Expression of the Fusarium graminearum galactose oxidase GaoA in Saccharomyces cerevisiae.

Abstract

Galactose oxidase, produced by fungi of the genus Fusarium, is an enzyme of great biotechnological importance. The gaoA gene has been recombinantly expressed in several hosts but has yet to be in Saccharomyces cerevisiae. This work aimed to express the Fusarium graminearum GaoA enzyme in S. cerevisiae. The full-length and the truncated F. graminearum gaoA gene were subcloned into a yeast expression vector. The GaoA enzyme expression level in S. cerevisiae was higher when the truncated gene, which codes for the mature form of the enzyme, was used. After purification of the expressed enzyme on a Sepharose® 6B column, the obtained yield of the pure and active enzyme was 16.7 mg/L. The purified protein showed a K_M of 9.8 mM, lower than that of the wild-type enzyme, and a k_cat/K_M of 2.9 × 10⁷ M^-1s^-1, higher than that of the wild-type enzyme. The expressed recombinant protein used several common substrates for galactose oxidase, such as galactose, raffinose, and 1,3-dihydroxyacetone dimer. In addition, it had increased activity on guar gum, lactose, and Arabic gum compared with the wild-type enzyme. The obtained enzyme's characteristics are compatible with the galactose oxidase biotechnological applications.