Purification and characterization of an extracellular alkaline cold-adapted serine metalo-protease from the cold tolerant bacterium, Stenotrophomonas sp. BTR88

444-450Proteases are the most economical enzymes in biotechnology and industry. Nowadays, a lot of attention is being paid to extremophiles microorganisms owing to the diversity of their enzymes. One hundred and Six proteolytic bacteria were isolated from Binaloud Mountain; one of them (strain BTR88) was selected as the best producer of extracellular protease and was used for further studies. This bacterium belongs to Stenotrophomonas sp., which were identified by the 16S rDNA sequence. Maximal protease production was detected at the beginning of exponential growth phase in the presence of starch and skim milk at 20°C and pH 9. This protease was purified to electrophoretic homogeneity with a fold: 27.5, yield: 33% and specific enzyme: 12.6 U/mg. SDS-PAGE and zymography analyses revealed a protein band of 22 kDa. The maximum activity was at pH 9 and in the range of 20-30°C; while, the enzyme exhibited a broad range of activity from 20-80°C as well as the pH of 5-10. Enzyme inhibition in the presence of phenylmethane sulfonyl fluoride (PMSF) and Ethylenediaminetetraacetic acid (EDTA) showed that the purified enzyme belongs to serine metallo-enzymes. Kinetic parameters of km, Vmax and kcat for the cold tolerant enzyme were determined to be 7.2 mg/mL, 1.45 mM/min and 33.2 sec−1, respectively. The characteristics of activity in cold and alkaline conditions and the broad range of pH and temperature suggest that serine metalloprotease has potential use in the detergent industry

Purification and characterization of an extracellular alkaline cold-adapted serine metalo-protease from the cold tolerant bacterium, Stenotrophomonas sp. BTR88

Proteases are the most economical enzymes in biotechnology and industry. Nowadays, a lot of attention is being paid to extremophiles microorganisms owing to the diversity of their enzymes. One hundred and Six proteolytic bacteria were isolated from Binaloud Mountain; one of them (strain BTR88) was selected as the best producer of extracellular protease and was used for further studies. This bacterium belongs to Stenotrophomonas sp., which were identified by the 16S rDNA sequence. Maximal protease production was detected at the beginning of exponential growth phase in the presence of starch and skim milk at 20°C and pH 9. This protease was purified to electrophoretic homogeneity with a fold: 27.5, yield: 33% and specific enzyme: 12.6 U/mg. SDS-PAGE and zymography analyses revealed a protein band of 22 kDa. The maximum activity was at pH 9 and in the range of 20-30°C; while, the enzyme exhibited a broad range of activity from 20-80°C as well as the pH of 5-10. Enzyme inhibition in the presence of phenylmethane sulfonyl fluoride (PMSF) and Ethylenediaminetetraacetic acid (EDTA) showed that the purified enzyme belongs to serine metallo-enzymes. Kinetic parameters of km, Vmax and kcat for the cold tolerant enzyme were determined to be 7.2 mg/mL, 1.45 mM/min and 33.2 sec−1, respectively. The characteristics of activity in cold and alkaline conditions and the broad range of pH and temperature suggest that serine metalloprotease has potential use in the detergent industry

Optimization of Protease Production by Psychrotrophic Rheinheimera sp. with Response Surface Methodology

Background and Objectives: Psychrotrophic bacteria can produce enzymes at low temperatures; this provides a wide biotechnological potential, and offers numerous economical advantages over the use of mesophilic bacteria. In this study, extracellular protease production by psychrotrophic Rheinheimera sp. (KM459533) was optimized by the response surface methodology.Materials and Methods: The culture medium was tryptic soy broth containing 1% (w v -1 ) skim milk. First, the effects of variables were independently evaluated on the microbial growth and protease production by one-factor-at-a-time method within the following ranges: incubation time 24-120 h, temperature 15-37°C, pH 6- 11, skim milk concentration 0-2% (w v -1 ), and inoculum size 0.5-3% (v v -1 ). The combinational effects of the four major variable including temperature, pH, skim milk concentration, and inoculum size were then evaluated within 96 h using response surface methodology through 27 experiments.Results and Conclusion: In one-factor-at-a-time method, high cell density was detected at 72h, 20°C, pH 7, skim milk 2% (w v -1 ), and inoculum size 3% (v v -1 ), and maximum enzyme production (533.74 Uml-1 ) was achieved at 96h, 20°C, pH 9, skim milk 1% (w v -1 ), and inoculum size 3% (v v -1 ). The response surface methodology study showed that pH is the most effective factor in enzyme production, and among the other variables, only temperature had significant interaction with pH and inoculum size. The determination coefficient (R2 =0.9544) and non-significant lack of fit demonstrated correlation between the experimental and predicted values. The optimal conditions predicted by the response surface methodology for protease production were defined as: 22C, pH 8.5, skim milk 1.1% (w v -1 ), and inoculum size 4% (v v -1 ). Protease production under these conditions reached to 567.19 Uml-1 . The use of response surface methodology in this study increased protease production by eight times as compared to the observed before optimization.Conflict of interests: The authors declare no conflict of interest

Communauté bactérienne de sols alpins et filtres environnementaux

Bacteria play a key role in biogeochemical cycles. While the effect of winter snow cover in function and composition of soil bacterial communities has been reported, the effect of spatio-temporal variation of snow cover remains to be studied. In this study, we characterised the spatio-temporal dynamics of bacterial communities from two sites at the extremes of a snow cover gradient. We used molecular (SSCP and cloning/sequencing) and traditional (bacterial isolation by culture) approaches. The presented results show that the overall bacterial diversity, composition and phylogenetic structure are strongly related to snow cover duration. Moreover, these effects are also detectable during the plant productive season. The biotic and abiotic factors (i.e. plant senescence and soil pH) play an essential role leading to the clustering of certain bacterial clades (Acidobacteria, Actinobacteria, α- and β-Proteobacteria). During the plant productive season, the bacterial clades are overdispersed. The preset study shows that, at a fine taxonomic level, the temporal variation is more important than the change over space. At higher taxonomic levels (i.e. sub-phylum), the space are more important than temporal variations. Only a minor fraction of the total bacterial diversity is cultivable, and may bacterial groups be overrepresented in culture plates. This study provides new insights in role of snow cover in bacterial communities' distribution and role of winter. This study may be useful in predicting of bacterial behaviour in nutrient cycle in a context of global warming.Les bactéries jouent un rôle clé dans les cycles biogéochimiques. Bien que l'effet du manteau neigeux en hiver dans la fonction et la composition des communautés bactériennes du sol ait été signalé, l'effet de la variation spatio-temporelle du manteau neigeux reste à étudier. Dans cette étude, nous avons caractérisé la dynamique spatio-temporelle des communautés bactériennes à partir de deux sites extrêmes selon un gradient de couvert neigeux. Pour cela, nous avons utilisé des approches moléculaires (SSCP et clonage / séquençage) et traditionnel (isolation par culture bactérienne). Les résultats présentés montrent que l'ensemble de la diversité bactérienne, sa composition et sa structure phylogénétique sont fortement liés à la durée de la couverture de neige. En outre, ces effets sont détectables au cours de la saison de végétation des plantes. Les facteurs biotiques et abiotiques (i.e. la sénescence des plantes et le pH du sol) jouent un rôle essentiel conduisant au regroupement de certaines bactéries en clades spécifiques (Acidobactéries, Actinobactéries, α-et β-Proteobactéries). Au cours de la saison de végétation des plantes, les clades de bactéries sont plus dispersés. La présente étude montre que, à un niveau taxonomique fin, la variation temporelle est un facteur plus important que la variation spatiale sur la diversité bactérienne. A un niveau taxonomique supérieur (i.e. sousphylum), la conclusion est inverse. Seule une petite fraction du total de la diversité bactérienne est cultivable et il se peut que certains groupes bactériens soient surreprésentés dans les plaques de culture. Cette étude apporte un nouvel éclairage sur le rôle de l'hiver et de la couverture neigeuse dans les distributions des communautés bactériennes. Cette étude peut-être utile pour prédire le comportement des bactéries dans les cycles des éléments nutritifs dans un contexte de réchauffement de la planète

Communautés bactériennes de sols alpins et filtres environnementaux

Les bactéri es jouent un rôle clé dans les cycles biogéochimiques. Bien que l'effet du manteau neigeux en hiver dans la fonction et la composition des communautés bactériennes du sol ait été signalé, l'effet de la variation spatio-temporelle du manteau neigeux reste à étudier. Dans cette étude, nous avons caractérisé la dynamique spatio-temporelle des communautés bactériennes à partir de deux sites extrêmes selon un gradient de couvert neigeux. Pour cela, nous avons utilisé des approches moléculaires (SSCP et clonage / séquençage) et traditionnel (isolation par culture bactérienne). Les résultats présentés montrent que l'ensemble de la diversité bactérienne, sa composition et sa structure phylogénétique sont fortement liés à la durée de la couverture de neige. En outre, ces effets sont détectables au cours de la saison de végétation des plantes. Les facteurs biotiques et abiotiques (i.e. la sénescence des plantes et le pH du sol) jouent un rôle essentiel conduisant au regroupement de certaines bactéries en clades spécifiques (Acidobactéries, Actinobactéries, a-et b-Proteobactéries). Au cours de la saison de végétation des plantes, les clades de bactéries sont plus dispersés. La présente étude montre que, à un niveau taxonomique fin, la variation temporelle est un facteur plus important que la variation spatiale sur la diversité bactérienne. A un niveau taxonomique supérieur (i.e. sousphylum), la conclusion est inverse. Seule une petite fraction du total de la diversité bactérienne est cultivable et il se peut que certains groupes bactériens soient surreprésentés dans les plaques de culture. Cette étude apporte un nouvel éclairage sur le rôle de l hiver et de la couverture neigeuse dans les distributions des communautés bactériennes. Cette étude peut-être utile pour prédire le comportement des bactéries dans les cycles des éléments nutritifs dans un contexte de réchauffement de la planète.Bacteria play a key role in biogeochemical cycles. While the effect of winter snow cover in function and composition of soil bacterial communities has been reported, the effect of spatiotemporal variation of snow cover remains to be studied. In this study, we characterised the spatio-temporal dynamics of bacterial communities from two sites at the extremes of a snow cover gradient. We used molecular (SSCP and cloning/sequencing) and traditional (bacterial isolation by culture) approaches. The presented results show that the overall bacterial diversity, composition and phylogenetic structure are strongly related to snow cover duration. Moreover, these effects are also detectable during the plant productive season. The biotic and abiotic factors (i.e. plant senescence and soil pH) play an essential role leading to the clustering of certain bacterial clades (Acidobacteria, Actinobacteria, a- and b-Proteobacteria). During the plant productive season, the bacterial clades are overdispersed. The preset study shows that, at a fine taxonomic level, the temporal variation is more important than the change over space. At higher taxonomic levels (i.e. sub-phylum), the space are more important than temporal variations. Only a minor fraction of the total bacterial diversity is cultivable, and may bacterial groups be overrepresented in culture plates. This study provides new insights in role of snow cover in bacterial communities distribution and role of winter. This study may be useful in predicting of bacterial behaviour in nutrient cycle in a context of global warming.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF