title: Functional characterization of Penicillium occitanis Pol6 and Penicillium funiculosum GH11 xylanases.
abstract: 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.
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: Jean Guy
authors.initials: JG
authors.lastname: Berrin
authors.forename: Nathalie
authors.initials: N
authors.lastname: Juge
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; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases
keywords: Glycoside hydrolase 11; Penicillium funiculosum; Penicillium occitanis Pol6; Pichia pastoris expression; Thumb; Xylan; Xylanase