title: Cloning and constitutive expression of His-tagged xylanase GH 11 from Penicillium occitanis Pol6 in Pichia pastoris X33: purification and characterization.
abstract: 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.
authors.affiliation: Unité Enzymes et Bioconversion, Ecole Nationale d'Ingénieurs de Sfax, Université de Sfax, Sfax, Tunisia. dorra_driss@yahoo.fr
authors.forename: Dorra
authors.initials: D
authors.lastname: Driss
authors.forename: Fatma
authors.initials: F
authors.lastname: Bhiri
authors.forename: Raoudha
authors.initials: R
authors.lastname: Ghorbel
authors.forename: Semia Ellouz
authors.initials: SE
authors.lastname: Chaabouni
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D043364:Endo-1,4-beta Xylanases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature
chemical_list: D005656:Fungal Proteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D043364:Endo-1,4-beta Xylanases