Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris

• Published in: Biochemical and biophysical research communications 2007-05, Vol.356 (4), p.925-929
• Main Authors: Pan, Heng-Yen, Whittaker, Mei M., Bouveret, Romaric, Berna, Anne, Bernier, François, Whittaker, James W.
• Format: Article
• Language: English
• Subjects: 60 APPLIED LIFE SCIENCES; BIOTECHNOLOGY; Cupin; ENZYME ACTIVITY; FERMENTATION; Germin; Glycan; Glycoprotein; GLYCOPROTEINS; Glycoproteins - chemistry; Glycoproteins - genetics; Glycoproteins - isolation & purification; Glycoproteins - metabolism; MATING; Oxalate oxidase; OXALATES; OXIDASES; Oxidoreductases - chemistry; Oxidoreductases - genetics; Oxidoreductases - isolation & purification; Oxidoreductases - metabolism; PEPTIDES; PERIODATES; Pichia - enzymology; Pichia - genetics; Pichia pastoris; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - isolation & purification; Plant Proteins - metabolism; Protein Engineering - methods; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; SODIUM; Triticum - enzymology; Triticum - genetics; Triticum aestivum; WHEAT
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2007.03.097

High-level secretory expression of wheat ( Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an α-mating factor signal peptide fusion, based on the native wheat cDNA coding sequence. The oxalate oxidase activity of the recombinant enzyme is substantially increased (7-fold) by treatment with sodium periodate, followed by ascorbate reduction. Using these methods, approximately 1 g (4 × 10 4 U) of purified, activated enzyme was obtained following eight days of induction of a high density Pichia fermentation culture, demonstrating suitability for large-scale production of oxalate oxidase for biotechnological applications. Characterization of the recombinant protein shows that it is glycosylated, with N-linked glycan attached at Asn47. For potential biomedical applications, a nonglycosylated (S49A) variant was also prepared which retains essentially full enzyme activity, but exhibits altered protein–protein interactions.