Dorra Driss

Cloning and constitutive expression of His-tagged xylanase GH 11 from Penicillium occitanis Pol6 in Pichia pastoris X33: Purification and characterization

High-level constitutive expression of xylanase GH11 from Penicillium occitanis Pol6 termed PoXyn2 was achieved using the methylotrophic yeast Pichia pastoris. The PoXyn2 cDNA encoding for a mature xylanase of 320 amino acids was subcloned into the pGAPZαA vector, to construct recombinant xylanse with six histidine residues at the N-terminal and further integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase (GAP) constitutive promoter. Activity assay and SDS-PAGE demonstrate that the His-tagged xylanase was extracellularly expressed in P. pastoris and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity chromatography (Ni-NTA resin). The purified PoXyn2 showed a single band on SDS-PAGE with an apparent molecular weight of 30 kDa. The xylanase activity was optimal at pH 3.0 and 50°C. The specific activity measured for Oat Spelt Xylan was 8549.85 U mg(-1). The apparent The K(M) and V(max) values were 8.33±0.7 mg ml(-1)and 58.82±0.9 μmol min(-1) ml(-1), respectively, as measured on Oat Spelt Xylan. This is the first report demonstrating the possibility of mass production of P. occitanis xylanase using P. pastoris.

Structural data and biological properties of almond gum oligosaccharide: application to beef meat preservation.

Enzymatic hydrolysis of almond gum generates low molecular weight oligosaccharides (OAG) with a yield of 33.5%. The generated oligosaccharides were purified and identified. OAG analyses show that the most prominent residues were galactose and arabinose with traces of xylose, rhamnose, glucose and mannose. The glycosyl linkage positions were analyzed using gas chromatography-mass spectrometry showing a main chain composed of galactose units [ → 3)-Gal-(1 → ] branched mainly with arabinose residues [Ara-(1 → ]. The antioxidant and antimicrobial activities of OAG were investigated. As regards the in vitro antioxidant activities, the OAG showed a high total antioxidant activity (347 μg ascorbic acid equivalent/mL), an important DPPH (1,1-diphenyl-2-picrylhydrazyl) scavenging activity (IC50 = 0.64 mg/mL) and a high reducing capacity (RP0.5AU = 3.6 mg/mL). Furthermore, OAG had a high antimicrobial activity against Salmonella thyphimirium, Bacillus cereus, Actinomycetes sp, Klebsiella pneumoniae, Escherichia coli, Alternaria alternate and Candidat albicans. Finally, OAG efficiency was tested using 0.5%; 0.75% and 1% concentrations in beef meat preservation. Microbial growth and lipid oxidation were monitored during 9 days at 4 °C. The results showed significant inhibitions (p < 0.05) of lipid oxidation and microbial growth in ground beef meat containing OAG.

Polysaccharide from garlic straw: extraction, structural data, biological properties and application to beef meat preservation

A novel polysaccharide (GSP) was isolated from garlic straw (Allium sativum L.) by hot water technique. The structural characterization, antioxidant and antimicrobial activities were investigated. The results showed that GSP was mainly composed of glucose, mannose, galactose and xylose and the major functional groups identified from FT-IR spectrum includes 1631.38 cm−1 (–COO–) and 3193 cm−1 (–OH). In addition, GSP had high DPPH radical scavenging activity, a strong reducing power and inhibited the peroxidation of linoleic acid. The antimicrobial activity of GSP was evaluated against a panel of 7 bacteria and 2 fungal strains using agar diffusion method. Results have shown that GSP exhibited moderate to strong antimicrobial activity against the tested species. These interesting results incite the experimental inoculation of GSP in minced beef meat preservation amended with different concentrations of the GSP and stored at 4 °C for 9 days. The obtained results showed significant inhibitions (p ≤ 0.05) of lipid oxidation over 9 days of aerobic storage and also improvement of meat colour stability while differences in total aerobic cell populations did not change noticeably over storage. Finally, sensory characteristics, e.g. colour, odour and texture, of treated meat with GSP, were higher than the control.

Water-soluble polysaccharides from Opuntia stricta Haw. fruit peels: recovery, identification and evaluation of their antioxidant activities

Abstract Opuntia stricta Haw. is considered as one of the most common cactus plant growing in Tunisia. Extracting valuable compounds from its fruit peel, considered as by-product, is drawing more and more attention, making it on the verge of commercialization. Water-soluble polysaccharides were extracted from Opuntia stricta Haw. peels, and their chemical composition assessed using thin layer chromatography. The antioxidant activities of the extracted polysaccharides were assessed using 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity, total antioxidant activity and reducing power capacity. The extraction yield of water-soluble polysaccharides was 7.53±0.86%. The chemical composition revealed the presence of rhamnose, arabinose, glucose, mannose, galactose and galacturonic acid. The infra-red spectroscopic analysis showed a similar structure to that of Opuntia ficus-indica polysaccharide peels. Additionally, the extracted polysaccharides exhibited high antioxidant activities. In fact, the free radical scavenging activity (half inhibition concentration = 6.5 mg ml−1 with 94.9% inhibition at 50 mg ml−1), the total antioxidant activity (100 μg ascorbic acid equivalent at 50 mg polysaccharides) and the reducing power capacity (absorbance 700 nm = 0.7 at 50 mg ml−1), appeared to be interesting compared to natural and synthetic antioxidants. Therefore, water-soluble polysaccharides from Opuntia stricta Haw. fruit peels could be a natural alternative to replace synthetic antioxidants.

Healing efficiency of oligosaccharides generated from almond gum (Prunus amygdalus) on dermal wounds of adult rats

Almond gum is a naturally occurring polymer produced by almond trees and shrubs. Its abundance, as well as its low cost production makes it a potential feedstock for use in food and pharmaceuticals. In this regard, almond gum oligosaccharides were enzymatically generated, purified and their monosaccharide composition assessed using gas chromatography-flame ionization detector. Oligosaccharide analyses show that the most prominent residues were galactose and arabinose with traces of xylose, rhamnose, glucose and mannose. The glycosyl linkage positions were analyzed using gas chromatography - mass spectrometry showing a main chain composed of galactose units [→3)-Gal-(1→] branched mainly with arabinose residues [Ara-(1→]. The potent role of the generated oligosaccharides on rats wound healing was investigated. They have been applied either alone or supplemented, as active substance, with cream formulation, on full-thickness wound created on the dorsum of the rats. The effect of oligosaccharides was assessed by measuring the wound closure percentage, reaching an average of around 100% when applied alone or supplemented to cream formulation. The healing percentage for the control group was only 74.3% at the same day. The histological evaluation of skin sections visualized by light microscopy revealed an improved collagen deposition and an increased fibroblast and vascular densities.

Functional characterization of Penicillium occitanis Pol6 and Penicillium funiculosum GH11 xylanases

Xylanases are hemicellulolytic enzymes, which are responsible for the degradation of heteroxylans constituting the lignocellulosic plant cell wall. Xylanases from the GH11 family are considered as true xylanases because of their high substrate specificity. In order to study in depth a crucial difference in the thumb region between two closely related xylanases from Penicillium in terms of kinetic parameters and inhibition sensitivity, the GH11 xylanases from Penicillium occitanis Pol6 (PoXyn3) and from Penicillium funiculosum (PfXynC) were heterologously expressed in Pichia pastoris. The PoXyn3 and PfXynC cDNAs encoding mature xylanases were cloned into pGAPZαA vectors and integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase constitutive promoter. PfXynC was expressed as a His-tagged recombinant protein and purified from the supernatant homogeneity by a one-step purification protocol using immobilized metal affinity chromatography. The recombinant PoXyn3 was purified using a single anion-exchange chromatography. The purified recombinant enzymes were optimally active at 45°C and pH 4.0 for PoXyn3 and 40°C and pH 3.0 for PfXynC. The measured kinetic parameters (k(cat) and Vmax) showed that PfXynC was five times more active than PoXyn3 irrespective of the substrate whereas the apparent affinity (K(m)) was similar. The recombinant enzymes showed distinct sensitivity to the Triticum aestivum xylanase inhibitor TAXI-I.

Antibacterial and antioxidant properties of mixed linkage beta-oligosaccharides from extracted β-glucan hydrolysed by Penicillium occitanis EGL lichenase

The aim of this study was first to ascertain the chemical composition and the physicochemical properties of cereal extracted β-glucan from barley flour. Secondly, to assess the antioxidant properties and the antibacterial properties of extracted β-glucan hydrolysates. The proximate composition, FT-IR and scanning electron microscopy of extracted β-Glucan were studied. Hydrolysates from extracted β-glucan, obtained by lichenase EGL from Penicillium occitanis, were a mixed linkage beta-oligosaccharides (MLBO) of trisaccharides and tetrasaccharides. MLBO showed a DPPH radical scavenger with IC50 about 1.8 ± 0.01 mg/mL whereas the IC50 of extracted β-glucan was about 5 ± 0.01 mg/mL. MLBO showed a high antioxidative capacity (175 μmol/mL α-tocopherol equivalents) at 5 mg/mL. The antimicrobial activity was confirmed against all tested bacteria especially at 20 mg/mL of MLBO while no inhibition was observed for all the strains used after the addition of either EGL or extracted β-glucan.

Altered hepatic mRNA expression of immune response-associated DNA damage in mice liver induced by potassium bromate: Protective role of vanillin.

Chronic exposure to potassium bromate (KBrO3), a toxic halogen existing widely in the environment, environment through contaminated drinking water, has become a global problem of public health. The present study investigates the protective role of vanillin against KBrO3 induced oxidative stress, distruption in inflammatory cytokines expression, DNA damage, and histopathological changes. Adult mice were exposed orally to KBrO3 (2g/L of drinking water) for 2 weeks The co‐administration of vanillin to the KBrO3‐treated mice significantly prevented the plasma transaminases increase in. Furthermore, it inhibited hepatic lipid peroxidation (malondialdehyde), advanced oxidation protein product (AOPP) and protein carbonyl (PCO) formation and attenuated the KBrO3‐mediated depletion of enzymatic and non enzymatic antioxidants catalase, superoxide dismutase, and glutathione peroxidase activities and glutathione level in the liver. In addition, vanillin markedly attenuated the expression levels of proinflammatory cytokines, including tumor necrosis factor‐α, interleukin‐1β, interleukin‐6, and COX2 and prevented KBrO3‐induced hepatic cell alteration and necrosis, as indicated by histopathological data. DNA damage, as assessed by the alkaline comet assay, was also found to be low in the co‐treated group. Thus, these findings show that vanillin acts as potent chemopreventive agent against KBrO3‐mediated liver oxidative stress and genotoxicity through its antioxidant properties.

Immobilization of His-tagged recombinant xylanase from Penicillium occitanis on Nickel-chelate Eupergit C for increasing digestibility of poultry feed

Recombinant xylanase 2 from Penicillium occitanis expressed with an His-tag in Pichia pastoris, termed PoXyn2, was immobilized on nickel-chelate Eupergit C by covalent coupling reaction with a high immobilization yield up to 93.49%. Characterization of the immobilized PoXyn2 was further evaluated. The optimum pH was not affected by immobilization, but the immobilized PoXyn2 exhibited more acidic and large optimum pH range (pH 2.0–4.0) than that of the free PoXyn2 (pH 3.0). The free PoXyn2 had an optimum temperature of 50 °C, whereas that of the immobilized enzyme was shifted to 65 °C. Immobilization increased both pH stability and thermostability when compared with the free enzyme. Thermodynamically, increase in enthalpy and free energy change after covalent immobilization could be credited to the enhanced stability. Immobilized xylanase could be reused for 10 consecutive cycles retaining 60% of its initial activity. It was found to be effective in releasing reducing sugar from poultry feed. Immobilization on Eupergit C is important due to its mechanical resistance at high pH and temperature. Hence, considerable stability and reusability of bound enzyme may be advantageous for its industrial application.

Effects of vanillin on potassium bromate-induced neurotoxicity in adult mice: impact on behavior, oxidative stress, genes expression, inflammation and fatty acid composition

Abstract Context: Vanillin is known to possess important antioxidant activity. Objective: The current study was conducted to establish the therapeutic efficiency of vanillin against potassium bromate (KBrO3)-induced depression-like behavior and oxidative stress in mice. Material and methods: Mice were exposed during 15 days either to potassium bromate (KBrO3), KBrO3+ vanillin or to only vanillin. Results: Our results revealed a significant modification in the fatty acid composition of the KBrO3-treated mice. In addition, KBrO3 induced a significant reduction in enzymatic activities and gene expressions, Na+ –K+  and Mg2+-ATPases, acetylcholinesterase and butylcholinesterase activities. The gene expression of tumor necrosis factor-α, interleukin-1β, interleukin-6 and COX2, significantly increased in the cerebrum of KBrO3-treated group. Histopathological observations were consistent with these effects. Co-treatment with vanillin significantly attenuated KBrO3-induced oxidative stress and inflammation. Conclusion: This work suggests that vanillin mitigates KBrO3-induced depression, and that this neuroprotective effect proceeds through anti-oxidant and anti-inflammatory activities.