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Heterologous expression of a novel galactose-1-phosphate uridylyltransferase from Thermodesulfatator indicus and its application for bioproduction of Gal-β-1,4-GlcNAc-X.
- Source :
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Protein expression and purification [Protein Expr Purif] 2024 Oct; Vol. 222, pp. 106538. Date of Electronic Publication: 2024 Jun 29. - Publication Year :
- 2024
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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 Mg <superscript>2+</superscript> 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.<br /> (Copyright © 2024 Elsevier Inc. All rights reserved.)
- Subjects :
- Bacterial Proteins genetics
Bacterial Proteins chemistry
Bacterial Proteins metabolism
Bacterial Proteins biosynthesis
Bacterial Proteins isolation & purification
UTP-Hexose-1-Phosphate Uridylyltransferase genetics
UTP-Hexose-1-Phosphate Uridylyltransferase metabolism
UTP-Hexose-1-Phosphate Uridylyltransferase chemistry
Gene Expression
Nucleotidyltransferases genetics
Nucleotidyltransferases metabolism
Nucleotidyltransferases chemistry
Cloning, Molecular
Galactosephosphates metabolism
Galactosephosphates genetics
Galactosyltransferases genetics
Galactosyltransferases metabolism
Galactosyltransferases chemistry
Escherichia coli genetics
Escherichia coli metabolism
Recombinant Proteins genetics
Recombinant Proteins metabolism
Recombinant Proteins chemistry
Recombinant Proteins biosynthesis
Recombinant Proteins isolation & purification
Subjects
Details
- Language :
- English
- ISSN :
- 1096-0279
- Volume :
- 222
- Database :
- MEDLINE
- Journal :
- Protein expression and purification
- Publication Type :
- Academic Journal
- Accession number :
- 38950762
- Full Text :
- https://doi.org/10.1016/j.pep.2024.106538