Monia Mezghani

Purification and sequence analysis of the atypical maltohexaose-forming α-amylase of the B. stearothermophilus US100

The maltohexaose-forming alpha-amylase, of B. stearothermophilus US100, was purified to homogeneity by a combination of osmotic shock, starch adsorption and anion exchange chromatography. This enzyme has a relative molecular mass of 59 kDa. The analysis of the nucleotide sequence, of the corresponding gene, allowed the identification of a single open reading frame encoding a 549 amino acid protein, exhibiting a large homology to the other B. stearothermophilus alpha-amylases. This homology reaches a maximum with those of DY-5 and DN1792 strains with respectively 3 and 4 aa different over 549. The relatively small differences, between Amy US100 and that of DN1792 strain, take in more importance since we have demonstrated that these enzymes differ essentially by their starch hydrolysis pattern.

A thermostable α-amylase producing maltohexaose from a new isolated Bacillus sp. US100 : study of activity and molecular cloning of the corresponding gene

A Bacillus sp. US100 strain was isolated from Tunisian hot spring soil. This strain secretes a thermoactive amylase having an optimal temperature of 82°C and a remarkable thermostability with a half time of 40 min at 110°C in the presence of 20% (w/v) of substrate. The main end products of its action on starch are maltohexaose and maltopentaose which is the smallest substrate hydrolysed by this enzyme. The molecular cloning, in E. coli, of a 3-kb DNA fragment encoding for this activity is also reported.

Characterization of glucansucrase and dextran from Weissella sp. TN610 with potential as safe food additives.

Pear-derived Weissella sp. TN610 produced extracellular glycosyltransferase activity responsible for the synthesis of soluble exopolysaccharide from sucrose. Acid and dextranase-catalyzed hydrolysis revealed that the synthesized polymer was a glucan. According to (1)H and (13)C NMR analysis, the glucan produced by TN610 was a linear dextran made of 96% α-(1→6) and 4% α-(1→3) linkages. Zymogram analysis confirmed the presence of a unique glucansucrase of approximately 180 kDa in the cell-free supernatant from TN610. The crude enzyme, optimally active at 37°C and pH 5, has promising potential for application as a food additive since it catalyzes dextran synthesis in sucrose-supplemented milk, allowing its solidification. A 4257-bp product corresponding to the mature glucansucrase gene was amplified by PCR from TN610. It encoded a polypeptide of 1418 residues having a calculated molecular mass of 156.089 kDa and exhibiting 96% and 95% identity with glucansucrases from Lactobacillus fermentum Kg3 and Weissella cibaria CMU, respectively.

Improvement of Trichoderma reesei xylanase II thermal stability by serine to threonine surface mutations.

Three simple mutants, S80T, S146T, and S149T, and a double mutant, S80T-S149T, were constructed and expressed in Escherichia coli to replace Serine on the surface of the Trichoderma reesei xylanase protein with Threonine residues. While the Wild-type (WT) xylanase showed a half-life time (t1/2) of 20 min at 55 °C, the double mutant was more thermostable exhibiting a t1/2 value of 37 min, followed by the S80T and S149T mutants whose t1/2 values were 25 and 23 min, respectively. At 55 °C, the S146T mutant showed a decrease in thermostability with a t1/2 value of 3 min. While the WT enzyme retained only 32% of residual activity after incubation for 5 min at 60°C, the S80T, S149T, and the S80T-S149T mutant enzymes retained 45%, 41%, and 60%, respectively. Molecular modeling attributed the increase in the thermostability of the S80T and S149T mutants to a new hydrogen bond formation and a packing effect, respectively.

Characterization and molecular cloning of thermostable alpha-amylase from Streptomyces sp.To1

SummaryA new thermophilic Streptomyces sp. TO1, isolated from Tunisian soil, produced a thermostable alpha-amylase and pullulanase. The gene encoding for the alpha-amylase activity was cloned into the multicopy cloning plasmid pLM1 using S. lividans ZX1 as host strain. The ZX1 / pLM1 strain has the same activity than the initial TO1 strain and about 25 fold higher activity than the ZX1 strain. This alpha-amylase has an optimum of pH and temperature at 6 and 70 °C respectively.

Two new gene clusters involved in the degradation of plant cell wall from the fecal microbiota of Tunisian dromedary

Dromedaries are capable of digesting plant cell wall with high content of lignocellulose of poor digestibility. Consequently, their intestinal microbiota can be a source of novel carbohydrate-active enzymes (CAZymes). To the best of our knowledge, no data are available describing the biochemical analysis of enzymes in dromedary intestinal microbiota. To investigate new hydrolytic enzymes from the dromedary gut, a fosmid library was constructed using metagenomic DNA from feces of non-domestic adult dromedary camels living in the Tunisian desert. High-throughput functional screening of 13756 clones resulted in 47 hit clones active on a panel of various chromogenic and non-chromogenic glycan substrates. Two of them, harboring multiple activities, were retained for further analysis. Clone 26H3 displayed activity on AZO-CM-cellulose, AZCL Carob galactomannan and Tween 20, while clone 36A23 was active on AZCL carob galactomannan and AZCL barley β-glucan. The functional annotation of their sequences highlighted original metagenomic loci originating from bacteria of the Bacteroidetes/Chlorobi group, involved in the metabolization of mannosides and β-glucans thanks to a complete battery of endo- and exo-acting glycoside hydrolases, esterases, phosphorylases and transporters.