Heterologous expression of a novel galactose-1-phosphate uridylyltransferase from Thermodesulfatator indicus and its application for bioproduction of Gal-β-1,4-GlcNAc-X

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS Protein expression and purification Pub Date : 2024-06-29 DOI:10.1016/j.pep.2024.106538

Kaiqi Li

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Abstract

Nucleotide sugars (UDP-Sugars) are essential for the production of polysaccharides and glycoconjugates utilized in medicines, cosmetics, and food industries. The enzyme Galactose-1-phosphate uridylyltransferase (GalU; EC 2.7.7.12) is responsible for the synthesis of UDP-galactose from α-d-galactose-1-phosphate (Gal-1P) and UTP. A novel bacterial GalU (TiGalU) encoded from a thermophilic bacterium, Thermodesulfatator indicus, was successfully purified using the Ni-NTA column after being expressed in Escherichia coli. The optimal pH for recombinant TiGalU was determined to be 5.5. The optimum temperature of the enzyme was 45 °C. The activity of TiGalU was not dependent on Mg²⁺ and was strongly inhibited by SDS. When coupled with galactose kinase (GALK1) and β-1,4-galactosyltransferase 1 (B4GALT1), the enzyme enabled the one-pot synthesis of Gal-β-1,4-GlcNAc-X by utilizing galactose and UTP as substrates. This study reported the in vitro biosynthesis of Gal-β-1,4-GlcNAc-X for the first time, providing an environmentally friendly way to biosynthesis glycosides and other polysaccharides.

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一种新型半乳糖-1-磷酸尿苷酰转移酶的异源表达及其在生物生产 Gal-β-1,4-GlcNAc-X 中的应用。

核苷酸糖（UDP-糖）是生产用于医药、化妆品和食品工业的多糖和糖结合物所必需的。半乳糖-1-磷酸尿苷酰转移酶（GalU；EC 2.7.7.12）负责从α-D-半乳糖-1-磷酸（Gal-1P）和UTP合成 UDP-半乳糖。一种新型细菌 GalU（TiGalU）由嗜热细菌 Thermodesulfatator indicus 编码，在大肠杆菌中表达后成功地用 Ni-NTA 柱纯化。重组 TiGalU 的最佳 pH 值被确定为 5.5。酶的最适温度为 45°C。TiGalU 的活性不依赖于 Mg2+，但受到 SDS 的强烈抑制。当与半乳糖激酶（GALK1）和β-1,4-半乳糖基转移酶1（B4GALT1）结合使用时，该酶可以利用半乳糖和UTP作为底物，实现Gal-β-1,4-GlcNAc-X的一锅合成。该研究首次报道了体外生物合成 Gal-β-1,4-GlcNAc-X，为生物合成糖苷和其他多糖提供了一种环境友好型方法。

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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.