Donor substrate promiscuity of bacterial β1-3-N-acetylglucosaminyltransferases and acceptor substrate flexibility of β1-4-galactosyltransferases.

•N. meningitidis and H. pylori β3GlcNAcTs were actively expressed in E. coli.•Bovine β4GalT 1 was expressed in E. coli as a soluble active enzyme.•Thirteen UDP-sugars could be used as donor substrates for NmLgtA and Hpβ3GlcNAcT.•Ten trisaccharides could be used as acceptors for one of the three β4GalTs.•GalNAcβ1–3LacβProN3 and Galβ1–3Lac were produced by OPME reaction containing NmLgtA.