title: Cloning and expression of a xylanase xynB from Aspergillus niger IA-001 in Pichia pastoris.
abstract: The high-level expression of the xylanase GH11 gene from Aspergillus niger IA-001 called xynB was successfully completed in Pichia pastoris. The xynB gene encoding a mature xylanase of 225 amino acid was subcloned into the pPICZαA vector and was transformed into P. pastoris X-33 under the control of the alcohol oxidase I (AOX1) promoter. The xynB gene was ligated with a sequence encoding modified α-factor signal peptide (pPICZαmA) and the recombinant xylanase activity, which was measured 1280 U ml(-1), was 1.5-fold higher than when it was inserted into pPICZαA and was 19.39-fold greater than the native xylanase in the original strain. In a 10 L fermenter, the recombinant xylanase activity measured 10,035 U ml(-1) after 114 h. The SDS-PAGE analysis revealed that the purified xynB protein migrated as a single band with an apparent molecular weight of 24 kDa. The specific activity, using beechwood xylan as a substrate, was 1916 U mg(-1). The xylanase activity was optimal at pH 5.0 and at 50 °C. In addition, the xynB was active over a pH range of 2.2 to 10.0. The apparent Km and Vmax values were 4.429 mg ml(-1) and 1429 U mg(-1), respectively.
authors.affiliation: Institute of Animal Nutrition, Northeast Agricultural University, Harbin, P. R., China.
authors.forename: Wei
authors.initials: W
authors.lastname: Fang
authors.forename: He
authors.initials: H
authors.lastname: Gao
authors.forename: Yunhe
authors.initials: Y
authors.lastname: Cao
authors.forename: Anshan
authors.initials: A
authors.lastname: Shan
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D015870:Gene Expression; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature; D014990:Xylans / Q000378:metabolism; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D004271:DNA, Fungal; D011994:Recombinant Proteins; D014990:Xylans; D001617:beta-Glucosidase; D043364:Endo-1,4-beta Xylanases; C090001:XynB xylanase
keywords: Aspergillus niger; Expression; Pichia pastoris; Xylanase