Activités amylase et lichenase d'une nouvelle souche de Bacillus. Production sur milieu solide et caractérisation.

L objectif de cette thèse était d isoler de nouvelles glycoside-hydrolases à partir d une souche de Bacillus issue d un Biotope sud-tunisien. Cette souche a montré des potentialités à produire une amylase et une lichenase à 45C et à pH 9. La production de ces deux hydrolases a été optimisée en fermentation solide sur millet, une agro-ressource de faible coût. Cette optimisation a été conduite en adoptant la méthodologie des plans d expériences. Nous avons ainsi obtenu des niveaux de production de l ordre de 540 Unités d activités amylase par gramme de substrat solide et 503 U/g d activité lichenase. Ces deux protéines ont été par la suite purifiées et caractérisées biochimiquement. L amylase présente un pH et une température d activité optimaux de 5 et 70C, respectivement. La lichenase a montré une thermoactivité et une thermostabilité remarquables qui la distinguent des lichenases précédemment décrites. En effet, l enzyme conserve plus de 20% de son activité à 100C, et plus de 60% de son activité après une incubation de 30 min à 90C. Le gène codant pour cette protéine a été isolé par la construction d une banque fosmidique dans E. coli. La comparaison de sa séquence avec la banque de données NCBI a montré que le gène de la lichenase UEB-S possède une très forte homologie avec celle de Bacillus subtilis 168, avec les positions de deux acides aminés seulement qui divergent. Un modèle de la lichenase construit au cours de cette étude laisse supposer que l un de ces deux acides aminés (Val 69) pourrait être impliqué dans sa thermostabilité, et ce en modifiant la géométrie du site de fixation au calciumThe aim of this thesis was to isolate new glycoside hydrolases from a Bacillus strain isolated from a Biotope in the south of Tunisia. This strain was able to produce a lichenase and an amylase at 45 C and pH 9. The production of these two hydrolases was optimized in solid state fermentaion using millet, a low cost. agro-resource as solid substrate. This optimization was carried out using response surface methodology (RSM) based on Doehlert design. We obtained production levels of around 540 units of amylase activity per gram of solid substrate and 503 U / g of lichenase activity.Both proteins were subsequently purified and characterized biochemically. The amylase has a pH and a temperature optimum of activity of 5 and 70 C, respectively. The lichenase showed a remarkable thermostability which distinguish it from described lichenases. Indeed, the enzyme retained more than 20% of its activity at 100 C, and more than 60% of its activity after incubation for 30 min at 90 C. The gene encoding this protein was isolated by the construction of genomic a library in E. coli. Comparison of its sequence with the NCBI database showed that the gene coding for UEB-S lichenase has a very high homology with that of Bacillus subtilis 168, with a difference in the position of only two amino acids A model for UEB-S lichenase built during this study suggests that one of these two amino acids (Val 69) could be involved in its thermostability probabely by changing the geometry of the calcium binding siteTOULOUSE-INSA-Bib. electronique (315559905) / SudocSudocFranceF

Investigation of Antimicrobial Activity and Statistical Optimization of Bacillus subtilis SPB1 Biosurfactant Production in Solid-State Fermentation

During the last years, several applications of biosurfactants with medical purposes have been reported. Biosurfactants are considered relevant molecules for applications in combating many diseases. However, their use is currently extremely limited due to their high cost in relation to that of chemical surfactants. Use of inexpensive substrates can drastically decrease its production cost. Here, twelve solid substrates were screened for the production of Bacillus subtilis SPB1 biosurfactant and the maximum yield was found with millet. A Plackett-Burman design was then used to evaluate the effects of five variables (temperature, moisture, initial pH, inoculum age, and inoculum size). Statistical analyses showed that temperature, inoculum age, and moisture content had significantly positive effect on SPB1 biosurfactant production. Their values were further optimized using a central composite design and a response surface methodology. The optimal conditions of temperature, inoculum age, and moisture content obtained under the conditions of study were 37°C, 14 h, and 88%, respectively. The evaluation of the antimicrobial activity of this compound was carried out against 11 bacteria and 8 fungi. The results demonstrated that this biosurfactant exhibited an important antimicrobial activity against microorganisms with multidrug-resistant profiles. Its activity was very effective against Staphylococcus aureus, Staphylococcus xylosus, Enterococcus faecalis, Klebsiella pneumonia, and so forth

Investigation of Antimicrobial Activity and Statistical Optimization of Bacillus subtilis SPB1 Biosurfactant Production in Solid-State Fermentation

During the last years, several applications of biosurfactants with medical purposes have been reported. Biosurfactants are considered relevant molecules for applications in combating many diseases. However, their use is currently extremely limited due to their high cost in relation to that of chemical surfactants. Use of inexpensive substrates can drastically decrease its production cost. Here, twelve solid substrates were screened for the production of Bacillus subtilis SPB1 biosurfactant and the maximum yield was found with millet. A Plackett-Burman design was then used to evaluate the effects of five variables (temperature, moisture, initial pH, inoculum age, and inoculum size). Statistical analyses showed that temperature, inoculum age, and moisture content had significantly positive effect on SPB1 biosurfactant production. Their values were further optimized using a central composite design and a response surface methodology. The optimal conditions of temperature, inoculum age, and moisture content obtained under the conditions of study were 37 • C, 14 h, and 88%, respectively. The evaluation of the antimicrobial activity of this compound was carried out against 11 bacteria and 8 fungi. The results demonstrated that this biosurfactant exhibited an important antimicrobial activity against microorganisms with multidrug-resistant profiles. Its activity was very effective against Staphylococcus aureus, Staphylococcus xylosus, Enterococcus faecalis, Klebsiella pneumonia, and so forth

Amylase and lichenase activities from a new strain of Bacillus production in solid medium and characterization

L’objectif de cette thèse était d’isoler de nouvelles glycoside-hydrolases à partir d’une souche de Bacillus issue d’un Biotope sud-tunisien. Cette souche a montré des potentialités à produire une amylase et une lichenase à 45°C et à pH 9. La production de ces deux hydrolases a été optimisée en fermentation solide sur millet, une agro-ressource de faible coût. Cette optimisation a été conduite en adoptant la méthodologie des plans d’expériences. Nous avons ainsi obtenu des niveaux de production de l’ordre de 540 Unités d’activités amylase par gramme de substrat solide et 503 U/g d’activité lichenase. Ces deux protéines ont été par la suite purifiées et caractérisées biochimiquement. L’amylase présente un pH et une température d’activité optimaux de 5 et 70°C, respectivement. La lichenase a montré une thermoactivité et une thermostabilité remarquables qui la distinguent des lichenases précédemment décrites. En effet, l’enzyme conserve plus de 20% de son activité à 100°C, et plus de 60% de son activité après une incubation de 30 min à 90°C. Le gène codant pour cette protéine a été isolé par la construction d’une banque fosmidique dans E. coli. La comparaison de sa séquence avec la banque de données NCBI a montré que le gène de la lichenase UEB-S possède une très forte homologie avec celle de Bacillus subtilis 168, avec les positions de deux acides aminés seulement qui divergent. Un modèle de la lichenase construit au cours de cette étude laisse supposer que l’un de ces deux acides aminés (Val 69) pourrait être impliqué dans sa thermostabilité, et ce en modifiant la géométrie du site de fixation au calciumThe aim of this thesis was to isolate new glycoside hydrolases from a Bacillus strain isolated from a Biotope in the south of Tunisia. This strain was able to produce a lichenase and an amylase at 45 ° C and pH 9. The production of these two hydrolases was optimized in solid state fermentaion using millet, a low cost. agro-resource as solid substrate. This optimization was carried out using response surface methodology (RSM) based on Doehlert design. We obtained production levels of around 540 units of amylase activity per gram of solid substrate and 503 U / g of lichenase activity.Both proteins were subsequently purified and characterized biochemically. The amylase has a pH and a temperature optimum of activity of 5 and 70 ° C, respectively. The lichenase showed a remarkable thermostability which distinguish it from described lichenases. Indeed, the enzyme retained more than 20% of its activity at 100 ° C, and more than 60% of its activity after incubation for 30 min at 90 ° C. The gene encoding this protein was isolated by the construction of genomic a library in E. coli. Comparison of its sequence with the NCBI database showed that the gene coding for UEB-S lichenase has a very high homology with that of Bacillus subtilis 168, with a difference in the position of only two amino acids A model for UEB-S lichenase built during this study suggests that one of these two amino acids (Val 69) could be involved in its thermostability probabely by changing the geometry of the calcium binding sit

Filamentous fungi isolated from Tunisian olive mill wastes: use of solid-state fermentation for enzyme production

International audienceOlive mill wastewaters and olive cake are effluents generated during olive oil production process. They represent a major disposal and potentially severe pollution problem for the industry, also promising source of substances of high value. The aim of this study is the valorization of olive mill wastes (OMWW, olive cake, olive twigs and leaves) to produce enzymes with high industrial and biotechnological potential, by the solidstate fermentation technique (SSF), from isolated fungi present in olive mill wastewater and olive cake. A total of 47 strains were isolated and purified from these two residues. The metabolic potential of isolated strains was study by testing the hydrolytic enzymes activities of lipase, protease, amylase, cellulase, invertase, phytase and tannase on agar plate media containing different substrate. The monitoring of SSF has shown that the metabolic activity of these strains is extremely rapid using this technique. Our fungi collection contains a diversity of strains capable to producing a variety of enzymes of biotechnological interest

A highly thermostable lichenase from <em>Bacillus</em> sp UEB-S: biochemical and molecular characterization

International audienceA highly thermostable and alkaline lichenase was isolated from the newly isolated strain Bacillus UEB-S. Single step purification was achieved by heating the enzyme extract for 30 min at 90 degrees C. The enzyme was a monomeric protein with a molecular weight of 28 kDa. The optimal temperature and pH for UEB-S lichenases activity were 60 degrees C and 6.0, respectively. More remarkably, the purified lichenase was stable over a broad range of temperature and pH. It retained more than 60% of its activity after incubation at 90 degrees C for 30min. Substrate specificity studies revealed that the enzyme is a true lichenase. A genomic library was screened. It allows the identification of a gene that encodes a putative lichenase showing 98% identity with the lichenase from Bacillus subtilis 168. Sequence comparison revealed that the two enzymes differed by two mutations at positions 69 and 83, where Va169 and Ser83 are replaced by Met and Ala amino acids, respectively. Therefore, a theoretical structural model was built using the lichenase from B. subtilis 168 Pdb code (3 o5sA) structure as template. Comparison of the two 3D structures suggested that Va169 stabilizes a calcium-binding site and could be involved in the higher stability of the enzyme

Carbon and nitrogen balances and CO2 emission after exogenous organic matter application in arid soil

Addition of organic amendments (OAs) could be a means to sequester carbon (C) in soils. However, the efficiency of C sequestration depends on how OAs evolve in the soil. A field study was installed in arid soil to which was added one of five OAs – olive husk-based compost (CM), palm leaf-based compost (CP), crushed olive pruning (GW), fresh olive mill wastewater (OMW) or fermented ovine manure (OM) – at an equivalent fixed level of 350 g of C/m2. C and N mineralization were followed for 112 days by measuring the evolution of released CO2 and mineral N evolution. The results showed that CM and CP did not disturb soil respiration, with a very low CO2 emission or almost no respiration for the CM, while CO2 release reached 7.6 g of CO2/m2 for GW and OMW. Soil organic carbon (SOC) was sustainably improved by 0.54% and 0.50%, respectively, for CM and CP. N mineralization showed no significant difference between amended and untreated soils. Based on these results, compost amendment was the most efficient for C sequestration to enhance soil fertility and consequently reduce the rate of CO2 emission