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See detailExploring the Antarctic soil metagenome as a source of novel cold-adapted enzymes and genetic mobile elements
Berlemont, Renaud ULg; Pipers; Delsaute, Maud ULg et al

in Revista Argentina de Microbiologia (2011)

Metagenomic library PP1 was obtained from Antarctic soil samples. Both functional and genotypic metagenomic screening were used for the isolation of novel cold-adapted enzymes with potential applications ... [more ▼]

Metagenomic library PP1 was obtained from Antarctic soil samples. Both functional and genotypic metagenomic screening were used for the isolation of novel cold-adapted enzymes with potential applications, and for the detection of genetic elements associated with gene mobilization, respectively. Fourteen lipase/esterase-, 14 amylase-, 3 protease-, and 11 cellulase-producing clones were detected by activity-driven screening, with apparent maximum activities around 35 °C for both amylolytic and lipolytic enzymes, and 35-55 °C for cellulases, as observed for other cold-adapted enzymes. However, the behavior of at least one of the studied cellulases is more compatible to that observed for mesophilic enzymes. These enzymes are usually still active at temperatures above 60 °C, probably resulting in a psychrotolerant behavior in Antarctic soils. Metagenomics allows to access novel genes encoding for enzymatic and biophysic properties from almost every environment with potential benefits for biotechnological and industrial applications. Only intI- and tnp-like genes were detected by PC R, encoding for proteins with 58-86%, and 58-73% amino acid identity with known entries, respectively. Two clones, BAC 27A-9 and BAC 14A-5, seem to present unique syntenic organizations, suggesting the occurrence of gene rearrangements that were probably due to evolutionary divergences within the genus or facilitated by the association with transposable elements. The evidence for genetic elements related to recruitment and mobilization of genes (transposons/integrons) in an extreme environment like Antarctica reinforces the hypothesis [less ▲]

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See detailStepwise adaptations to low temperature as revealed by multiple mutants of a psychrophilic alpha-amylase from an Antarctic bacterium
Cipolla, Alexandre ULg; D'Amico, Salvino ULg; Barumandzadeh, Roya et al

in Journal of Biological Chemistry (2011), 286(44), 3834838355

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See detailPsychrophilic enzymes: cool responses to chilly problems
Roulling, Frédéric ULg; Piette, Florence ULg; Cipolla, Alexandre ULg et al

in Horikoshi, K.; Antranikian, G.; Bull, A. (Eds.) et al Extremophiles Handbook (2011)

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See detailCold adaptation of proteins: a biophysical study of a psychrophilic alpha-amylase and its stabilized mutants
Cipolla, Alexandre ULg; D'Amico, Salvino ULg; Feller, Georges ULg

Poster (2010, September 28)

Habitats of permanently cold temperature, like polar regions for example, have been colonized by a great variety of psychrophilic organisms producing enzymes adapted to function efficiently in these cold ... [more ▼]

Habitats of permanently cold temperature, like polar regions for example, have been colonized by a great variety of psychrophilic organisms producing enzymes adapted to function efficiently in these cold environments. According to the hypothesis developed in our laboratory, the adaptation to cold temperature involves relationships between activity, flexibility and stability. Even if activity and stability are not physically linked in proteins 1, the consensus for the adaptive strategy is to take advantage of the lack of selective pressure for stable proteins to lose stability, therefore increasing the flexibility or mobility of the enzyme at low temperatures that restrict molecular motions. 2 Working on alpha-amylase, we have investigated the role of weak interactions in thermal adaptation of proteins by site-directed mutagenesis. We have built two multiple-mutants (Mut5 and Mut5CC) of the psychrophilc alpha-amylase (AHA) from the Antarctic bacterium, Pseudoalteromonas haloplanktis. The single mutations were selected by comparison of the presence of weak interactions in a mesophilic chloride-dependant homolog from pig pancreas, PPA. The study of selected single mutations prompt us to construct two multiple-mutants, Mut5 and Mut5CC, carrying 5 and 6 additional weak interactions found in PPA, that showed an increased stability and a lower activity at 25 °C.3 We have compared AHA, Mut5 and Mut5CC with additional methods like differential scanning calorimetry, thermal and chemical unfolding in order to determine the gain in stability. We also studied the flexibility or breathing of the enzymes by acrylamide-induced fluorescence quenching and we determined the optimum activity temperature for the three amylases. In order to investigate the kinetic origin of the gain in stability 4 for the two multiple-mutants, we studied in a first step the kinetic unfolding and refolding by GdmCl of the three amylases by manual methods following fluorescence signal at 15°C. The newly introduced weak interactions stabilized the proteins, protected them against heat and chemical unfolding and also induced an effective loss of flexibility. In addition, the two multiple-mutants exhibit a different optimum activity temperature than AHA. The first result in manual kinetic studies seems to show a similar refolding phase but a difference between the three amylases in the unfolding phase. This is in correlation with results of Dieter, P et al 4. These results and those of the previous work 3, unambiguously support the capital role of weak interactions in the balance between activity, flexibility and stability and provide a better knowledge of the adaptation of enzymes to cold temperatures. [less ▲]

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See detailProteomic study of cold-repressed proteins in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125
Piette, Florence; D'Amico, Salvino; Leprince, Pierre ULg et al

Poster (2010)

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See detailThermodynamic and Kinetic Contributions to Stability Adaptation in a Psychrophilic Alpha-amylase
Cipolla, Alexandre; D'Amico, Salvino; Feller, Georges ULg

Poster (2010)

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See detailProtein stability and enzyme activity at extreme biological temperatures
Feller, Georges ULg

in Journal of Physics : Condensed Matter (2010), 22

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See detailCoordination sphere of the third metal site is essential to the activity and metal selectivity of alkaline phosphatases
Koutsioulis, D.; Lyskowski, A.; Maki, S. et al

in Protein Science : A Publication of the Protein Society (2010), 19(1), 75-84

Alkaline phosphatases (APs) are commercially applied enzymes that catalyze the hydrolysis of phosphate monoesters by a reaction involving three active site metal ions. We have previously identified H135 ... [more ▼]

Alkaline phosphatases (APs) are commercially applied enzymes that catalyze the hydrolysis of phosphate monoesters by a reaction involving three active site metal ions. We have previously identified H135 as the key residue for controlling activity of the psychrophilic TAB5 AP (TAP). In this article, we describe three X-ray crystallographic structures on TAP variants H135E and H135D in complex with a variety of metal ions. The structural analysis is supported by thermodynamic and kinetic data. The AP catalysis essentially requires octahedral coordination in the M3 site, but stability is adjusted with the conformational freedom of the metal ion. Comparison with the mesophilic Escherichia coli, AP shows differences in the charge transfer network in providing the chemically optimal metal combination for catalysis. Our results provide explanation why the TAB5 and E. coli APs respond in an opposite way to mutagenesis in their active sites. They provide a lesson on chemical fine tuning and the importance of the second coordination sphere in defining metal specificity in enzymes. Understanding the framework of AP catalysis is essential in the efforts to design even more powerful tools for modern biotechnology. [less ▲]

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See detailBiotechnological applications of psychrophiles
Margesin, R.; Feller, Georges ULg

in Environmental Technology (2010), 31

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See detailTemperature Adaptation of Proteins: Stability, Folding and Flexibility in Mesophilic-like Engineered Alpha-Amylases
Cipolla, Alexandre ULg; D'Amico, Salvino ULg; Feller, Georges ULg

Poster (2009, July 02)

Habitats of permanently cold temperature, like polar regions for example, have been colonized by a great variety of psychrophilic organisms producing enzymes adapted to function efficiently in these cold ... [more ▼]

Habitats of permanently cold temperature, like polar regions for example, have been colonized by a great variety of psychrophilic organisms producing enzymes adapted to function efficiently in these cold environments. According to the hypothesis developed in our laboratory, the adaptation to cold temperature involves relationships between activity, flexibility and stability. Even if activity and stability are not physically linked in proteins 1, the consensus for the adaptive strategy is to take advantage of the lack of selective pressure for stable proteins to lose stability, therefore increasing the flexibility or mobility of the enzyme at low temperatures that restrict molecular motions. 2 Working on alpha-amylase, we have investigated the role of weak interactions in thermal adaptation of proteins by site-directed mutagenesis. We have built two multiple-mutants (Mut5 and Mut5CC) of the psychrophilc alpha-amylase (AHA) from the Antarctic bacterium, Pseudoalteromonas haloplanktis. The single mutations were selected by comparison of the presence of weak interactions in a mesophilic chloride-dependant homolog from pig pancreas, PPA. The study of selected single mutations prompt us to construct two multiple-mutants, Mut5 and Mut5CC, carrying 5 and 6 additional weak interactions found in PPA, that showed an increased stability and a lower activity at 25 °C.3 We have compared AHA, Mut5 and Mut5CC with additional methods like differential scanning calorimetry, thermal and chemical unfolding and circular dichroism in order to determine the gain in stability. We also studied the flexibility or breathing of the enzymes by acrylamide-induced fluorescence quenching. The newly introduced weak interactions stabilized the proteins, protected them against heat and chemical unfolding and also induced an effective loss of flexibility. These results and those of the previous work 3, unambiguously support the capital role of weak interactions in the balance between activity, flexibility and stability and provide a better knowledge of the adaptation of enzymes to cold temperatures. [less ▲]

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See detailStability-activity relationships in natively unstable proteins
Feller, Georges ULg

Conference (2009)

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See detailInsights into bacterial cellulose biosynthesis by functional metagenomics on Antarctic soil samples.
Berlemont, Renaud ULg; Delsaute, Maud ULg; Pipers, Delphine ULg et al

in ISME Journal (The) (2009), 3(9), 1070-1081

In this study, the mining of an Antarctic soil sample by functional metagenomics allowed the isolation of a cold-adapted protein (RBcel1) that hydrolyzes only carboxymethyl cellulose. The new enzyme is ... [more ▼]

In this study, the mining of an Antarctic soil sample by functional metagenomics allowed the isolation of a cold-adapted protein (RBcel1) that hydrolyzes only carboxymethyl cellulose. The new enzyme is related to family 5 of the glycosyl hydrolase (GH5) protein from Pseudomonas stutzeri (Pst_2494) and does not possess a carbohydrate-binding domain. The protein was produced and purified to homogeneity. RBcel1 displayed an endoglucanase activity, producing cellobiose and cellotriose, using carboxymethyl cellulose as a substrate. Moreover, the study of pH and the thermal dependence of the hydrolytic activity shows that RBcel1 was active from pH 6 to pH 9 and remained significantly active when temperature decreased (18% of activity at 10 degrees C). It is interesting that RBcel1 was able to synthetize non-reticulated cellulose using cellobiose as a substrate. Moreover, by a combination of bioinformatics and enzyme analysis, the physiological relevance of the RBcel1 protein and its mesophilic homologous Pst_2494 protein from P. stutzeri, A1501, was established as the key enzymes involved in the production of cellulose by bacteria. In addition, RBcel1 and Pst_2494 are the two primary enzymes belonging to the GH5 family involved in this process.The ISME Journal advance online publication, 21 May 2009; doi:10.1038/ismej.2009.48. [less ▲]

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See detailA nondetergent sulfobetaine improves protein unfolding reversibility in microcalorimetric studies
D'Amico, Salvino ULg; Feller, Georges ULg

in Analytical Biochemistry (2009), 385(2), 389-91

A nondetergent sulfobetaine (NDSB) was found to improve unfolding reversibility of several proteins by inhibiting heat-induced aggregation. As a consequence, DeltaH(cal)/DeltaH(vH) ratios were also ... [more ▼]

A nondetergent sulfobetaine (NDSB) was found to improve unfolding reversibility of several proteins by inhibiting heat-induced aggregation. As a consequence, DeltaH(cal)/DeltaH(vH) ratios were also improved to values close to 1 for a two-state unfolding. NDSB is effective in a wide range of pH values and especially at acidic pH generally used to calculate DeltaC(p) values by the Kirchhoff relation. The sulfobetaine also allows recording protein refolding by protecting the heat-induced unfolded state against aggregation. [less ▲]

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See detailSelection of a cold-adapted bacterium for bioremediation of wastewater at low temperatures
Gratia, E.; Weekers, F.; Margesin, R. et al

in Extremophiles : Life Under Extreme Conditions (2009), 13(5), 763-8

Amongst more than 1000 isolates collected in various cold environments, the strain Arthrobacter psychrolactophilus Sp 31.3 has been selected for its ability to grow and to produce exoenzymes at low ... [more ▼]

Amongst more than 1000 isolates collected in various cold environments, the strain Arthrobacter psychrolactophilus Sp 31.3 has been selected for its ability to grow and to produce exoenzymes at low temperatures, its inability to grow at 37 degrees C, its non-halophilic character and its growth versatility on various media. This non-pathogenic strain displays a strong resistance to desiccation and storage at room temperature and is suitable for the production of freeze-dried bacterial starters. When grown in a synthetic wastewater at 10 degrees C, the strain induces a complete clarification of the turbid medium and efficiently hydrolyses proteins, starch and lipids in the broth. Furthermore, this strain has a remarkable capacity to improve the biodegradability of organic compounds in wastewater as indicated by a BOD(5)/COD ratio of 0.7. [less ▲]

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