Expression of the Fusarium graminearum galactose oxidase GaoA in Saccharomyces cerevisiae

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2024-11-29 DOI:10.1016/j.pep.2024.106637

Lucas Yudai Nozaki, Nathalia Rodrigues Bulka, Karina Lima dos Reis, Damaris Batistão Martim, Fausto Fernandes de Castro, Ione Parra Barbosa-Tessmann

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引用次数: 0

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.

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谷草镰刀菌半乳糖氧化酶gaa在酿酒酵母中的表达。

半乳糖氧化酶是由镰刀菌属真菌产生的一种具有重要生物技术意义的酶。gaoA基因已在几种宿主中重组表达，但尚未在酿酒酵母中表达。本研究的目的是在酿酒酵母中表达谷草镰刀菌GaoA酶。将小麦赤霉病菌gaoA基因全长和截短片段亚克隆到酵母表达载体上。当截断的基因编码成熟形式的GaoA酶时，酿酒酵母中GaoA酶的表达量更高。表达酶经Sepharose 6B柱纯化后，得到纯活性酶的产率为16.7 mg/L。纯化后的蛋白KM为9.8 mM，低于野生型酶，kcat/KM为2.9 × 107 M-1s-1，高于野生型酶。所表达的重组蛋白使用了几种常见的半乳糖氧化酶底物，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增加。所得酶的特性与半乳糖氧化酶的生物技术应用相适应。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.