title: Expression of recombinant galactose oxidase by Pichia pastoris.
abstract: Galactose oxidase catalyzes the oxidation of a variety of primary alcohols, producing hydrogen peroxide as a product. Among hexose sugars, the enzyme exhibits a high degree of specificity for the C6-hydroxyl of galactose and its derivatives, underlying a number of important bioanalytical applications. Galactose oxidase cDNA has been cloned for expression in Pichia pastoris both as the full-length native sequence and as a fusion with the glucoamylase signal peptide. Expression of the full-length native sequence results in a mixture of partly processed and mature galactose oxidase. In contrast, the fusion construct directs efficient secretion of correctly processed galactose oxidase in high-density, methanol-induced fermentation. Culture conditions (including induction temperature and pH) have been optimized to improve the quality and yield (500 mg/L) of recombinant enzyme. Lowering the temperature from 30 to 25 degrees C during the methanol induction phase results in a fourfold increase in yield. A simple two-step purification and one-step activation produce highly active galactose oxidase suitable for a wide range of biomedical and bioanalytical applications.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland, Oregon 97291, USA.
authors.forename: M M
authors.initials: MM
authors.lastname: Whittaker
authors.forename: J W
authors.initials: JW
authors.lastname: Whittaker
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary; D005285:Fermentation; D005692:Galactose Oxidase / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D005692:Galactose Oxidase