Previously, GH20 hexosaminidases have been extensively used for transglycosylation . Therefore, herein we focused on BbHI from Bifidobacterium bifidum, sp. infantis . This enzyme has the ability to catalyse the synthesis of LNT2 (β-D-GlcpNAc-(1→3)-β-D-Galp-(1→4)-D-Glc, also known as lacto-N-triose II), a widely sought-after glycomotif that is present in human milk oligosaccharides . Moreover, BbHI-D746E -D746 being involved in the oxazolinium ion stabilization -has been recently reported as the first glycosynthase with high enzymatic activity, thus requiring only moderate enzyme loadings variants and BbHI-WT were analysed for their ability to synthesize LNT2 from 10 mM β-D-GlcpNAcOpNP and 40 mM lactose (Fig. ). BbHI-H603F displayed the highest T/H ratio (data not schown), but also a drastically reduced activity. With the highest enzyme loading (15 µM), the reaction was incomplete after 12 h, thus further characterization of BbHI-H603F was abandoned. The variants R577K and W882H displayed high yields of LNT2 (66% for W882H), although the latter was also poorly active. Nevertheless, BbHI-R577K represents an interesting compromise, since compared to BbHI-WT, it generated a markedly higher yield (36 vs 16%) of LNT2 at a reasonable enzyme loading (1.3 μM). Importantly, these results demonstrate that our strategy is not restricted to retaining GHs catalysing transglycosylation through a covalent glycosyl-enzyme intermediate. Conversely, the application of our approach to another GH20, from Ewingella americana failed to generate variants able to perform transglycosylation using β-D-GlcpNAcOpNP as donor and lactose as an acceptor (data not shown). Although this might indicate that the strategy is not fully generic, we believe that this was unsuccessful due to the fact that the WT GH20 from Ewingella americana displays no detectable ability to transglycosylate lactose (no other acceptors were tested). This emphasises that the existence of innate, albeit weak, transglycosylation activity is possibly a prerequisite for success when using our approach.