Multiple sequence alignment and residue conservation. The method described herein relies on identifying conserved residues. While the concept of amino acid conservation is central to molecular evolution theory, to date there is no agreed, precise definition of what it signifies. To circumvent this obstacle, we have devised a methodology that pinpoints residues that are significantly more conserved compared to others within a given sequence. For this approach to be successful, a ranking method is required that is robust even in extreme cases where the target sequences are either highly diverse, meaning that the overall stringency of conservation is low, or alternatively where they are highly homogenous. The threestep methodology begins with the collection of a large number of sequences, which are then clustered to reduce redundancy . After clustering, iterative multiple sequence alignments (MSA) are performed to select sequences that share at least 5% sequence identity. This minimum of sequence identity (termed "ID% threshold") is then iteratively increased, while at each step the residues are ranked by decreasing conservation. Their conservation is then plotted against the logarithm of their rank (Fig. ). When a significant conservation drop is observed after rank 10-15, the sequences are kept and the ID% threshold is no longer increased. The corresponding MSA is analysed to identify the residues hereafter designated as "conserved". Iterative MSA and conservation analysis are performed using ClustalΩ and an in-house script. To test the methodology, GHs from families 2, 10, 20, 29 and 51 were selected. These cover a wide range of glycosidic bond features, each representing a distinctive synthetic challenge. The outcomes of the sequence analysis are summarized in Table . Comparing GH10 and GH51 shows that similar sequence pool sizes and identical stringency criteria generate different heterogeneity levels (21 vs 35% average identity within sequences kept -termed "mean ID%"), while the cases of GH2 and GH20 reveal that similar heterogeneity can be obtained from a variable number of selected sequences (9301 vs 585 sequences retained from 20 000 retrieved). Nevertheless, in all cases the methodology yields 6-12 candidates for experimental evaluation. . Overall, the challenge of synthesizing β-mannosides has led to interest in enzymatic synthesis and several GH2 mannosidases have been successfully tested . Furthermore, a glycosynthase variant of the Cellulomonas fimi GH2 βmannosidase (CfMan2A) proved to be a proficient synthetic catalyst when fed with donor α-D-mannosyl fluoride and various acceptors . Herein, we investigate whether CfMan2Amediated transmannosylation can be enhanced without using the radical glycosynthase strategy.