title: Production and characterization of recombinant glucose oxidase from Aspergillus niger expressed in Pichia pastoris.
abstract: Recombinant glucose oxidase from Aspergillus niger expressed in Pichia pastoris by fed-batch fermentation was purified and assessed with 1·26 purification fold to homogeneity using Q-Sepharose F.F. chromatography. The enzyme was determined by SDS-PAGE and gradient PAGE, which showed a dimeric form of 150 kDa. The purified rGOD was proved to be a glycoprotein, and the content of which was estimated to be 36·7 and 25·14% by phenol-sulfuric acid and anthrone-sulfuric acid methods. Characteristics demonstrated that the highest activity was in pH 6·0 at 40°C and was stable at a broad pH range from 4·0 to 9·0 at 55°C or below. The optimum substrate for this enzyme was d-glucose, and the Km was 21·06 mmol l(-1) as well as the Vmax was 359 μmol min(-1) mg(-1). rGOD possessed high resistance to various chemicals except for Hg(2+), Fe(2+), Ag(+), Cu(2+), 1,4-dithiothreitol, sodium dodecyl sulfate and ascorbic acid. In addition, the inhibitors also exhibited intensive fluorescence quenching effect on rGOD.
  
  
  CONCLUSIONS
  Glucose oxidase is a very important enzyme produced by several species. However, large-scale applications have always been postponed by its complexity in fermentation and purification. Our research focused on developing new purification strategy of recombinant GOD from A. niger expressed in P. pastoris. Here, we described this novel one-step purification method and subsequent research in the characteristics of rGOD which showed different results from previous work. These can open new opportunities to increase its application.
authors.affiliation: School of Medical Laboratory Science, Chengdu Medical College, Chengdu, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Meng
authors.forename: M
authors.initials: M
authors.lastname: Zhao
authors.forename: M
authors.initials: M
authors.lastname: Yang
authors.forename: Q
authors.initials: Q
authors.lastname: Zhang
authors.forename: J
authors.initials: J
authors.lastname: Hao
authors.forename: Y
authors.initials: Y
authors.lastname: Meng
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology*; D005285:Fermentation; D005949:Glucose Oxidase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D005949:Glucose Oxidase
keywords: Aspergillus niger; Pichia pastoris; characterization; glucose oxidase; purification