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Author
Fargas Gutiérrez, Ignacio
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Abstract
To date, one of the main challenges faced by companies producing infant formula milk is to obtain a product whose properties mimic those of breast milk as much as possible, whose unique composition in oligosaccharides provides hardly replaceable features. The promotion of a correct development of the gut microbiota, modulating the immune system and protecting the body against infections by blocking the adhesion of pathogenic bacteria are just a few examples of the many benefits associated with the consumption of these Human Milk Oligosaccharides, more commonly known as HMOs.
Nowadays, the structure of more than 200 HMOs in breast milk has been discovered and identified, whose basic elements that make up the majority are the following 5 monosaccharides: D-Glucose (Glc), D-Galactose (Gal), N-acetyl-D-glucosamine (GlcNAc), L-fucose (Fuc), and N-acetylneuraminic or sialic acid (Neu5Ac). Due to the lack of natural sources that provide these molecules in sufficient amounts for use as an additive in infant milk powder, numerous investigations have been carried out in order to produce HMOs artificially and on a large scale in order to address the huge demand for this type of compounds. The high complexity of the chemical synthesis processes, as well as the low production yields, make this, not a viable option to be transferred to an industrial scale. On the other hand, it has been discovered that the enzymatic synthesis could suppose a promising methodology due to the fact that it allows the formation of glycosidic bonds with a high enantio- and stereo- specificity, giving rise to complex structures; as it happens in nature.
In this context, the objective of this project is to use the native Lacto-N-biosidase of Bifidobacterium bifidum (LnbB) as a biocatalyst for the synthesis of Lacto-N-tetraose (LNT). In its wild type form, this enzyme catalyzes the hydrolysis of LNT into Lacto-N-biose (LNB) and lactose. Previous studies have shown that the application of protein engineering techniques on specific LnbB residues allows the hydrolytic reaction to be reversed, shifting the balance towards the transglycosilation reaction.
Throughout this research, protein engineering methodologies have been used to modify specific positions of the LnbB to generate the mutants W394F/H263R and W394F/Q190L in order to be compared against the W394F. The expression of the resulting proteins and the characterization of their enzymatic activity revealed a reduction in the hydrolytic activity and in the transglycosilation activity of the W394F/Q190L mutant.
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