Sucrose-utilizing transglucosidases are valuable enzymatic tools for the diversification of carbohydrate-based molecules. Among them, recombinant amylosucrase from Neisseria polysaccharea is a glucansucrase that naturally catalyzes the synthesis of an amylose-like polymer as well as the transglucosylation of exogenous hydroxylated acceptors. A semirational engineering approach was recently undertaken to redesign the enzyme active site and adapt it to the glucosylation of a nonnatural acceptor, allyl 2-N-acetyl-2-deoxy-α-D-glucopyranoside (α-D-GlcpNAcOAll), to produce a key building block in the chemoenzymatic synthesis of Shigella flexneri 1b serotype O-antigen repeating unit. This prior work shows the beneficial effect of single amino acid mutations at two positions (228 and 290) on the recognition of the acceptor by amylosucrase. On the basis of these first results, a library of about 8000 amylosucrase variants combining mutations at these two positions is constructed by saturation mutagenesis. The library is prescreened using a novel pH-sensitive colorimetric screening method for the detection of sucrose-utilizing amylosucrase variants, thereby reducing by about 95 % the size of the library to be subsequently screened for acceptor glucosylation. Active clones (5 % of the initial library) are then screened for acceptor recognition, leading to the isolation of 20 variants of potential interest for the production of the target disaccharide α-D-Glcp-(1→4)-α-D-GlcpNAc.