Reductase activity involved in the synthesis of gold nanoparticles has been previously reported to be present in others microorganisms. This reduction using NADH as substrate was tested in ID17. Crude extracts of the microorganism could catalyze the NADH-dependent Au3+ reduction. Conclusions Our results strongly suggest that the biosynthesis of gold nanoparticles by ID17 is mediated by enzymes and NADH as a cofactor for this biological transformation.