Heterologous expression, biochemical characterization and transglycosylation potential of a novel GH70 glucansucrase from Lactobacillus delbrueckii subsp. delbrueckii

Enzymatic synthesis of functional biomolecules relevant to food and health industries calls for characterization of enzymes producing such compounds. Among these, enzymes of the GH70 CAZy family have attracted attention due to their ability to synthesize oligosaccharides through transglycosylation. These enzymes operate via a double displacement mechanism, involving the transfer of a glycosyl unit either to water, or to an acceptor molecule determining whether hydrolysis or transglycosylation occurs.

A variant of the GH70 family was expressed in different E.coli strains to determine the most efficient for protein expression. To establish optimal conditions, activity assays were used to evaluate the effect of different reaction conditions in terms of temperature, pH and the presence of various salts. Substrate specificity in terms of both hydrolysis and transglycosylation was assayed using maltose, lactose, sucrose, and cellobiose. Finally, transglycosylation assays were performed and the results were analyzed with thin-layer chromatography (TLC) and high-performance anion-exchange chromatography (HPAEC) to determine optimal timing and conditions for transglycosylation.

The results of the study contribute to a better understanding of GH70 transglycosylation mechanisms while highlighting the need for further investigation of acceptor molecules and transglycosylation portfolios. Ultimately, this research facilitates the production of specialized biomolecules while transforming industrial waste into high added value bioactive compounds.