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

Journal Article · Fri May 18 04:00:00 EDT 2007 · Biochemical and Biophysical Research Communications

DOI:https://doi.org/10.1016/j.bbrc.2007.03.097· OSTI ID:20991343

Pan, Heng-Yen ^[1]; Whittaker, Mei M ^[1]; Bouveret, Romaric ^[2]; Berna, Anne ^[2]; Bernier, Francois ^[2]; Whittaker, James W ^[1]

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1. Department of Environmental and Biomolecular Systems, Oregon Health and Sciences University, 20000 N.W. Walker Road, Beaverton, OR 97006-8921 (United States)
2. IBMP-Institut de Botanique, Strasbourg (France)

High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an {alpha}-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 x 10{sup 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.

• OSTI ID: 20991343
• Journal Information: Biochemical and Biophysical Research Communications, Journal Name: Biochemical and Biophysical Research Communications Journal Issue: 4 Vol. 356; ISSN BBRCA9; ISSN 0006-291X
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
BIOTECHNOLOGY
ENZYME ACTIVITY
FERMENTATION
GLYCOPROTEINS
MATING
OXALATES
OXIDASES
PEPTIDES
PERIODATES
SODIUM
WHEAT