title: [Cloning and expression of Aspergillus niger glucose oxidase gene in methylotrophic yeast].
abstract: The DNA fragment encoding A. niger glucose oxidase was amplified by PCR using A. niger genomic DNA as template, and was cloned into vector of pPIC9 for expression in Pichia pastoris. When transformed into methylotrophic yeast Pichia pastoris GS115, The constructed plasmid pPICGOD1 directed the synthesis and secretion of functionally active GOD. After induction in MM medium for 4 days, the GOD activity in the medium reached 30-40 u/mL. SDS-PAGE revealed that recombinant yeast GOD was expressed up to 60%-70% of the total soluble protein, and the secreted GOD could be purified to electrophoretic homogeneity with one purification step using Q Sepharose Fast Flow ion exchange chromatography. The recombinant yeast GOD had very high catalytic activity, showed about 1.6-fold increase of specific activity over the commercial A. niger GOD. Kinetic analysis clearly demonstrated that recombinant yeast GOD showed similar substrate affinity for glucose to A. niger GOD, but the turnover number of the GOD from yeast was determined to be much higher than that of A. niger GOD. In addition, the linear range of glucose electrode made with recombinant yeast GOD was efficiently widened due to the high catalytic activity of yeast GOD.
authors.affiliation: Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
authors.forename: Y F
authors.initials: YF
authors.lastname: Zhou
authors.forename: X E
authors.initials: XE
authors.lastname: Zhang
authors.forename: H
authors.initials: H
authors.lastname: Liu
authors.forename: C G
authors.initials: CG
authors.lastname: Zhang
authors.forename: A E
authors.initials: AE
authors.lastname: Cass
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics*; D015374:Biosensing Techniques; D003001:Cloning, Molecular; D005949:Glucose Oxidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D005949:Glucose Oxidase