Infant formula milk tries to mimic human milk as closely as possible, since it is well-known that breast milk has considerable implications in the development of the infant intestinal microbiota in the first years of life. Human milk is considered to be unique in terms of its content of complex oligosaccharides. They are abundant in human milk and their role in the development of intestinal flora blocking the attachment of pathogens and modulating immune system of the infant make them of great interest. To date, 150 chemical structures of human milk oligosaccharides (HMO) had been identified. Two tetrasaccharides are the core structures: the lacto-N-tetraose (type 1) characterized by lacto-N-biose (Galbeta1,3GlcNAc) and lactose units, and lacto-neo-N-tetraose (type 2) formed by N-acetyllactosamine (Galbeta1,4GlcNAc) and lactose. They are elongated to decaose and then, decorated with fucosyl and sialic residues. Humans are the only species in which type 1 HMO dominate over type 2. Until now, large scale synthesis of type 1 HMO has not been possible by any synthetic methodology.
With our expertise on engineering CAZYmes, now we are eager to reach efficient synthesis of the type 1 HMO. For this, we focus on the GH20 family hexosaminidases where lacto-N-biosidases enzymes are present in the gut microbiota of breast-fed infants and able to hydrolyse the type 1 HMO core structures. We have defined the minimal functional structure of Bifidobacterium bifidum lacto-N-biosidase and identified the hot-spots positions on the sugar binding sites using rational design and in silico methods to modulate synthase/hydrolase activities ratio and promote transglycosylation. In this framework, this project aims at obtaining lacto-N-biosidase variants library using random mutagenesis to identify other positions (hot spots) of the catalytic site not discovered by rational design. Then this library will be screened to analyse the mutants with the best transglycosylation/hydrolysis ratio.