Methods for Improving Enzymatic Trans-glycosylation for Synthesis of Human Milk Oligosaccharide Biomimetics

Recently, significant progress has been made within enzymatic synthesis of biomimetic, functional glycans, including, for example, human milk oligosaccharides. These compounds are maminly composed of N-acetylglucosamine, fucose, sialic acid, galactose, and glucose, and their controlled enzymatic synthesis is novel field of research in advanced food ingredient chemistry, involving the use of rare enzymes, which have until now mianly been studied for their biochemical significance, not for targeted biosynthesis applications. For the enzymatic synthesis of biofunctional glycans reaction parameter optimization to promote reverse catalysis with glycosidades is currently preferred over the use of glycosyl transferases. Numerous methods exist for minimizing the undersirable glycosidase-catalyzed hydrolysis and for improving the trans-glycosylation yields. This review provides an overview of the approaches and data available concerning optimization of enzymatic trans-glycosylation for novel synthesis of compled bioactive carbohydraets using sialidases, alpha-L-fucosidase, and beta-galactosidases as examples, The use of an adequately high acceptor/donor ratio, reaction time control, continuous product removal, enzyme recyling, and/or the use of cosolvents may significantly improve trans-glycosylation and biocatalytic productivity of the enzymatic reactions. Protein engineering is also a promising technique for obtaining high trans-glycosylation yields, and proof-of-concept for reversing sialidase activity to trans-sialidase action has been established. However, the protein engineering route currently requires significant research efforts in each case because the structure-function relationship of the enzymes is presently poorly understood.