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See detailCold adaptation of a phosphoglycerate kinase affects the stability of only one domain
Zecchinon, Laurent; Bentahir, M.; Feller, Georges ULg et al

Poster (2000)

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See detailCalorimetric characterization of mutants from a cold-active alpha-amylase
D'Amico, Salvino; Gerday, Charles ULg; Feller, Georges ULg

Poster (2000)

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See detailPurification and characterization of a 43.5 kDa keratinase from Microsporum canis
Brouta, F.; Descamps, F.; Fett, Thomas ULg et al

Conference (2000)

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See detailPurification and Characterization of the Heat-Labile Alpha-Amylase Secreted by the Psychrophilic Bacterium Tac 240b
Chessa, Jean-Pierre; Feller, Georges ULg; Gerday, Charles ULg

in Canadian Journal of Microbiology (1999), 45(6), 452-7

A total of 59 bacteria samples from Antarctic sea water were collected and screened for their ability to produce alpha-amylase. The highest activity was recorded from an isolate identified as an ... [more ▼]

A total of 59 bacteria samples from Antarctic sea water were collected and screened for their ability to produce alpha-amylase. The highest activity was recorded from an isolate identified as an Alteromonas species. The purified alpha-amylase shows a molecular mass of about 50,000 Da and a pI of 5.2. The enzyme is stable from pH 7.5 to 9 and has a maximal activity at pH 7.5. Compared with other alpha-amylases from mesophiles and thermophiles, the "cold enzyme" displays a higher activity at low temperature and a lower stability at high temperature. The psychrophilic alpha-amylase requires both Cl- and Ca2+ for its amylolytic activity. Br- is also quite efficient as an allosteric effector. The comparison of the amino acid composition with those of other alpha-amylases from various organisms shows that the cold alpha-amylase has the lowest content in Arg and Pro residues. This could be involved in the principle used by the psychrophilic enzyme to adapt its molecular structure to the low temperature of the environment. [less ▲]

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See detailThermodynamic Stability of a Cold-Active Alpha-Amylase from the Antarctic Bacterium Alteromonas Haloplanctis
Feller, Georges ULg; d'Amico, D.; Gerday, Charles ULg

in Biochemistry (1999), 38(14), 4613-9

The thermal stability of the cold-active alpha-amylase (AHA) secreted by the Antarctic bacterium Alteromonas haloplanctis has been investigated by intrinsic fluorescence, circular dichroism, and ... [more ▼]

The thermal stability of the cold-active alpha-amylase (AHA) secreted by the Antarctic bacterium Alteromonas haloplanctis has been investigated by intrinsic fluorescence, circular dichroism, and differential scanning calorimetry. It was found that this heat-labile enzyme is the largest known multidomain protein exhibiting a reversible two-state unfolding, as demonstrated by the recovery of DeltaHcal values after consecutive calorimetric transitions, a DeltaHcal/DeltaHeff ratio close to unity, and the independence of unfolding thermodynamic parameters of scan rates. By contrast, the mesophilic alpha-amylases investigated here (from porcine pancreas, human salivary glands, yellow meal beetle, Bacillus amyloliquefaciens, and Bacillus licheniformis) unfold irreversibly according to a non-two-state mechanism. Unlike mesophilic alpha-amylases, the melting point of AHA is independent of calcium and chloride binding while the allosteric and structural functions of these ions are conserved. The thermostability of AHA at optimal conditions is characterized by a Tm of 43.7 degrees C, a DeltaHcal of 238 kcal mol-1, and a DeltaCp of 8.47 kcal mol-1 K-1. These values were used to calculate the Gibbs free energy of unfolding over a wide range of temperatures. This stability curve shows that (a) the specific DeltaGmax of AHA [22 cal (mol of residue)-1] is 4 times lower than that of mesophilic alpha-amylases, (b) group hydration plays a crucial role in the enzyme flexibility at low temperatures, (c) the temperature of cold unfolding closely corresponds to the lower limit of bacterial growth, and (d) the recombinant heat-labile enzyme can be expressed in mesophilic hosts at moderate temperatures. It is also argued that the cold-active alpha-amylase has evolved toward the lowest possible conformational stability of its native state. [less ▲]

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See detailPsychrophilic organisms and their enzymes as new tools in biotechnology
Gerday, Charles ULg; Chessa, Jean Pierre; Feller, Georges ULg

Conference (1999)

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See detailThermodynamic stability of a cold-active alpha-amylase
Feller, Georges ULg; D'Amico, Salvino; Gerday, Charles ULg

Conference (1999)

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See detailEngineering the stability of a cold-active alpha-amylase
D'Amico, Salvino; Gerday, Charles ULg; Feller, Georges ULg

Poster (1999)

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See detailModification of the Thermoresistance to Spray-Drying of a Cold-Adapted Subtilisin by Genetic Engineering
Bare, G.; Diakiese, A.; Zgoulli, S. et al

in Applied Biochemistry and Biotechnology (1999), 77-79(Spring), 857-65

The thermoresistance of a cold-adapted subtilisin dried by spray-drying was studied. Proteolytic activity of this enzyme was measured before and after spray-drying. Without chemical additives, spray ... [more ▼]

The thermoresistance of a cold-adapted subtilisin dried by spray-drying was studied. Proteolytic activity of this enzyme was measured before and after spray-drying. Without chemical additives, spray-drying yields ranged from 2-13%. The use of arabic gum and lactose in the composition of the enzyme solutions allowed the strengthening of the enzyme structures and increased water mobility in the product. Increase of water mobility led to a shorter residence time of the product in the spray-drier and a net yield increase was obtained (yield higher than 50%). The effect of two selective mutations on the thermoresistance to spray-drying of the cold-adapted subtilisin was also investigated. Mutation T85D (introduction of an additional link with an ion Ca2+ necessary for enzyme activity, by substitution of Asp for Thr 85) had no effect on the thermoresistance of the subtilisin to spray-drying. Mutation H121W (introduction of an additional aromatic link by substitution of Trp for His 121) reduced the drying yield from 66% (not modified subtilisin) to 52%. This higher thermosensitivity could be explained by an increase of the hygroscopic character of the modified subtilisin (mutation H121W). [less ▲]

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See detailCold enzymes : a hot topic
Gerday, Charles ULg; Aittaleb, M.; Arpigny, J. L. et al

in Margesin, R.; Schinner, F. (Eds.) Cold-adapted Organisms : Ecology, Physiology, Enzymology and Molecular Biology (1999)

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See detailPurification and Characterization of a 315 Kda Keratinolytic Subtilisin-Like Serine Protease from Microsporum Canis and Evidence of Its Secretion in Naturally Infected Cats
Mignon, Bernard ULg; Swinnen, M.; Bouchara, J. P. et al

in Medical Mycology (1998), 36(6), 395-404

A keratinolytic protease, secreted as the major component by a feline clinical isolate of Microsporum canis cultivated in a minimal medium containing cat keratin, was purified by affinity chromatography ... [more ▼]

A keratinolytic protease, secreted as the major component by a feline clinical isolate of Microsporum canis cultivated in a minimal medium containing cat keratin, was purified by affinity chromatography on bacitracin agarose and gel filtration. The apparent molecular mass of the enzyme was 31.5 kDa and the pI was 11.8. The enzyme was not glycosylated and its first 15 N-terminal amino acids showed numerous similarities with other fungal subtilisins. The optimum pH was around 9 while inactivation of the enzyme was reversible at pH 4, but not at pH 11. The enzyme was stable at 37 degrees C with an apparent optimum temperature around 55 degrees C. PMSF, soybean trypsin inhibitor (SBTI) and chymostatin strongly inhibited the proteinase. The highest affinity (Km of 0.37 mM) and physiological efficiency (k(cat)/Km) were obtained for the synthetic substrate N-Suc-Ala-Ala-Pro-Phe-p-nitroanilide. These results indicate that the keratinase belongs to the subtilisin-like serine protease family. Purified rabbit immunoglobulins G prepared against the keratinase and used in an immunohistochemical test allowed the detection of the keratinase produced by the fungus invading hair structures in naturally infected cats. The in vitro keratinolytic activity of the enzyme and its production in vivo suggest that it may contribute to pathogenicity. [less ▲]

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See detailCharacterization of the C-Terminal Propeptide Involved in Bacterial Wall Spanning of Alpha-Amylase from the Psychrophile Alteromonas Haloplanctis
Feller, Georges ULg; D'Amico, Salvino ULg; Benotmane, A. M. et al

in Journal of Biological Chemistry (1998), 273(20), 12109-15

The antarctic psychrophile Alteromonas haloplanctis secretes a Ca2+- and Cl--dependent alpha-amylase. The nucleotide sequence of the amy gene and the amino acid sequences of the gene products indicate ... [more ▼]

The antarctic psychrophile Alteromonas haloplanctis secretes a Ca2+- and Cl--dependent alpha-amylase. The nucleotide sequence of the amy gene and the amino acid sequences of the gene products indicate that the alpha-amylase precursor is a preproenzyme composed by the signal peptide (24 residues), the mature alpha-amylase (453 residues, 49 kDa), and a long C-terminal propeptide or secretion helper (192 residues, 21 kDa). In cultures of the wild-type strain, the 70-kDa precursor is secreted at the mid-exponential phase and is cleaved by a nonspecific protease into the mature enzyme and the propeptide. The purified C-terminal propeptide displays several features common to beta-pleated transmembrane proteins. It has no intramolecular chaperone function because active alpha-amylase is expressed by Escherichia coli in the absence of the propeptide coding region. In E. coli, the 70-kDa precursor is directed toward the supernatant. When the alpha-amylase coding region is excised from the gene, the secretion helper can still promote its own membrane spanning. It can also accept a foreign passenger, as shown by the extracellular routing of a beta-lactamase-propeptide fusion protein. [less ▲]

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See detailModification of the thermoresistance to spray-drying of a cold-adapted subtilisin by genetic engineering
Bare, G.; Diakiese, A.; Zgoulli, S. et al

Poster (1998, May 08)

Detailed reference viewed: 18 (2 ULg)