Nozaki Lucas Yudai, Bulka Nathalia Rodrigues, Dos Reis Karina Lima, Martim Damaris Batistão, Fernandes de Castro Fausto, Barbosa-Tessmann Ione Parra
Department of Biochemistry, State University of Maringá, Av. Colombo, 5790, 87020-900, Maringá, PR, Brazil.
Department of Biochemistry, State University of Maringá, Av. Colombo, 5790, 87020-900, Maringá, PR, Brazil.
Protein Expr Purif. 2025 Mar;227:106637. doi: 10.1016/j.pep.2024.106637. Epub 2024 Nov 29.
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 of 9.8 mM, lower than that of the wild-type enzyme, and a k/K of 2.9 × 10 Ms, 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.
由镰刀菌属真菌产生的半乳糖氧化酶是一种具有重要生物技术意义的酶。高A基因已在多种宿主中进行重组表达，但尚未在酿酒酵母中表达。这项工作旨在在酿酒酵母中表达禾谷镰刀菌的高A酶。将全长和截短的禾谷镰刀菌高A基因亚克隆到酵母表达载体中。当使用编码该酶成熟形式的截短基因时，酿酒酵母中高A酶的表达水平更高。在琼脂糖6B柱上对表达的酶进行纯化后，获得的纯活性酶产量为16.7mg/L。纯化后的蛋白质的K值为9.8mM，低于野生型酶，k/K值为2.9×10Ms，高于野生型酶。表达的重组蛋白使用了几种半乳糖氧化酶的常见底物，如半乳糖、棉子糖和1,3-二羟基丙酮二聚体。此外，与野生型酶相比，它对瓜尔胶、乳糖和阿拉伯胶的活性有所增加。所获得的酶的特性与半乳糖氧化酶的生物技术应用相兼容。
