An Engineered Biocatalyst for the Synthesis of Glycoconjugates: Utilization of β1,3-N-Acetyl-D-glucosaminyltransferase from Streptococcus agalactiae Type Ia Expressed in Escherichia coli as a Fusion with Maltose-Binding Protein

Abstract

A fusion protein composed of β1,3-N-acetyl-D-glucosaminyltransferase (β1,3-GlcNAcT) from Streptococcus agalactiae type Ia and maltose-binding protein (MBP) was produced in Escherichia coli as a soluble and highly active form. Although this fusion protein (MBP-β1,3-GlcNAcT) did not show any sugar-elongation activity to some simple low-molecular weight acceptor substrates such as galactose, Galβ(1→4)Glc (lactose), Galβ(1→4)GlcNAc (N-acetyllactosamine), Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4)Glc (lacto-N-tetraose), and Galβ(1→4)GlcβCer (lactosylceramide, LacCer), the multivalent glycopolymer having LacCer-mimic branches (LacCer mimic polymer, LacCer primer) was found to be an excellent acceptor substrate for the introduction of a β-GlcNAc residue at the O-3 position of the non-reducing galactose moiety by this engineered enzyme. Subsequently, the polymer having GlcNAcβ(1→3)Galβ(1→4)Glc was subjected to further enzymatic modifications by using recombinant β1,4-D-galactosyltransferase (β1,4-GalT), α2,3-sialyltransferase (α2,3-SiaT), α1,3-L-fucosyltransferase (α1,3-FucT), and ceramide glycanase (CGase) to afford a biologically important ganglioside; Neu5Aα(2→3)Galβ(1→4)[Fucα(1→3)]GlcNAcβ(1→3)Galβ(1→4)GlcCerα(IV3Neu5Acα,III3Fucα-nLc4Cer) in 40% yield (4 steps). Interestingly, it was suggested that MBP-β1,3-GlcNAcT could also catalyze a glycosylation reaction of the LacCer mimic polymer with N-acetyl-D-galactosamine served from UDP-GalNAc to afford a polymer carrying trisaccharide branches, GalNAcβ(1→3)Galβ(1→4)Glc. The versatility of the MBP-β1,3-GlcNAcT in the practical synthesis was preliminarily demonstrated by applying this fusion protein as an immobilized biocatalyst displayed on the amylose resin which is known as a solid support showing potent binding-affinity with MBP.