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Author
Gayet Mas, Helena
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Abstract
Human milk oligosaccharides (HMOs) are known not only for shaping the gut microbiota of infants but for being beneficial for immunology, allergies, and cognitive development of newborns. Thus, there is a great interest to create formula milks supplemented with this type of oligosaccharides. There is no animal source of such carbohydrates and, despite the outstanding progress in the field, their synthesis is still challenging and tedious because, mainly, of its highly specific glycosidic bonds. The most abundant HMO in milk is tetrasaccharide lacto-N-tetraose (LNT, Galβ1,3GlcNAcβ1,3Galβ1,4Glc), which is also the core of type 1 HMOs.
An alternative for the preparation of complex carbohydrates such as HMOs consists in the use of engineered enzymes, either evolved transglycosylases or glycosynthases. Combining organic synthesis and biocatalysis is a promising avenue for preparation of oligosaccharides by shorten multistep pathways. The aim of this project is to perform synthesis of LNT through an enzymatic cascade reaction by using two enzymes. The first one, an engineered GH35 galactosidase (BgaC) to perform the synthesis of lacto-N-biose 1,2-oxazoline (LNB-oxa), and the second, an engineered GH20 lacto-N-biosidase to complete the synthesis of LNT.
The laboratory work of this project can be divided in three parts: (i) first, the synthesis of the required both donor and acceptor substrates for the enzymatic reaction catalyzed by BgaC E233G : α-D-galactopyranosyl fluoride (α-D-GalF) and α-D-NAG-oxa; (ii) Then, the expression and purification of the glycosynthase mutant BgaC E233G; and (iii) finally, the evaluation of the enzymatic reaction by using BgaC E233G with three different acceptor substrates, to control its substrate specificity, and varying operating conditions, pH and substrates concentrations.
Although the previously reported substrates specificity of BgaC E233G has been confirmed, this project has leaded us to conclude that α-D-NAG-oxa is not a suitable acceptor for this enzyme in the tested conditions. Indeed, α-D-NAG-oxa was hydrolyzed faster than the kinetics of the enzymatic glycosynthase reaction. Hence, further research is needed, such as other operating conditions (pH, enzyme concentration, etc.) or enzyme engineering, to obtain an efficient chemo-enzymatic synthesis pathway for LNT.
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