Semia Ellouz Chaabouni

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.

Production of Xylooligosaccharides by Immobilized His-tagged Recombinant Xylanase from Penicillium occitanis on Nickel-Chelate Eupergit C

Penicillium occitanis xylanase 2 expressed with a 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 93.49xa0%. 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 (pHxa02.0–4.0) than that of the free PoXyn2 (pHxa03.0). The free PoXyn2 had an optimum temperature of 50xa0°C, whereas that of the immobilized enzyme was shifted to 65xa0°C. Immobilization increased both pH stability and thermostability when compared with the free enzyme. Time courses of the xylooligosaccharides (XOS) produced from corncob xylan indicated that the immobilized enzyme tends to use shorter xylan chains and to produce more xylobiose and xylotriose initially. At the end of 24-h reaction, XOS mixture contained a total of 21.3 and 34.2xa0% (w/w) of xylobiose and xylotriose with immobilized xylanase and free xylanase, respectively. The resulting XOS could be used as a special nutrient for lactic bacteria.

Purification and Properties of a Thermostable Xylanase GH 11 from Penicillium occitanis Pol6

An extracellular, endo-β-1,4-xylanase was purified to homogeneity from the culture filtrate of the filamentous fungus Penicillium occitanis Pol6, grown on oat spelt xylan. The purified enzyme (PoXyn2) showed a single band on SDS–PAGE with an apparent molecular weight of 30xa0kDa. The xylanase activity was optimal at pHxa03.0 and 65xa0°C. The specific activity measured for oat spelt xylan was 2,368xa0Uu2009mg−1. The apparent Km and Vmax values were 8.33xa0mgu2009ml−1 and 58.82xa0μmolu2009min−1u2009ml−1, respectively, as measured on oat spelt xylan. Thin-layer chromatography experiments revealed that purified PoXyn2 degrades xylan in an endo-fashion releasing xylobiose as main end product. The genomic DNA and cDNA encoding this protein were cloned and sequenced. This PoXyn2 presents an open reading frame of 962xa0bp, not interrupted by any introns and encoding for a mature protein of 320 amino acids and 29.88xa0kDa.

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.

Effect of xylan oligosaccharides generated from corncobs on food acceptability, growth performance, haematology and immunological parameters of Dicentrarchus labrax fingerlings.

The objective of this study was to determine the effect of two levels of inclusion of xylan oligosaccharides (XOS) extracted from corncob on growth, feed utilization, immune status and disease resistance of Mediterranean sea bass (Dicentrarchus labrax) fingerlings. Specimens of 4.75xa0±xa00.69xa0g at initial density of 2.7xa0±xa00.13xa0kg/m3 were fed during 12xa0weeks at 0xa0gxa0kg−1 diet, 5xa0gxa0kg−1 diet and 10xa0gxa0kg−1 diet, dietary XOS level of inclusion in a commercial sea bass diet. Feeding the fish at both XOS dietary inclusion levels significantly increased weight gain, protein efficiency ratio and feed conversion ratio. Feeding of supplemented diets to fish led to reducing mortalities after challenging with A. hydrophila. The haematological and immunological parameters were assayed in both pre-challenged and post-challenged groups. There was an increased trend in red blood corpuscles, white blood corpuscles, pack cell volume, haemoglobin (Hb %) and serum protein content in treated groups over the control as time elapsed with the feeding trials. The serum immunoglobulin level and lysozyme activity showed an increased trend in the fed groups. Histological features of the liver showed lower lipid vacuolization and regular-shaped morphology of hepatocytes around the sinusoidal spaces denoting a better utilization of dietary nutrients supported with the morphometric data. In conclusion, XOS added at a designated dose (5xa0gxa0kg−1 diet) in the diet improves growth and stimulates the immunity and makes D. labrax fingerlings more resistant to infection by A. hydrophila.

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.

Synthesis and antibacterial activity of new peptides from Alfalfa RuBisCO protein hydrolysates and mode of action via a membrane damage mechanism against Listeria innocua

In this work we evaluated the mode of action of six new synthesized peptides (Met-Asp-Asn; Glu-leu-Ala-Ala-Ala-Cys; Leu-Arg-Asp-Asp-Phe; Gly-Asn-Ala-Pro-Gly-Ala-Val-Ala; Ala-Leu-Arg-Met-Ser-Gly and Arg-Asp-Arg-Phe-Leu), previously identified, from the most active peptide fractions of RuBisCO peptic hydrolysate against Listeria innocua via a membrane damage mechanism. Antibacterial effect and the minimum inhibitory concentrations (MIC) of these peptides were evaluated against six strains and their hemolytic activities towards bovine erythrocytes were determined. Prediction of the secondary structure of peptides indicated that these new antibacterial peptides are characterized by a short peptide chains (3-8 amino acid) and a random coli structure. Moreover, it was observed that one key characteristic of antibacterial peptides is the presence of specific amino acids such as cysteine, glycine, arginine and aspartic acid. In addition the determination of the extracellular potassium concentration revealed that treatment with pure RuBisCO peptides could cause morphological changes of L. innocua and destruction of the cell integrity via irreversible membrane damage. The results could provide information for investigating the antibacterial model of antibacterial peptides derived from RuBisCO protein hydrolysates.