Abstract

The glycoside hydrolase family 20 (GH20) predominantly features N‐acetylhexosaminidases (EC 3.2.1.52), with only few known lacto‐N‐biosidases (EC 3.2.1.140; LNBases). LNBases catalyze the degradation of lacto‐N‐tetraose (LNT), a prominent component of human milk oligosaccharides, thereby supporting a healthy infant gut microbiome development. We investigated GH20 diversity to discover novel enzymes that release disaccharides such as lacto‐N‐biose (LNB). Our approach combined peptide clustering, sequence analysis, and 3D structure model evaluation to assess active site topologies, focusing on the presence of a subsite ‐2. Five LNBases were active on pNP‐LNB and four showed activity on LNT. One enzyme displayed activity on both pNP‐LacNAc and pNP‐LNB, establishing the first report of N‐acetyllactosaminidase (LacNAcase) activity. Exploration of this enzyme cluster led to the identification of four additional enzymes sharing this dual substrate specificity. Comparing the determined crystal structure of a specific LNBase (TrpyGH20) and the first crystal structure of an enzyme with dual LacNAcase/LNBase activity (TrdeGH20) revealed a highly conserved subsite ‐1, common to GH20 enzymes, while the ‐2 subsites varied significantly. TrdeGH20 had a wider subsite ‐2, accommodating Gal with both β1,4‐ and β1,3‐linkages to the GlcNAc in subsite ‐1. Biotechnological applications of these enzymes may include structural elucidation of complex carbohydrates and glycoengineering.