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Protein Expr Purif. 2000 Oct;20(1):105-11.

Expression of recombinant galactose oxidase by Pichia pastoris.

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  • 1Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland, Oregon 97291, USA.


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.

Copyright 2000 Academic Press.

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