Characterization of GH2 and GH29 Enzymes in Bifidobacterium: Exploring Their Interactions with Human Milk Oligosaccharides Through Bioinformatics and Experimental Approaches

Abstract

Breast milk should serve as the primary source of nourishment for infants, providing the optimal balance of macronutrients, including proteins, lipids, and carbohydrates, essential for healthy growth and development. In addition to these fundamental nutrients, breast milk contains bioactive components that enhance the immune system and offer protection against a wide range of illnesses. Beyond providing nutritional support, breastfeeding significantly contributes to the physical and cognitive development of infants by supporting gut health and brain maturation. This study investigates the enzymatic activity of glycoside hydrolases, specifically GH2 and GH29, derived from Bifidobacterium species. These enzymes are crucial for the digestion of human milk oligosaccharides (HMOs) and play a significant role in maintaining a healthy gut microbiota in infants. The research involved the expression, purification, and functional analysis of these enzymes, with thin-layer chromatography (TLC) employed to evaluate their activity. The GH2 enzyme demonstrated considerable activity on galactosidase substrates such as lactose, lacto-N-tetraose, and lacto-N-neotetraose, indicating its effectiveness in breaking down sugars present in breast milk. In contrast, the GH29 enzyme exhibited limited activity on fucosidase substrates, which may be attributed to suboptimal experimental conditions or insufficient protein expression. Phylogenetic analysis suggests an evolutionary adaptation in these enzymes, enhancing their ability to degrade complex sugars essential for the development of a healthy infant gut microbiome. The study's findings underscore the importance of GH2 in promoting the growth of beneficial gut bacteria, contributing to a favorable intestinal environment. The limited activity observed for GH29 highlights the need for further research to optimize experimental conditions and explore potential interactions between different enzymes. These insights have important implications for improving infant formula by incorporating enzymes that enhance the digestion of sugars, thereby supporting gut health in both breastfed and formula-fed infants. Future research should focus on refining experimental methodologies and investigating the synergistic effects between various enzymes to better understand their roles in infant nutrition and gut microbiota development.