Characterizing Family 1 Glycosyltransferases (GT1, UGT) by Reverse Glycosylation: Fast Determination of Acceptor Specificity, Donor Specificity, Hydrolysis, and Enzyme Stability.

Manufacturing glycoside conjugates through conventional chemistry is a labor-intensive process that involves multiple reaction steps, generates substantial toxic waste, and often results in low yields with poor control over regioselectivity. In contrast, glycosyltransferase family 1 enzymes offer an efficient alternative, enabling the transfer of glycosyl groups in a single step with excellent regioselective control and high yields. However, the factors governing acceptor specificity and the scope of individual enzymes remain unclear, limiting their broader application. To date, there is no widely implemented, low-cost approach to the high-throughput screening of these enzymes. Here, we use reverse glycosylation with 2-chloro-4-nitrophenyl-β-d-glycosides as a generic high-throughput screening method for UGT characterization. We investigate reverse glycosylation kinetics of 34 UGTs and their donor specificity toward the glucose and galactose moieties. Then, the method is utilized in an acceptor screening with 44 acceptors against 32 enzymes, and we determined the