Expression of the His-tagged r-XAn11 gene in P. pastoris
In order to express the gene encoding the His-tagged r-XAn11 in Pichia pastoris, the His-tagged r-XAn11 gene was amplified using the recombinant plasmid pET28a-XAn11  with primers Xyl-his-tag- PstI (AGC TGC AGA AAT GGG CAG CAG CCA TCA TCA TCA TCA T) and Xyl- XbaI (GCT CTA GAG CAG TGG AG ATC GTG ACA CTG GC) or Xyl-NotI (TTG CGG CCG CAA TTA AGT GGA GAT CGT GAC ACT GGC) supplemented with PstI and XbaI or NotI restriction sites (underlined).
The thermal profiles involve 30 cycles of denaturation at 94°C for 30 s, primer annealing at 65°C for 60 s and extension at 72°C for 90 s, with a final extension at 72°C for 10 min. The PCR products were purified and ligated into pCR®-Blunt vectors and the resulting plasmids were transformed into E. coli Top10. The transformants were screened on LB agar medium supplemented with 50 µg mL-1 of kanamycin.
The recombinant plasmids were digested with PstI and XbaI or PstI and NotI and ligated into pGAPZαB vectors predigested with the same restriction enzymes. The resulting plasmids, pGAPZαB-His-tagged r-XAn11, were transformed into Pichia pastoris SMD1168H strain (pep4, Mut+). Electrocompetent P. pastoris cells were prepared using standard methods . The recombinant yeast clones were selected on YPDS plates (1% yeast extract, 2% peptone, 2% dextrose, 1 M sorbitol and 1.5% agar ) containing 100 µg mL-1 zeocin. Selected positive colonies were cultured in 5 mL of YPD medium (1% yeast extract, 2% peptone and 2% dextrose) with zeocin. These cell cultures were further used to inoculate 250 mL Erlenmeyer flasks containing 50 mL YPD medium with zeocin, at 30°C for 72 h under shaking at 100 rpm. All yeast cultures had an initial optical density A600nm of 0.2. The most efficient xylanase secreting clones were selected for larger cultures performed in a 1 L baffled Erlenmeyer flask containing 200 mL YPD medium and were stopped after 72 h. Culture supernatants obtained after centrifugation (4500 rpm, 10 min, 4 °C) were dialyzed against 1×Phosphate Buffer by frontal flow ultrafiltration on a 10 kDa membrane (Millipore, USA). The concentrated enzymes were applied to 1 mL HisTrap Chelating Ni-affinity columns (Amersham Pharmacia Biotech, USA) equilibrated with 1×Phosphate Buffer containing 20 mM of imidazole. The adsorbed proteins were eluted using a linear gradient of imidazole (50 mM-200 mM). The fractions containing the xylanase activity were pooled and concentrated. Protein purity was checked using SDS-PAGE electrophoresis . Protein bands were visualized by Coomassie brilliant blue R-250 (Bio-Rad) staining.
Protein concentration was measured according to Bradford’s method with Bovine Serum Albumin as standard,.
Assay of xylanolytic activity
0.5 mL of the enzyme solution, diluted in citrate buffer (0.1 M, pH 5), was incubated for 20 min 50 °C with 0.5 mL of 1% soluble beechwood xylan (Sigma-Aldrich Co., St. Louis, MO, USA). The amount of reducing sugars released was determined by the dinitrosalicylic acid (DNS) method , using D-xylose as standard.
One unit of xylanase activity was defined as the amount of enzyme which produces one micromole of xylose equivalents per minute.
Effect of metal ions and EDTA on recombinant xylanase activity
Xylanase activities were measured under optimal conditions in the presence of EDTA or several metallic ions (Co2+, Zn2+, Mg2+, Ca2+, Cu2+, Fe2+ and Mn2+) at 5 mM.
Effects of pH and temperature on recombinant xylanase
The effect of pH on the recombinant xylanases activities was determined at 50 °C using the different buffer solutions at 100 mM. Buffers used were citrate buffer (pH 3 - 5.5), phosphate (pH 5.5 - 8), Tris-HCl (pH 8 - 9) and glycine-NaOH (9 - 10).
The optimum temperatures for the recombinant xylanases activities were determined by carrying out the enzymes assays at different temperatures (40 - 60 °C) at pH 5. The thermal stabilities of the recombinant enzymes were determined by incubating the pure enzymes at different temperatures (55 °C and 60 °C) at pH 5. Aliquots were drawn at regular time intervals and immediately cooled in ice-cold water. The residual activity was determined, after centrifugation, under standard assay conditions.
The kinetic parameters of the two recombinant xylanases were studied by determining the hydrolysis rates of beechwood xylan used at various concentrations ranging from 0 to 2 mg mL-1. Assays were performed for 5 min at 50°C. The maximum velocity (Vmax) and the Michaelis-Menten constant (Km) values were determined from the Lineweaver-Burk curves.
The molecular modeling of the Non-C-terminal tagged-His-tagged r-XAn11 and the C-terminal tagged-His-tagged r-XAn11 were performed with the SWISS-MODEL server (http://www.expasy.org/swissmod/) using the crystal structure of Xyn1 from A. niger (PDB accession code 1UKR) as a template with which the XAn11 possess 93.48% of sequence identity. The C and N terminal His-tag were generated using Swiss-Pdb Viewer 4.0.1 . The constructed models were subjected to energy minimization using GROMACS software  and single point energy computation. The PyMol Molecular Graphics System (DeLano Scientific, SanCarlos, CA. http://www.pymol.org) was used to visualize and analyze the generated model structures.
Prediction of protein flexibility of Non-C-terminal tagged-His-tagged r-XAn11 and the C-terminal tagged-His-tagged r-XAn11
The structural flexibility simulations of the two enzymes were presented as Root Mean Square Fluctuation plots and were performed by the CABS-FLEX tool. For each simulation, 10 clustered structure models were obtained and analyzed through residue fluctuation and dynamic movements. CABS-flex is based on the CABS model, a well-established coarse-grained protein modeling tool .