697 research outputs found

    Etude de la production d'hydrogène en bioréacteur par la bactérie photosynthétique Rhodobacter capsulatus 2. Transformation du lactate et bilans carbonés

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    La d√©gradation du lactate par la bact√©rie photosynth√©tique Rhodobacter capsulatus cultiv√©e dans un photobior√©acteur en ana√©robiose, √† la lumi√®re, en limitation d'azote, a √©t√© mesur√©e ainsi que la production d'hydrog√®ne par la nitrog√©nase. En r√©acteur ouvert √† faible taux de dilution (D = 0,04 h-1), le lactate initialement √† 30 mM est d√©grad√© √† pr√®s de 90 % avec une faible accumulation (< 1-2 mM) des produits de d√©gradation (formiate et ac√©tate). C'est dans ces conditions qu'on observe la meilleure production d'hydrog√®ne (44 ml ‚ÄĘ h-1 ‚ÄĘ h-1). Lorsque la culture bact√©rienne devient trop dense en r√©acteur ferm√© ou an recyclage des bact√©ries, Il y a apparemment passage √† un m√©tabolisme de type fermentatif, chute de la production d'hydrog√®ne et accumulation d'acides organiques (formique, ac√©tique, propionique) provenant du lactate. Dans ces conditions, il n'y a pas diminution de la charge de carbone organique. Outre la pr√©sence de ces acides √† courtes cha√ģnes, les bilans carbon√©s; font appara√ģtre la pr√©sence, dans le surnageant de la culture, de substances carbon√©es exocellulaires non identifi√©es pouvant repr√©senter jusqu'√† 50 % du flux carbon√© sortant.A 10 l photobioreactor, consisting of a PVC tubing, spiral-coiled so as to form a plane light captor (DELACHAPELLE et al., 1990), was used to study the degradation of lactate by the photosynthetic bacterium Rhodobacter capsulatus strain B10. The bacterial culture was continuously circulated in the reactor so as to maintain a homogeneous suspension, to optimize illumination of the cells, and make a well-mixed reactor for optimal nutrient transfers and degas ing of the medium. The bacterium was cultivated anaerobically under photoheterotrophic and N-limited conditions. The bioreactor was operated in three modes¬†: as an open system (chemostat), as a closed system (batch), and as an open system with phases of bacteria recycling through the use of an ultrafiltration cell.When the bioreactor functioned as a chemostat, at low dilution rate (D < 0.04 h-1) and at relatively low bacterial density (A660 nm < 2.5), up to 90 % of the added lactate (initial concentration 30 mM) was degraded; the concentration of degradation products (formate, acetate) remained low (<2 mM). Under these conditions nitrogenase-mediated H2 production was maximal (44 ml ‚ÄĘ h-1 ‚ÄĘ l-1). In batch cultures or when the cells were ¬ę¬†recycled¬†¬Ľ in the chemostat by ultrafiltration, the increase in absorbancy led to a decrease in cell illumination and the bacteria apparently switched from a photosynthetic of a fermentative type of metabolism; lactate was converted into formate + acetate + propionate, no H2 was produced. From the determination of the carbon balance between the influent and the effluent medium, the presence of non identified extracellular carbon compounds (up to 50 % of the C content of the effluent) was proved

    Etude de la production d'hydrogène en bioréacteur par une bactérie photosynthétique Rhodobacter capsulatus 1. Photobioréacteur et conditions optimales de production d'hydrogène

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    Un r√©acteur de 10 litres, automatis√© pour la culture continue en ana√©robiose de bact√©ries photosynth√©tiques, a √©t√© r√©alis√© et mis au point. Ce r√©acteur parfaitement agit√© a √©t√© utilis√© dans diff√©rentes conditions de fonctionnement en syst√®me ferm√© (batch), en syst√®me semi-ouvert (atouts de substrats concentr√©s en discontinu), en syst√®me ouvert (ch√©mostat) avec et sans recyclage de la biomasse, afin d'√©tudier la consommation, par les bact√©ries, d'un substrat carbon√©, le lactate. La production d'hydrog√®ne par la bact√©rie photosynth√©tique Rhodobacter capsulatus, souche B10, r√©sultant de la d√©gradation du lactate, est optimale pour des cultures en continu dilu√©es, limit√©es en source azot√©e. Ainsi, √† un taux de dilution de 0,04 h-1, avec 5 mM glutamate dans le milieu nutritif, la densit√© bact√©rienne √©tant de 2,1 √® 660 nm, on a observ√© une production continue moyenne de 65 ml ‚ÄĘ h-1‚ÄĘ l-1 pendant une p√©riode de 200 heures. Pour des concentrations bact√©riennes √©lev√©es, la limitation d'√©nergie lumineuse entrain une perte d'activit√© nitrog√©nase et, de ce fait, une chute de la production d'hydrog√®ne.A photobioreactor was set up to cultivate a photosynthetic bacterium in continuous cultures. The bioreactor was designed so as to 1) allow the capture of light energy by bacteria through a spiral transparent flexible tube placed under the light, in a water bath maintaining the growth temperature at 30 ¬įC; 2) male the suspension of bacteria circulate continuously in the reactor with a volumetric pump to maintain the medium homogeneous; 3) allow degassing of the suspension in a degassing chamber; 4) feed the culture with nutritive media, add neutralizing solution (pH 7) and withdrax aliquots white maintaining constant the volume of the culture; 5) recycle the bacteria by filtration when the bioreactor was used as e closed system (batch).The photosynthetic bacterium was Rhodobacter capsulatus strain B10 is known to lie a good H2 producer [Hillmer and Gest (1977) J. Bacteriol. 129, 724-731]. The bioreactor was run using 10 l of a synthetic medium containing lactate as carbon source and glutamate as nitrogen source. It was studied for its capacity to degrade lactate. Glutamate was the growth-liliting substrate allowing a maximum derepression of nitrogenase, the enzyme catalysing the reduction of protons to H2. The bacterial suspension was continuously circulated in the photoreactor, conceived as a plane light captor of 1 m2, to avoid bacterial self-shading and allow regeneration of ATP by photophosphorylation at high rates. The circuit was tightly closed to avoid air entry, which would prevent H2 production due to respiration of the bacteria.To run it under automated conditions, the bioreactor was equipped with two temperature sensors, two pH electrodes, a water level detector, a manometer and a computer-controlled electric valve. The bioreactor, of the well-mixed type, was used under various working conditions, namely as a closed (batch) system, as a fed-batch system (discontinuous additions of concentrated substrates), and as an open system (chemostat) with or without biomass recycling.Control of key parameters (pH, temperature, dilution rates) allowed us to define the culture conditions producing maximal amounts of molecular hydrogen. The production of H2 accompanying lactate degradation was maximal in diluted nitrogen-limited continuous cultures. It was observed at a dilution rate of 0.04 h-1 with 5 mM glutamate in the influent medium, the optical density of the culture being 2.1 at 660 nm. Under these conditions an average production H2 of 85 ml ‚ÄĘ h-1 ‚ÄĘ l-1 was observed over a 200 h period. At higher bacterial concentrations, the limitation of light energy resulted in a decrease in nitrogenase activity and therefore in a drop in the production of hydrogen.The interdependence of various parameters (pH, dilution rates, N and C sources light intensity) renders the system complex and not easily controlled by computer. Indeed, we observed that during recycling of the bacteria by the use of an ultrafiltration cell, the bacteria became a fermentative-type of metabolism accompanied by a decrease in nitrogenase activity and therefore in a drop in the production of hydrogen

    Cell-Free Synthesis of the Mitochondrial ADP/ATP Carrier Protein of Neurospora crassa

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    ADP/ATP carrier protein was synthesized in heterologous cell-free systems programmed with Neurospora poly(A)-containing RNA and homologous cell-free systems from Neurospora. The apparent molecular weight of the product obtained in vitro was the same as that of the authentic mitochondrial protein. The primary translation product obtained in reticulocyte lysates starts with formylmethionine when formylated initiator methionyl-tRNA (fMet-tRNAfMet) was present. The product synthesized in vitro was released from the ribosomes into the postribosomal supernatant. The evidence presented indicates that the ADP/ATP carrier is synthesized as a polypeptide with the same molecular weight as the mature monomeric protein and does not carry an additional sequence

    Requirement of a Membrane Potential for the Posttranslational Transfer of Proteins into Mitochondsria

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    Posttranslational transfer of most precursor proteins into mitochondria is dependent on energization of the mitochondria. Experiments were carried out to determine whether the membrane potential or the intramitochondrial ATP is the immediate energy source. Transfer in vitro of precursors to the ADP/ATP carrier and to ATPase subunit 9 into isolated Neurospora mitochondria was investigated. Under conditions where the level of intramitochondrial ATP was high and the membrane potential was dissipated, import and processing of these precursor proteins did not take place. On the other hand, precursors were taken up and processed when the intramitochondrial ATP level was low, but the membrane potential was not dissipated. We conclude that a membrane potential is involved in the import of those mitochondrial precursor proteins which require energy for intracellular translocatio

    Transport of Proteins into Mitochondria

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    The mitochondrial ADP/ATP carrier is an integral transmembrane protein of the inner membrane. It is synthesized on cytoplasmic ribosomes. Kinetic data suggested that this protein is transferred into mitochondria in a posttranslational manner. The following results provide further evidence for such a mechanism and provide information on its details. 1. In homologous and heterologous translation systems the newly synthesized ADP/ATP carrier protein is present in the postribosomal supernatant. 2. Analysis by density gradient centrifugation and gel filtration shows, that the ADP/ATP carrier molecules in the postribosomal fraction are present as soluble complexes with apparent molecular weights of about 120000 and 500000 or larger. The carrier binds detergents such as Triton X-100 and deoxycholate forming mixed micelles with molecular weights of about 200000‚Äď400000. 3. Incubation of a postribosomal supernatant of a reticulocyte lysate containing newly synthesized ADP/ATP carrier with mitochondria isolated from Neurospora spheroplasts results in efficient transfer of the carrier into mitochondria. About 20‚Äď30% of the transferred carrier are resistant to proteinase in whole mitochondria. The authentic mature protein is also largely resistant to proteinase in whole mitochondria and sensitive after lysis of mitochondria with detergent. Integrity of mitochondria is a prerequisite for translocation into proteinase resistant position. 4. The transfer in vitro into a proteinase-resistant form is inhibited by the uncoupler carbonyl-cyanide m-chlorophenylhydrazone but not the proteinase-sensitive binding. These observations suggest that the posttranslational transfer of ADP/ATP carrier occurs via the cytosolic space through a soluble oligomeric precursor form. This precursor is taken up by intact mitochondria into an integral position in the membrane. These findings are considered to be of general importance for the intracellular transfer of insoluble membrane proteins. They support the view that such proteins can exist in a water-soluble form its precursors and upon integration into the membrane undergo a conformational change. Uptake into the membrane may involve the cleavage of an additional sequence in some proteins, but this appears not to be a prerequisite as demonstrated by the ADP/ATP carrier protein

    A [4Fe-4S]-Fe(CO)(CN)-L-cysteine intermediate is the first organometallic precursor in [FeFe] hydrogenase H-cluster bioassembly.

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    Biosynthesis of the [FeFe] hydrogenase active site (the 'H-cluster') requires the interplay of multiple proteins and small molecules. Among them, the radical S-adenosylmethionine enzyme HydG, a tyrosine lyase, has been proposed to generate a complex that contains an Fe(CO)2(CN) moiety that is eventually incorporated into the H-cluster. Here we describe the characterization of an intermediate in the HydG reaction: a [4Fe-4S][(Cys)Fe(CO)(CN)] species, 'Complex A', in which a CO, a CN- and a cysteine (Cys) molecule bind to the unique 'dangler' Fe site of the auxiliary [5Fe-4S] cluster of HydG. The identification of this intermediate-the first organometallic precursor to the H-cluster-validates the previously hypothesized HydG reaction cycle and provides a basis for elucidating the biosynthetic origin of other moieties of the H-cluster

    An Electrochemical Study of Frustrated Lewis Pairs: A Metal-free Route to Hydrogen Oxidation

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    [Image: see text] Frustrated Lewis pairs have found many applications in the heterolytic activation of H(2) and subsequent hydrogenation of small molecules through delivery of the resulting proton and hydride equivalents. Herein, we describe how H(2) can be preactivated using classical frustrated Lewis pair chemistry and combined with in situ nonaqueous electrochemical oxidation of the resulting borohydride. Our approach allows hydrogen to be cleanly converted into two protons and two electrons in situ, and reduces the potential (the required energetic driving force) for nonaqueous H(2) oxidation by 610 mV (117.7 kJ mol(‚Äď1)). This significant energy reduction opens routes to the development of nonaqueous hydrogen energy technology

    The exchange activities of [Fe]¬†hydrogenase (iron‚Äďsulfur-cluster-free hydrogenase) from methanogenic archaea in comparison with the exchange activities of [FeFe] and [NiFe]¬†hydrogenases

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    [Fe]¬†hydrogenase (iron‚Äďsulfur-cluster-free hydrogenase) catalyzes the reversible reduction of methenyltetrahydromethanopterin (methenyl-H4MPT+) with H2 to methylene-H4MPT, a reaction involved in methanogenesis from H2 and CO2 in many methanogenic archaea. The enzyme harbors an iron-containing cofactor, in which a low-spin iron is complexed by a pyridone, two CO and a cysteine sulfur. [Fe]¬†hydrogenase is thus similar to [NiFe] and [FeFe]¬†hydrogenases, in which a low-spin iron carbonyl complex, albeit in a dinuclear metal center, is also involved in H2 activation. Like the [NiFe] and [FeFe]¬†hydrogenases, [Fe]¬†hydrogenase catalyzes an active exchange of H2 with protons of water; however, this activity is dependent on the presence of the hydride-accepting methenyl-H4MPT+. In its absence the exchange activity is only 0.01% of that in its presence. The residual activity has been attributed to the presence of traces of methenyl-H4MPT+ in the enzyme preparations, but it could also reflect a weak binding of H2 to the iron in the absence of methenyl-H4MPT+. To test this we reinvestigated the exchange activity with [Fe]¬†hydrogenase reconstituted from apoprotein heterologously produced in Escherichia coli and highly purified iron-containing cofactor and found that in the absence of added methenyl-H4MPT+ the exchange activity was below the detection limit of the tritium method employed (0.1¬†nmol min‚ąí1¬†mg‚ąí1). The finding reiterates that for H2 activation by [Fe]¬†hydrogenase the presence of the hydride-accepting methenyl-H4MPT+ is essentially required. This differentiates [Fe]¬†hydrogenase from [FeFe] and [NiFe]¬†hydrogenases, which actively catalyze H2/H2O exchange in the absence of exogenous electron acceptors

    Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase

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    Background: Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies. Principal Findings: We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus, led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L‚ąí1 of culture from E. coli with specific activities of 1000 U (U = 1 ¬Ķmol hydrogen evolved mg‚ąí1 min‚ąí1). Significance: The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.United States. Dept. of Energy. (contract DE-AC36-08-GO28308
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