title: Cloning and optimized expression of a GH-11 xylanase from Fusarium oxysporum in Pichia pastoris.
abstract: The endo-1,4-β-xylanase gene xyn11a from Fusarium oxysporum, member of the fungal glycosyl hydrolase (GH) family 11, was cloned and expressed in Pichia pastoris. The mature xylanase gene, which generates after the excision of one intron and the secreting signal peptide, was placed under the control of an alcohol oxidase promoter (AOX1) in the plasmid pPICZαC. The final construction was integrated into the genome of the methylotrophic yeast P. pastoris X33 and the ability to produce xylanase activity was evaluated in flask cultures. Recombinant P. pastoris efficiently secreted xylanase into the medium and produced high level of enzymatic activity (110 U/ml) after 216 hours of growth, under methanol induction. To achieve higher enzyme production, the influence of initial pH, methanol concentration, agitation and flask design was evaluated. Under optimum culture conditions, production of the recombinant xylanase increased by 50%, reaching a final yield of 170 U/ml, underpinning aeration as the most important factor in improving enzyme production.
authors.affiliation: BIOtechMASS Unit, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str, Zografou Campus, 15700 Athens, Greece.
authors.forename: Maria
authors.initials: M
authors.lastname: Moukouli
authors.forename: Evangelos
authors.initials: E
authors.lastname: Topakas
authors.forename: Paul
authors.initials: P
authors.lastname: Christakopoulos
mesh_terms: D003001:Cloning, Molecular; D043364:Endo-1,4-beta Xylanases / Q000096:biosynthesis* / Q000235:genetics*; D005670:Fusarium / Q000502:physiology*; D010843:Pichia / Q000201:enzymology* / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases