References of "Gerday, Charles"
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See detailExtremophiles
Berlemont, Renaud ULg; Gerday, Charles ULg

in Comprehensive Biotechnology (2010)

Extremophiles are organisms which inhabit environments characterized by properties harsh enough to hinder the survival of common cells. They are highly diversified and are classified on the basis of the ... [more ▼]

Extremophiles are organisms which inhabit environments characterized by properties harsh enough to hinder the survival of common cells. They are highly diversified and are classified on the basis of the main extreme property that prevails in the habitat. Six main categories can be distinguished: the thermophiles found in high temperature sites and which can tolerate temperatures sometimes close to that of the boiling point of water; the psychrophiles living in permanently cold habitats with temperatures sometimes well below the freezing point of water; the piezophiles, which tolerate pressure as high as 1000 atm; the halophiles supporting salt concentrations, in some cases, higher than 300gl–1; the acidophiles thriving well at pH sometimes close to zero; and the alkaliphiles, which, on the contrary, tolerate pH largely exceeding neutrality. These organisms are mainly microorganisms and they notably produce enzymes that are adapted to work in unusual conditions often required in biotechnological processes. This confers upon these organisms a very high potential. They are the target of a steadily increasing interest and are nowadays largely used in various industrial applications. [less ▲]

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See detailFundamentals of Cold-Adapted Enzymes
Collins, T.; Roulling, Frédéric ULg; Piette, Florence ULg et al

in Margesin, R.; Schinner, F.; Gerday, Charles (Eds.) et al Psychrophiles: from Biodiversity to Biotechnology (2008)

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See detailThe linker region plays a key role in the adaptation to cold of the cellulase from an Antarctic bacterium
Sonan, Guillaume K; Receveur-Brechot, Véronique; Duez, Colette ULg et al

in Biochemical Journal (2007), 407(Part 2), 293-302

The psychrophilic cellulase, Cel5G, from the Antarctic bacterium Pseudoalteromonas haloplanktis is composed of a catalytic module (CM) joined to a carbohydrate-binding module (CBM) by an unusually long ... [more ▼]

The psychrophilic cellulase, Cel5G, from the Antarctic bacterium Pseudoalteromonas haloplanktis is composed of a catalytic module (CM) joined to a carbohydrate-binding module (CBM) by an unusually long, extended and flexible linker region (LR) containing three loops closed by three disulfide bridges. To evaluate the possible role of this region in cold adaptation, the LR was sequentially shortened by protein engineering, successively deleting one and two loops of this module, whereas the last disulfide bridge was also suppressed by replacing the last two cysteine residue by two alanine residues. The kinetic and thermodynamic properties of the mutants were compared with those of the full-length enzyme, and also with those of the cold-adapted CM alone and with those of the homologous mesophilic enzyme, Cel5A, from Erwinia chrysanthemi. The thermostability of the mutated enzymes as well as their relative flexibility were evaluated by differential scanning calorimetry and fluorescence quenching respectively. The topology of the structure of the shortest mutant was determined by SAXS (small-angle X-ray scattering). The data indicate that the sequential shortening of the LR induces a regular decrease of the specific activity towards macromolecular substrates, reduces the relative flexibility and concomitantly increases the thermostability of the shortened enzymes. This demonstrates that the long LR of the full-length enzyme favours the catalytic efficiency at low and moderate temperatures by rendering the structure not only less compact, but also less stable, and plays a crucial role in the adaptation to cold of this cellulolytic enzyme. [less ▲]

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See detailCold-Adapted Enzymes
Collins, T.; D'Amico, Salvino ULg; Marx, J. C. et al

in Gerday, Charles; Glansdorff, N. (Eds.) Physiology and biochemistry of extremophiles (2007)

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See detailAdaptation strategies and uses of cold adapted enzymes in biotechnological processes
Gerday, Charles ULg; D'Amico, Salvino ULg; Collins, T. et al

in JAMSTEC ERC (Ed.) Proceedings of the International Symposium on Extremophiles and their Applications 2005 (2007)

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See detailA novel family 8 psychrophilic xylanase: fundamentals and application
Collins, T.; Hoyoux, A.; Van Petegem, F. et al

in JAMSTEC E.R.C (Ed.) Proceedings of the International Symposium on Extremophiles and their Applications 2005 (2007)

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See detailCold-adapted enzymes from marine antarctic microorganisms
Marx, J. C.; Collins, T.; D'Amico, Salvino ULg et al

in Marine Biotechnology (2007), 9(3, May-Jun), 293-304

The Antarctic marine environment is characterized by challenging conditions for the survival of native microorganisms. Indeed, next to the temperature effect represented by the Arrhenius law, the ... [more ▼]

The Antarctic marine environment is characterized by challenging conditions for the survival of native microorganisms. Indeed, next to the temperature effect represented by the Arrhenius law, the viscosity of the medium, which is also significantly enhanced by low temperatures, contributes to slow down reaction rates. This review analyses the different challenges and focuses on a key element of life at low temperatures: cold-adapted enzymes. The molecular characteristics of these enzymes are discussed as well as the adaptation strategies which can be inferred from the comparison of their properties and three-dimensional structures with those of their mesophilic counterparts. As these enzymes display a high specific activity at low and moderate temperatures associated with a relatively high thermosensitivity, the interest in these properties is discussed with regard to their current and possible applications in biotechnology. [less ▲]

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See detailRole of lysine versus arginine in enzyme cold-adaptation: Modifying lysine to homo-arginine stabilizes the cold-adapted alpha-amylase from Pseudoalteramonas haloplanktis
Siddiqui, K. S.; Poljak, A.; Guilhaus, M. et al

in Proteins-Structure Function and Bioinformatics (2006), 64(2), 486-501

The cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis (AHA) is a multidomain enzyme capable of reversible unfolding. Cold-adapted proteins, including AHA, have been predicted to be ... [more ▼]

The cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis (AHA) is a multidomain enzyme capable of reversible unfolding. Cold-adapted proteins, including AHA, have been predicted to be structurally flexible and conformationally unstable as a consequence of a high lysine-to-arginine ratio. In order to examine the role of low arginine content in structural flexibility of AHA, the amino groups of lysine were guanidinated to form homoarginine (hR), and the structure-function-stability properties of the modified enzyme were analyzed by transverse urea gradient-gel electrophoresis. The extent of modification was monitored by MALDI-TOF-MS, and correlated to changes in activity and stability. Modifying lysine to hR produced a conformationally more stable and less active a-amylase. The k(cat) of the modified enzyme decreased with a concomitant increase in Delta H-# and decrease in K-m. To interpret the structural basis of the kinetic and thermodynamic properties, the hR residues were modeled in the AHA X-ray structure and compared to the X-ray structure of a thermostable homolog. The experimental properties of the modified AHA were consistent with K106hR forming an intra-Domain B salt bridge to stabilize the active site and decrease the cooperativity of unfolding. Homo-Arg modification also appeared to alter Ca2+ and Cl- binding in the active site. Our results indicate that replacing lysine with hR generates mesophilic-like characteristics in AHA, and provides support for the importance of lysine residues in promoting enzyme cold adaptation. These data were consistent with computational analyses that show that AHA possesses a compositional bias that favors decreased conformational stability and increased flexibility. [less ▲]

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See detailOligosaccharide binding in family 8 glycosidases: Crystal structures of active-site mutants of the beta-1,4-xylanase pXyl from Pseudoaltermonas haloplanktis TAH3a in complex with substrate and product
De Vos, D.; Collins, T.; Nerinckx, W. et al

in Biochemistry (2006), 45(15), 4797-4807

The structures of inactive mutants D144A and E78Q of the glycoside hydrolase family 8 (GH-8) endo-beta-1,4-D-Xylanase (pXyl) from the Antarctic bacterium Pseudoalteromonas haloplanktis TAH3a in complex ... [more ▼]

The structures of inactive mutants D144A and E78Q of the glycoside hydrolase family 8 (GH-8) endo-beta-1,4-D-Xylanase (pXyl) from the Antarctic bacterium Pseudoalteromonas haloplanktis TAH3a in complex with its substrate xylopentaose (at 1.95 angstrom resolution) and product xylotriose (at 1.9 angstrom resolution) have been determined by X-ray crystallography. A detailed comparative analysis of these with the apoenzyme and with other GH-8 structures indicates an induced fit mechanism upon ligand binding whereby a number of conformational changes and, in particular, a repositioning of the proton donor into a more catalytically competent position Occurs. This has also allowed for the description of protein-ligand interactions in this enzyme and for the demarcation of subsites -3 to +3. An in-depth analysis of each of these subsites gives an insight into the structure-function relationship of this enzyme and the basis of xylose/glucose discrimination in family 8 glycoside hydrolases. Furthermore, the structure of the -1/+1 subsite spanning complex reveals that the substrate is distorted from its ground state conformation. Indeed, structural analysis and in silico docking Studies indicate that substrate hydrolysis in GH-8 members is preceded by a conformational change, away from the substrate ground-state chair conformation, to a pretransition state local minimum S-2(O) conformation. [less ▲]

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See detailPsychrophilic microorganisms: challenges for life
D'Amico, Salvino ULg; Collins, T.; Marx, J. C. et al

in EMBO Reports (2006), 7(4), 385-389

The ability of psychrophiles to survive and proliferate at low temperatures implies that they have overcome key barriers inherent to permanently cold environments. These challenges include: reduced enzyme ... [more ▼]

The ability of psychrophiles to survive and proliferate at low temperatures implies that they have overcome key barriers inherent to permanently cold environments. These challenges include: reduced enzyme activity; decreased membrane fluidity; altered transport of nutrients and waste products; decreased rates of transcription, translation and cell division; protein cold- denaturation; inappropriate protein folding; and intracellular ice formation. Cold- adapted organisms have successfully evolved features, genotypic and/ or phenotypic, to surmount the negative effects of low temperatures and to enable growth in these extreme environments. In this review, we discuss the current knowledge of these adaptations as gained from extensive biochemical and biophysical studies and also from genomics and proteomics. [less ▲]

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See detailUse of glycoside hydrolase family 8 xylanases in baking
Collins, T.; Hoyoux, A.; Dutron, A. et al

in Journal of Cereal Science (2006), 43(1), 79-84

Xylanases have long been used in the baking industry for improving dough stability and flexibility and for increasing bread volume and crumb structure. Only xylanases from glycoside hydrolase families 10 ... [more ▼]

Xylanases have long been used in the baking industry for improving dough stability and flexibility and for increasing bread volume and crumb structure. Only xylanases from glycoside hydrolase families 10 and I I appear to have been tested in this application and only those from the latter family have as yet found application. Interestingly, enzymes with a putative xylanase activity are also found in glycoside hydrolase families 5, 7, 8 and 43, but apparently these have not, as yet, been tested in baking. Baking trials were used to determine the effectiveness of a psychrophilic and a mesophilic family 8 xylanolytic enzyme as well as a psychrophilic family 10 xylanase and a currently used family 11 commercial mesophilic xylanase. The potential of family 8 xylanases as technological aids in baking was clearly demonstrated as both the psychrophilic enzyme from Pseudoalteromonas haloplanktis TAH3a and the mesophilic enzyme from Bacillus halodurans C-125 had a positive effect on loaf volume. In contrast, the psychrophilic family 10 enzyme from Cryptococcus adeliae TAE85 was found to be ineffective. (c) 2005 Elsevier Ltd. All rights reserved. [less ▲]

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See detailA novel family 8 psychrophilic xylanase: fundamentals and applications.
Collins, Tony; Gerday, Charles ULg; Feller, Georges ULg

Poster (2006)

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See detailFundamentals and biotechnological aspects of cold-adapted enzymes
Gerday, Charles ULg; Collins, Tony; D'Amico, Salvino et al

Conference (2006)

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See detailCold-adapted xylanases: from Antarctica to the table.
Collins, Tony; Georis, Jacques; Dauvrin, Thierry et al

Poster (2006)

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See detailStudy of the active site residues of a glycoside hydrolase family 8 xylanase
Collins, T.; De Vos, D.; Hoyoux, A. et al

in Journal of Molecular Biology (2005), 354(2), 425-435

Site-directed mutagenesis and a comparative characterisation of the kinetic parameters, pH dependency of activity and thermal stability of mutant and wild-type enzymes have been used in association with ... [more ▼]

Site-directed mutagenesis and a comparative characterisation of the kinetic parameters, pH dependency of activity and thermal stability of mutant and wild-type enzymes have been used in association with crystallographic analysis to delineate the functions of several active site residues in a novel glycoside hydrolase family 8 xylanase. Each of the residues investigated plays an essential role in this enzyme: E78 as the general acid, D281 as the general base and in orientating the nucleophilic water molecule, Y203 in maintaining the position of the nucleophilic water molecule and in structural integrity and D144 in sugar ring distortion and transition state stabilization. Interestingly, although crystal structure analyses and the pH-activity profiles clearly identify the functions of E78 and D281, substitution of these residues with their amide derivatives results in only a 250-fold and 700-fold reduction in their apparent k(cat) values, respectively. This, in addition to the observation that the proposed general base is not conserved in all glycoside hydrolase family 8 enzymes, indicates that the mechanistic architecture in this family of inverting enzymes is more complex than is conventionally believed and points to a diversity in the identity of the mechanistically important residues as well as in the arrangement of the intricate microenvironment of the active site among members of this family. (c) 2005 Elsevier Ltd. All rights reserved. [less ▲]

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See detailThe active site is the least stable structure in the unfolding pathway of a multidomain cold-adapted alpha-amylase
Siddiqui, K. S.; Feller, Georges ULg; D'Amico, Salvino ULg et al

in Journal of Bacteriology (2005), 187(17), 6197-6205

The cold-active alpha-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis (AHA) is the largest known multidomain enzyme that displays reversible thermal unfolding (around 30 degrees C ... [more ▼]

The cold-active alpha-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis (AHA) is the largest known multidomain enzyme that displays reversible thermal unfolding (around 30 degrees C) according to a two-state mechanism. Transverse urea gradient gel electrophoresis (TUG-GE) from 0 to 6.64 M was performed under various conditions of temperature (3 degrees C to 70 degrees C) and pH (7.5 to 10.4) in the absence or presence of Ca2+ and/or Tris (competitive inhibitor) to identify possible low-stability domains. Contrary to previous observations by strict thermal unfolding, two transitions were found at low temperature (12 degrees C). Within the duration of the TUG-GE, the structures undergoing the first transition showed slow interconversions between different conformations. By comparing the properties of the native enzyme and the N12R mutant, the active site was shown to be part of the least stable structure in the enzyme. The stability data supported a model of cooperative unfolding of structures forming the active site and independent unfolding of the other more stable protein domains. In light of these findings for AHA, it will be valuable to determine if active-site instability is a general feature of heat-labile enzymes from psychrophiles. Interestingly, the enzyme was also found to refold and rapidly regain activity after being heated at 70 degrees C for 1 h in 6.5 M urea. The study has identified. fundamental new properties of AHA and extended our understanding of structure/stability relationships of cold-adapted enzymes. [less ▲]

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See detailRole of disulfide bridges in the activity and stability of a cold-active alpha-amylase
Siddiqui, K. S.; Poljak, A.; Guilhaus, M. et al

in Journal of Bacteriology (2005), 187(17), 6206-6212

The cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis unfolds reversibly and cooperatively according to a two-state mechanism at 30 degrees C and unfolds reversibly and sequentially with two ... [more ▼]

The cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis unfolds reversibly and cooperatively according to a two-state mechanism at 30 degrees C and unfolds reversibly and sequentially with two transitions at temperatures below 12 degrees C. To examine the role of the four disulfide bridges in activity and conformational stability of the enzyme, the eight cysteine residues were reduced with beta-mercaptoethanol or chemically modified using iodoacetamide or iodoacetic acid. Matrix-assisted laser desorption-time of flight mass spectrometry analysis confirmed that all of the cysteines were modified. The iodoacetamide-modified enzyme reversibly folded/unfolded and retained approximately one-third of its activity. Removal of all disulfide bonds resulted in stabilization of the least stable region of the enzyme (including the active site), with a concomitant decrease in activity (increase in activation enthalpy). Disulfide bond removal had a greater impact on enzyme activity than on stability (particularly the active-site region). The functional role of the disulfide bridges appears to be to prevent the active site from developing ionic interactions. Overall, the study demonstrated that none of the four disulfide bonds are important in stabilizing the native structure of enzyme, and instead, they appear to promote a localized destabilization to preserve activity. [less ▲]

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See detailStructure of a full length psychrophilic cellulase from Pseudoalteromonas haloplanktis revealed by X-ray diffraction and small angle X-ray scattering
Violot, S.; Aghajari, N.; Czjzek, M. et al

in Journal of Molecular Biology (2005), 348(5), 1211-1224

Pseudoalteromonas haloplanktis is a psychrophilic Gram-negative bacterium isolated in Antarctica, that lives on organic remains of algae. This bacterium converts the cellulose, highly constitutive of ... [more ▼]

Pseudoalteromonas haloplanktis is a psychrophilic Gram-negative bacterium isolated in Antarctica, that lives on organic remains of algae. This bacterium converts the cellulose, highly constitutive of algae, into an immediate nutritive form by biodegrading this biopolymer. To understand the mechanisms of cold adaptation of its enzymatic components, we studied the structural properties of an endoglucanase, Cel5G, by complementary methods, X-ray crystallography and small angle X-ray scattering. Using X-ray crystallography, we determined the structure of the catalytic core module of this family 5 endoglucanase, at 1.4 angstrom resolution in its native form and at 1.6 angstrom in the cellobiose-bound form. The catalytic module of Cel5G presents the (beta/alpha)(8)-barrel structure typical of clan GH-A of glycoside hydrolase families. The structural comparison of the catalytic core of Cel5G with the mesophilic catalytic core of Cel5A from Erwinia chrysanthemi revealed modifications at the atomic level leading to higher flexibility and thermolability, which might account for the higher activity of Cel5G at low temperatures. Using small angle X-ray scattering we further explored the structure at the entire enzyme level. We analyzed the dimensions, shape, and conformation of Cel5G full length in solution and especially of the linker between the catalytic module and the cellulose-binding module. The results showed that the linker is unstructured, and unusually long and flexible, a peculiarity that distinguishes it from its mesophilic counterpart. Loops formed at the base by disulfide bridges presumably add constraints to stabilize the most extended conformations. These results suggest that the linker plays a major role in cold adaptation of this psychrophilic enzyme, allowing steric optimization of substrate accessibility. (c) 2005 Elsevier Ltd. All rights reserved. [less ▲]

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See detailXylanases, xylanase families and extremophilic xylanases
Collins, T.; Gerday, Charles ULg; Feller, Georges ULg

in FEMS Microbiology Reviews (2005), 29(1), 3-23

Xylanases are hydrolytic enzymes which randomly cleave the beta 1,4 backbone of the complex plant cell wall polysaccharide xylan. Diverse forms of these enzymes exist, displaying varying folds, mechanisms ... [more ▼]

Xylanases are hydrolytic enzymes which randomly cleave the beta 1,4 backbone of the complex plant cell wall polysaccharide xylan. Diverse forms of these enzymes exist, displaying varying folds, mechanisms of action, substrate specificities, hydrolytic activities (yields, rates and products) and physicochemical characteristics. Research has mainly focused on only two of the xylanase containing glycoside hydrolase families, namely families 10 and 11, yet enzymes with xylanase activity belonging to families 5, 7, 8 and 43 have also been identified and studied, albeit to a lesser extent. Driven by industrial demands for enzymes that can operate under process conditions, a number of extremophilic xylanases have been isolated, in particular those from thermophiles, alkaliphiles and acidiphiles, while little attention has been paid to cold-adapted xylanases. Here, the diverse physicochemical and functional characteristics, as well as the folds and mechanisms of action of all six xylanase containing families will be discussed. The adaptation strategies of the extremophilic xylanases isolated to date and the potential industrial applications of these enzymes will also be presented. [less ▲]

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See detailActivity-stability relationships in extremophilic enzymes
D'Amico, Salvino; Collins, Tony; Georlette, Daphné et al

Poster (2005)

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