Human milk oligosaccharides (HMOs) are bioactive glycans that play a crucial role in infant health, yet their large-scale production remains a major challenge. HMOs have bactericidal, bacteriostatic and antibiofilm properties. They help in gut maturation and brain development in infants. Lacto-N-triose II (LNT II) is a key HMO core structure and an important precursor for the synthesis of more complex HMOs. Three N-acetylglucosaminyltransferases, BaGlcNAcT, RsGlcNAcT, and BiGlcNAcT, were evaluated for their ability to catalyze the enzymatic synthesis of LNT II from lactose using UDP-GlcNAc as the donor substrate. The genes encoding these enzymes were synthesized, and heterologously expressed in E. coli BL21(DE3). Recombinant proteins were purified using Ni–NTA affinity chromatography and functionally characterized. Product formation was confirmed by thin-layer chromatography, ion chromatography, and liquid chro-matography–mass spectrometry. All three enzymes were capable of producing LNT II; however, BaGlcNAcT exhibited superior performance, with the highest lactose conversion efficiency, the greatest LNT II yield, and minimal residual substrate compared to the other enzymes. In addition, BaGlcNAcT demonstrated a higher recombinant protein yield per litre of culture. These results identify BaGlcNAcT as a highly efficient catalyst for LNT II biosynthesis and highlight its potential for scalable enzymatic production of HMOs. This work provides a promising foundation for future biocatalytic and metabolic engineering strategies aimed at sustainable HMO manufacturing.

Oligosaccharides, Glycans, N-acetylglucosaminyltransferases, BiGlcNAcT, Chromatography, Recombinant