Efficient biosynthesis of lacto-N-neotetraose by a novel β-1,4-galactosyltransferase from Aggregatibacter actinomycetemcomitans NUM4039

Lacto-N-neotetraose (LNnT) is a unique tetrasaccharide naturally occurring in human milk, as an important member of human milk oligosaccharides. Because of promising beneficial effects, it has been commercially added as a functional fortifier in infant formula. β-1,4-Galactosyltransferase (β-1,4-GalT) catalyzes LNnT biosynthesis from uridine 5'-diphospho-galactose (UDP-Gal) to lacto-N-triose II (LNT II). There have been only two LNnT-producing bacterial β-1,4-GalTs, including the ones from Neisseria meningitidis and Histophilus somni. In this study, a novel LNnT-producing β-1,4-GalT was identified from Aggregatibacter actinomycetemcomitans. The enzyme was easily overexpressed in E. coli in soluble form. It displayed much higher transglycosylation versus hydrolysis activity, indicating its great potential in LNnT biosynthesis. The enzyme produced 13 mM LNnT from 20 mM LNT II and 60 mM UDP-Gal, with the yield of 65 % on LNT II and very low level of UDP-Gal hydrolysis. Therefore, it could be considered as a good candidate for the practical LNnT production.