Further analysis revealed that using X4 as substrate, RmXyn10A─CM variants synthesized XOS of DP 6-11 (Figs. S2 and S3, Table ). Importantly, compared to the WT enzyme, all variants display greater ability to form XOS exhibiting DP ≥ 8 (Table ), these being the result of multiple transglycosylation events. The findings reveal that the conserved sequence approach led to RmXyn10A variants with significantly enhanced transglycosylation abilities compared to the WT enzyme, primarily due to a drastic decrease of hydrolytic activity (Fig. ). Available structural data reveal that the three most effective mutational targets (H69, N118 and W284) are all closely interacting with the xylose moiety in the -1 subsite (Fig. ). GH20, β-hexosaminidase BbHI. During catalysis, most retaining GHs generate a glycosyl-enzyme intermediate whose anomeric configuration is opposite to that of both the substrate (donor) and the products. However, GH families 18, 20, 25, 56, 84, 85 and 123 use a substrate-assisted mechanism, in which an equatorial N-acetyl in position C-2 of the donor acts as the nucleophile . This leads to the formation of a non-covalent oxazoline-or oxazolinium ion-enzyme intermediate (oxazolinium ion in the case of GH20 ). Importantly, this type of mechanism is predominant in GH-catalyzed reactions involving β-D-GlcNAc or β-D-GalNAc, sugars that are highly prevalent in biological systems. Hence, to assess if the methodology presented here is also relevant when a glycosyl-enzyme intermediate is not formed, a GH20 enzyme was targeted.