Discovery of Sialidases with Transglycosylation Activity and Rational Engineering of Their Catalytic Pockets for Efficient Synthesis of 6′-Sialyllactose

Sialyloligosaccharides are essential components of human milk oligosaccharides. Sialidases with transglycosylation activity have shown great potential for industrial production of sialyloligosaccharides using cheaper glycosyl donors. Herein, five novel sialidases with transglycosylation activity were identified from human gut metagenomic DNA, among which BiS47 synthesized 6'-sialyllactose with the highest yield of 1.32 g/L. Molecular dynamics simulations revealed that BiS47 possessed an open conformation of the catalytic pocket which was then remodeled to narrow, a more favorable near-attack conformation, by site-directed mutagenesis and combinatorial mutations based on the design of entrance loops flexibility and bottom residues side chains. Two mutants G185C and G398H were obtained with 6'-sialyllactose yields increased 2.43-fold and 2.45-fold, respectively. "Property grafting" of G185C and G398H onto other four sialidases improved their transglycosylation activities, and eight mutants generated also exhibited narrowed catalytic pockets. This work provides efficient enzymatic tools for sialyloligosaccharide synthesis and important guidance for rational design of glycosidases.