Bassem Khemakhem

Morphological and biochemical behavior of fenugreek (Trigonella foenum-graecum) under copper stress.

The effects of copper on germination and growth of fenugreek (Trigonella foenum-graecum) was investigated separately using different concentrations of CuSO₄. The germination percentage and radical length had different responses to cupric ions: the root growth increased with increasing copper concentration up to 1 mM Cu²⁺ and was inhibited thereafter. In contrast, the germination percentage was largely unaffected by concentrations of copper below 10 mM. The reduction in root growth may have been due to inhibition of hydrolytic enzymes such as amylase. Indeed, the average total amylolytic activity decreased from the first day of treatment with [Cu²⁺] greater than 1 mM. Furthermore, copper affected various plant growth parameters. Copper accumulation was markedly higher in roots as compared to shoots. While both showed a gradual decrease in growth, this was more pronounced in roots than in leaves and in stems. Excess copper induced an increase in the rate of hydrogen peroxide (H₂O₂) production and lipid peroxidation in all plant parts, indicating oxidative stress. This redox stress affected leaf chlorophyll and carotenoid content which decreased in response to augmented Cu levels. Additionally, the activities of proteins involved in reactive oxygen species (ROS) detoxification were affected. Cu stress elevated the ascorbate peroxidase (APX) activity more than two times at 10 mM CuSO₄. In contrast, superoxide dismutase (SOD) and catalase (CAT) levels showed only minor variations, only at 1 mM Cu²⁺. Likewise, total phenol and flavonoid contents were strongly induced by low concentrations of copper, consistent with the role of these potent antioxidants in scavenging ROS such as H₂O₂, but returned to control levels or below at high [Cu²⁺]. Taken together, these results indicate a fundamental shift in the plant response to copper toxicity at low versus high concentrations.

Crucial role of Pro 257 in the thermostability of Bacillus phytases: biochemical and structural investigation.

We have previously cloned and characterized the thermostable phytase (PHY US417) from Bacillus subtilis US417. It differs with PhyC from B. subtilis VTTE-68013 by the R257P substitution. PHY US417 was shown to be more thermostable than PhyC. To elucidate the mechanism of how the Pro 257 changes the thermostability of Bacillus phytases, this residue was mutated to Arg and Ala. The experimental results revealed that the thermostability of the P257A mutants and especially P257R was significantly decreased. The P257R and P257A mutants recovered, respectively, 64.4 and 81.5% of the wild-type activity after incubation at 75 °C for 30 min in the presence of 5mM CaCl(2). The P257R mutation also led to a severe reduction in the specific activity and catalytic efficiency of the enzyme. Structural investigation, by molecular modeling of PHY US417 and PhyC focused on the region of the 257 residue, revealed that this residue was present in a surface loop connecting two of the six characteristic β sheets. The P257 residue is presumed to reduce the local thermal flexibility of the loop, thus generating a higher thermostability.

Effect of treated and untreated domestic wastewater on seed germination, seedling growth and amylase and lipase activities in Avena sativa L

BACKGROUND Oats (Avena sativa L.) are a potential economically viable source of lipids and starch for use in foods. The aim of this study was to determine the effect of treated and untreated urban wastewater on seed germination, growth parameters and lipase and amylase activities in A. sativa. RESULTS Untreated wastewater was highly toxic in nature and had an inhibitory effect on seed germination and seedling growth. However, after bacterial treatment, its toxicity was significantly reduced and it showed improved seed germination. It was observed that treated wastewater had no inhibitory effect on seedling growth parameters. However, A. sativa seeds treated with untreated effluent showed reduced lipase and amylase activities. CONCLUSION Treated wastewater could be used for irrigation purposes provided that it satisfies other conditions fixed by legislation.

Evaluation of anti-diabetic and anti-tumoral activities of bioactive compounds from Phoenix dactylifera L's leaf: In vitro and in vivo approach.

Among various chronic disorders, cancer and diabetes mellitus are the most common disorders. This study was designed to evaluate the effectiveness of hydroalcoholic extract of Phoenix dactylifera L. leaves (HEPdL) in animal models of type II diabetes in vitro/in vivo and in a human melanoma-derived cell line (IGR-39). A liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was also performed to determine the amount of phenolic and flavonoid compounds in this plant. The physicochemical results by LC-MS/MS analysis of HEPdL showed the presence of 10 phenolic compounds. The in vitro study showed that the extract exhibited a more specific and potent inhibitor of α-glucosidase than α-amylase with an IC50 value of 20±1μg/mL and 30±0.8μg/mL, respectively. More importantly, the in vivo study of the postprandial hyperglycemia activity with (20mg/kg) of HEPdL showed a decrease in plasma glucose levels after 60min in resemblance to the glucor (acarbose) (50mg/kg) effect. The oral administration of HEPdL (20mg/kg) in alloxan-induced diabetic mices for 28days showed a more significant anti-diabetic activity than that of the drug (50mg/kg). Moreover, cytotoxicity effects of HEPdL in IGR-39 cancer cell lines were tested by MTT assay. This extract was effective in inhibiting cancer cells growth (IGR-39) at dose 35 and 75μg/mL. These results confirm ethnopharmacological significance of the plant and could be taken further for the development of an effective pharmaceutical drug against diabetes and cancer.

Optimised amylases extraction from oat seeds and its impact on bread properties.

Statistical approaches were employed for the optimisation of the extraction of amylolytic activity from oat (Avena sativa) seeds. The application of the response surface methodology allows us to determine a set of optimal conditions (ratio seed weight/buffer volume 0.1, germination days 10 days, temperature 20 °C and pH 5.6). Experiments carried out under these conditions led to amylase production yield of 91 U/g. Its maximal activity was in the pH 5.6 and at 55 °C. Study of the incorporation of the optimised oat extract into the bread formulation revealed an improvement of the sensory quality and the textural properties of fresh and stored bread. Three-dimensional elaborations of Confocal Laser Scanning Microscopy (CLSM) images were performed on crumb of the different breads to evaluate the influence of amylase activity on microstructure. The result showed improved baking characteristics as well as overall microscopic and macroscopic appearance.

Purification and characterization of an amylase from Opuntiaficus‐indica seeds

BACKGROUND In Tunisia, prickly pear fruit grow spontaneously; it is consumed as fresh fruit, juice or jam. When the fruit is used for juice production, the seeds are discarded and go to waste. Our study aimed to extract biomolecules from seeds by producing value-added products from the fruits. RESULTS An amylase from Opuntia ficus-indica seeds was extracted and purified to homogeneity. An increase in specific activity of 113-fold was observed. The apparent molecular mass of the enzyme is 64 kDa. The optimum pH and temperature for enzyme activity were pH 5 and 60 °C, respectively. Under these conditions, the specific activity is 245.5 U mg(-1) . The enzyme was activated by Co(2+) and Mg(2+) (relative activity 117% and 113% respectively) at lower ion concentrations. It was strongly inhibited by Mn(2+) and Fe(2+) . Cu(2+) inhibited totally the activity of this enzyme, but Ca(2+) has an inhibitory effect which increases with ion concentration. CONCLUSION The extracted enzyme belongs to the exo type of amylases and is classified as a β-cyclodextrin glycosyltransferase since it generates mainly β-cyclodextrin from starch. It exhibits high thermal stability and a broad range of pH stability, making it a promising prospect for industrial and food applications.

Toward the smallest active subdomain of a TIM-barrel fold: insights from a truncated α-amylase.

AmyTM is a truncated mutant of the α-amylase of Bacillus stearothermophilus US100. It has been derived from the wild type amylase gene via a reading frame shift, following a tandem duplication of the mutant primer, associated to an Adenine base deletion. AmyTM was composed of 720 nucleotides encoding 240 amino acid residues out of 549 of the wild type. The AmyTM protein was devoided of the three catalytic residues but still retains catalytic activity. It is Ca-independent maltotetraose producing amylase, optimally active at pH 6 and 60°C, under monomeric or multimeric forms. AmyTM is the smallest functional truncated TIM barrel. It contains the βαβα unit as the minimal subdomain associated to an enzymatic function. The enzymatic activity can, until now, be attributed to the presence of the whole domain B, in the structure of AmyTM. This mutant revealed, for the first time, the regeneration of a catalytic site after its abolition. This fact may be considered as the restoration of a primitive active site, which was lost in the course of evolution toward more stable domains.

Extraction optimization and in vitro and in vivo anti-postprandial hyperglycemia effects of inhibitor from Phoenix dactylifera L. parthenocarpic fruit

Phoenix dactylifera L. plays an important role in social, economic, and ecological Tunisian sectors. Some date palms produce parthenocarpic fruit named Sish. The aqueous ethanolic extract from P. dactylifera parthenocarpic dates demonstrated a potent inhibition of the enzymes related to type II diabetes. In this work, extraction optimization of amylase inhibitors was carried out using Box-Behnken Design. Bioactivity-guided fractionation of the 70% aqueous ethanol extract was performed to identify the active compounds. The physicochemical results by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed the presence of 13 phenolic compounds. The in vitro study showed that the extract exhibited a more specific inhibitor of α-glucosidase than α-amylase with an IC50 value of 0.6 and 2.5mg/mL, respectively. The in vivo study of this extract effect on the postprandial hyperglycemia activity showed a decrease in plasma glucose levels after 30min stronger than the Acarbose effect. These results confirmed the anti-postprandial hyperglycemia activity of the aqueous ethanolic extract from P. dactylifera parthenocarpic dates, which could lend support for its pharmaceutical use.

Mutations affecting the activity of the cyclodextrin glucanotransferase of Paenibacillus pabuli US132: insights into the low hydrolytic activity of cyclodextrin glucanotransferases

The cyclodextrin glucanotransferase from Paenibacillus pabuli US132 (US132 CGTase) was engineered using a rational approach in an attempt to provide it with anti-staling properties comparable to those of the commercial maltogenic amylase (Novamyl). The study aimed to concurrently decrease the cyclization activity and increase the hydrolytic activity of US132 CGTase. A five-residue loop (PAGFS) was inserted, alone or with the substitution of essential residues for cyclization (G180, L194 and Y195), mimicking the case of Novamyl. The findings indicate that, unlike the case of the CGTase of Thermoanerobacterium thermosulfurigenes strain EM1 whose initial high hydrolytic activity was exceptional, these mutations completely abolished the cyclization and hydrolytic activities of the US132 CGTase. This suggests that those mutations are not able to convert conventional CGTases, whose hydrolytic activities are very weak, into hydrolases. Accordingly, and for the first time, a structural barrier at subsite −3 was advanced as an influential factor which might explain the low hydrolytic activity of conventional CGTases.

Identification of a new oat β-amylase by functional proteomics.

Oat (Avena sativa L.) seed extracts exhibited a high degree of catalytic activity including amylase activities. Proteins in the oat seed extracts were optimized for their amylolytic activities. Oat extract with amylolytic activity was separated by SDS-PAGE and a major protein band with an apparent molecular mass of 53 kDa was subjected to tryptic digestion. The generated amino acid sequences were analyzed by liquid chromatography–tandem mass spectrometry (LC/ESI/MS/MS) and database searches. These sequences were used to identify a partial cDNA from expressed sequence tags (ESTs) of A. sativa L. Based upon EST sequences, a predicted full-length gene was identified, with an open reading frame of 1464 bp encoding a protein of 488 amino acid residues (AsBAMY), with a theoretical molecular mass of 55 kDa identified as a β-amylase belonging to the plant β-amylase family. Primary structure of oat β-amylase (AsBAMY) protein indicated high similarity with other β-amylase from other cereals such as wheat (Triticum aestivum), barley (Hordeum vulgare), and rye (Secale cereale) with two conserved Glu residues (E184 and E378) assigned as the “putative” catalytic residues which would act as an acid and base pair in the catalytic process. In addition, a 3D-model of AsBAMY was built from known X-ray structures and sequence alignments. A similar core (β/α)8-barrel architecture was found in AsBAMY like the other cereal β-amylases with a specific location of the active site in a pocket-like cavity structure made at one end of this core (β/α)8-barrel domain suggesting an accessibility of the non-reducing end of the substrate and thus confirming the results of AsBAMY exo-acting hydrolase.