Branching pattern of gluco-oligosaccharides and 1.5 kDa dextran grafted by the α-1,2 branching sucrase GBD-CD2

GBD-CD2, an engineered sucrose-acting enzyme of glycoside hydrolase family 70, transfers d-glucopyranosyl (d-Glcp) units from sucrose onto dextrans or gluco-oligosaccharides (GOS) through the formation of α-(1 → 2) linkages leading to branched products of interest for health, food and cosmetic applications. Structural characterization of the branched products obtained from sucrose and pure GOS of degree of polymerization (DP) 4 or DP 5 revealed that highly α-(1 → 2) branched and new molecular structures can be synthesized by GBD-CD2. The formation of α-(1 → 2) branching is kinetically controlled and can occur onto vicinal α-(1 → 6)-linked d-Glcp residues. To investigate the mode of branching of 1.5 kDa dextran, simulations of various branching scenarios and resistance to glucoamylase degradation were performed. Analysis of the simulation results suggests that the branching process is stochastic and indicates that the enzyme acceptor site can accommodate both linear and poly-branched acceptors. This opens the way to the design of novel enzyme-based processes yielding carbohydrate structures varying in size and resistance to hydrolytic enzymes.