Glycans are amongst the most abundant biomolecules on the planet but we are far from elucidating their role in the design and regulation of biological systems. There is a dire need of tools to systematically synthesize well-defined glycan polymers as is currently possible with other biopolymers like DNA/RNA and proteins. Glycans synthesis is naturally performed by membrane associated glycosyl transferases (GTs), but GTs can be challenging to express sometimes for large-scale synthesis. Alternatively, transglycosidases can be selected from a large repertoire of glycosyl hydrolases (GH) and rationally engineered to enable chemoenzymatic synthesis of designer glycans. Various factors such as modulating substrate and product binding site affinities, regulating water accessibility, and reaction conditions all play important roles in engineering a glycosyl hydrolase to function efficiently as a transglycosidase. Here, we focus on engineering a multifunctional family 5 GH from <i>Clostridium thermocellum </i>(CelE) into a transglycosidase to synthesize diverse glucan-based polymers. We use molecular dynamics (MD) simulations to study how to both tweak substrate specificity and rationally design a highly efficient GH 5 based transglycosidase. We study the effect of active site mutations, presence of various carbohydrate binding modules (CBMs), and varying reaction conditions (such as pH and salt concentrations) on the engineered enzyme’s transglycosylation efficiency and specificity. All transglycosylation products are characterized using TLC, HPLC, and mass spectrometry to decode the synthesized glycan structures and reaction efficiency. In summary, we are developing a broadly applicable tool-kit and workflow to rationally convert glycosyl hydrolases into highly efficient and specific transglycosidases.

Engineering a multifunctional family 5 glycosyl hydrolase into a transglycosidase

256th National Meeting (2018)

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