The Cyclodextrin Glycosyltransferase of Paenibacillus pabuli US132 Strain: Molecular Characterization and Overproduction of the Recombinant Enzyme

The gene encoding the cyclodextrin glycosyltransferase (CGTase) of Paenibacillus pabuli US132, previously described as efficient antistaling agent and good candidate for cyclodextrins production, was cloned, sequenced, and expressed in Escherichia coli. Sequence analysis showed that the mature enzyme (684 amino acids) was preceded by a signal peptide of 34 residues. The enzyme exhibited the highest identity (94%) to the β-CGTase of Bacillus circulans no. 8. The production of the recombinant CGTase, as active form, was very low (about 1 U/mL) in shake flasks at 37°C. This production reached 22 U/mL after 22 hours of induction by mainly shifting the postinduction temperature from 37 to 19°C and using 2TY instead of LB medium. High enzyme production (35 U/mL) was attained after 18 hours of induction in fermentor using the same culture conditions as in shake flask. The recombinant enzyme showed Vmax and Km values of 253 ± 36 μmol of β-cyclodextrin/mg/min and 0.36 ± 0.18 g/L, respectively

Une porphyrine comme photosensibilisant des eaux d'irrigation, photostabilité et efficacité

Le processus de photosensibilisation (phototraitement) de l'eau suscite beaucoup d'intérêt, car il implique trois composants qui sont individuellement inoffensifs pour le milieu biologique, à savoir le photosensibilisant, la lumière et l'oxygène moléculaire. La photostabilité de la porphyrine tétra-méso-cationique (T4MPyP) dans l'eau usée et l’efficacité du phototraitement dépend de la concentration du photosensibilisant, de la qualité de l'eau (contaminants organiques, turbidité, pH, taux d'oxygène dissous et épaisseur de la lame d'eau) ainsi que de l'intensité et de la nature du rayonnement lumineux. L’étude expérimentale consistait à apporter à un sol sableux i) de l’eau usée secondaire traitée par boues activées, ii) la même eau usée, mais phototraitée avec 5 μM∙L-1 de T4MPyP pendant 6 h d’ensoleillement, et iii) une eau de puits. L’eau usée phototraitée était conforme aux normes tunisiennes (NT 106.03) pour l’irrigation sans restriction au niveau du taux de bactéries indicatrices de pollution fécale (l’abattement des coliformes fécaux avoisinait 99,99 %), mais renfermait encore une concentration résiduelle de porphyrine (environ 35 % de la concentration initiale). À la fin de l’expérimentation, la configuration saline du sol dépendait de la qualité des eaux d’irrigation. Dans le cas des eaux usées secondaires, la couche de surface (0-5 cm) se caractérisait par une concentration élevée de sels solubles et par une prolifération d’algues contribuant ainsi au colmatage superficiel et à un manque de continuité des macropores. En revanche, le colmatage chimique et biologique était réduit suite à l’utilisation des eaux usées phototraitées. La salinité était plus importante au niveau de la couche profonde (5-15 cm) due à une importante mobilité des ions chlorures et sodium suivie des sulfates et du calcium de la couche 0-5 cm vers la couche 5-15 cm, ce qui peut être attribué à une oxydation plus élevée de la matière organique induite par la présence de porphyrine. Il ressort de cette étude que la valorisation des eaux usées avec des concentrations micromolaires de T4MPyP dans le domaine agricole inhiberait le développement d'algues à la surface du sol et conduirait à une meilleure infiltration des sels vers les profondeurs évitant ainsi l’installation du colmatage à la surface. En outre, une meilleure rétention des ions ammonium et orthophosphates a été observée dans le sol sableux lors de la percolation des eaux usées phototraiteées.The process of photosensitization (phototreatment) of water is gaining much interest as it involves three components that are individually harmless to the biological environment, namely, the photosensitizer, light, and molecular oxygen. Laboratory experiments on study factors affecting photostability of T4MPyP (meso-tetra [4-N-methylpyridyl] porphyrin) in water show that wavelength and intensity of incident light, concentration of photosensitizer and water quality (presence of organics contaminants, turbidity, pH, dissolved oxygen, and thickness of the water sheet) all affects the rate of photostability and phototreatment. The experimental study consisted in bringing i) secondary treated (by activated sludge) wastewater, ii) the same type of wastewater but photosensitized with 5 μM∙L-1 of T4MPyP during 6h of sunshine, and iii) well water to a sandy soil. Phototreated wastewater met Tunisian standards (NT 106.03) indicating that the porphyrin was efficient in removing indicator bacteria (fecal coliforms removal averaged 99.99%) but still contained a residual concentration of porphyrin (about 35% of the initial concentration). At the end of the experiment, the saline configuration of the soil depended on the irrigation water quality. In the case of the secondary wastewater, the surface layer (0-5 cm) was characterized by high concentration of soluble salts and by proliferation of some algae which resulted in surface clogging and lack of macropore continuity. In contrast, biological, chemical and physical clogging processes were reduced by using phototreated wastewater with a residual photosensitizer. The absence of algae in the surface layer of soil and the decrease of nutrients (nitrogen and phosphorus) and bacteria could reduce clogging of irrigated water. The higher salinity was observed in the deep layer (5-15 cm) due to a high mobility of the sodium and chloride ions followed by sulfates and calcium from the layer 0-5 cm to the layer 5-15 cm which can be attributed to the higher oxidation of organic matter induced by the presence of porphyrin. The results revealed that using wastewater with micromolar concentrations of T4MPyP in agriculture would inhibit the development of algae at the soil surface and lead to a better infiltration of salts in depths, which would prevent surface clogging. In addition, better retention of ammonium and orthophosphate ions was observed in the sandy soil during percolation of phototreated wastewater

<span style="font-size:15.0pt;font-family: "Times New Roman";mso-fareast-font-family:"Times New Roman";mso-bidi-font-family: Mangal;mso-ansi-language:EN-GB;mso-fareast-language:EN-US;mso-bidi-language: HI" lang="EN-GB">Polymorphic study of chitinase catalytic domains from <i>Bacillus thuringiensis</i> isolates</span>

476-481<span style="font-size:9.0pt;font-family: " times="" new="" roman";mso-fareast-font-family:"times="" roman";mso-bidi-font-family:="" mangal;mso-ansi-language:en-gb;mso-fareast-language:en-us;mso-bidi-language:="" hi"="" lang="EN-GB">The coding regions of the chitinase catalytic domains (CDs) from eight Bacillus thuringiensis isolates from Tunisian soil were cloned and sequenced. The cloned sequences consist of 255 nucleotides encoding amino acid fragments of 85 residues. Nucleotide and amino acid sequences analysis showed the novelty of two of them and the identity of six to that of Chi255 chitinase from B. thuringiensis subsp. kurstaki BUPM255 taken as reference. In fact, the two CDs coding regions CDchiA85 and <i style="mso-bidi-font-style: normal">CDchiA173, obtained from isolates BUPM85 and BUPM173, respectively, present the highest identity of 99% to 26 published chitinases from B. thuringiensis and B. cereus. Indeed, two nucleotide transversions of G to T and of A to C, occurring at positions 156 and 109 of CDchiA85 and CDchiA173, respectively caused substitutions at the amino acid sequence level. They are mutation E192D in the CD of chitinase ChiA85 and mutation F177V in that of ChiA173. The occurrence of changes at these positions generates polymorphisms that, obviously, do not occur at the highly conserved amino acids reported to be essential for chitin hydrolysis. Insights on the correlation of this polymorphism with the corresponding chitinases were brought by means of molecular modelling. We supposed that while the polymorphism E192D could have indirect effect on the catalytic activity of the corresponding chitinase, the residue valine brought by the polymorphism F177V could interact with the aromatic residue W210 located between the most important catalytic amino acids (D209 & E211); thereby suggesting its effect on catalysis. Therefore, the study taken into account the overall folding of the subject chitinases is intended for further work to ensure the relationship.</span

French wine - a historical, social and economic phenomenon

The thesis aims to gather the information from various sources- the historic, specialized sources-books, from the Internet or from my personal consultations as well. The topic has been chosen because the wine stands for France; it has been an inseparable part of the French history and cultural traditions. In other words, it is a phenomenon which along with a language has been developing the identity of the nation. The thesis does not seek to cover the whole representation of the French wine but it provides to look into the world of the French winegrowing. The end of the thesis deals with the wine from an economic point of view. So far, the wine has largely been participant in a prosperous state economy. However, as socalled new wines are placed on the market, France is loosing its original standing is receding from its authentic positions. Powered by TCPDF (www.tcpdf.org

Secretion of cyclodextrin glucanotransferase in <i>E. coli</i> using <i>Bacillus subtilis</i> lipase signal peptide and optimization of culture medium

72-79The cyclodextrin glycosyltransferase (CGTase) of Paenibacillus pabuli US132 was fused to the secretive lipase signal peptide of B. subtilis. This leads to an efficient secretion of the recombinant enzyme into the culture medium of E. coli as an active and soluble form contrasting with the native construction leading to a periplasmic production. In order to enhance the yield of CGTase production, an experimental design methodology was applied for the optimization of the culture composition. Hence, the media components were submitted to preliminary screening using a Plakett-Burman design. The concentrations of the major operating ones were then optimized to enhance the secretion of CGTase using response surface methodology. The findings revealed that concentrations of 0.5% potato starch, 3% yeast extract, 3% tryptone, 1.5% casein hydrolysate, 0.5% NaCl, 0.2% KH2PO4, and 0.02% MgSO4 were the optimal conditions for CGTase production. The experimental value (9.43 U/ml) obtained for CGTase activity was very close to the predicted value (9.27 U/ml)

The Amino-Acid Sequence Analysis of <i>Aspergillus Oryzae</i> S2 α-amylases.

<p>(A, B) The Structure-based multiple sequence alignment of AmyA, AmyB₁, AmyB, AmyB₂, (Accession code Hx2000049571) of <i>Aspergillus oryzae</i> S2 with α-amylase of <i>Aspergillus niger</i> (Accession code 2GUY_A). The invariable residues among sequences are typed in white on a red background; differences between conserved groups are displayed on a yellow background; the numbers (1, 2, 3, and 4) correspond to the disulfide bonds.</p

The contact maps of AmyA and AmyB.

<p>The black dots show the common contacts, the pink dots show the contacts which are unique to the native structure and the green for the contacts unique to the truncated enzyme structure.</p

The recapitulation of kinetic constants and general physico-chemical parameters of the AmyA and AmyB of <i>Aspergillus oryzae</i> S2 isoforms.

<p>Kinetic constants were previously evaluated [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153868#pone.0153868.ref023" target="_blank">23</a>]. Physico-chemical parameters revelation was performed using the Swiss-ProtParam tool (<a href="http://www.expasy.org/tools/" target="_blank">http://www.expasy.org/tools/</a>).The instability index computed classified AmyA and AmyB as stable proteins.</p

The zymogram of α-amylase activities.

<p>(A) The zymogram (10%) of α-amylase activities produced in the culture medium from the crude extract of flask incubation containing individually protease inhibitors Leupeptine, PMSF, pepstatine A as well as TPCK (N α-p-Tosyl L- PhenylalanylChloromethyl Ketone). The α-amylases produced in the culture medium from crude extracts without antiproteases were taken as control. (B) The zymogram (20%) of α-amylase activities of 48h and 92h of incubation time.</p

Localization and expression analysis of a novel catalase from Triticum monococcum TmCAT1 involved in response to different environmental stresses

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