Biochemical characterization of β-galactosidases and engineering of their product specificity

The human intestine contains tens of trillions of microorganisms, and has a weight up to 2 kg. This gut microbiota is important for human health; it contributes to food digestion, produces some vitamins, and helps combating pathogenic bacteria. Therefore, a healthy and balanced gut microbiota benefits human wellbeing. Prebiotic compounds are special carbohydrates that promote growth of beneficial microorganisms which in turn inhibit harmful microorganisms, and thus stimulate a healthy gut microbiota. Human milk oligosaccharides (hMOS) are the special prebiotics that occur in mother milk and provide strong health benefits to babies. Breast feeding is not possible for every mother, however. In this case, they may choose to use infant formula to feed their babies. Infant formula is based on cow milk, which contains almost no prebiotics. As an alternative to hMOS – galactooligosaccharides (GOS), have been added into the infant formula to mimic the prebiotic functions of hMOS. GOS are carbohydrate mixtures produced by microbial β-galactosidase enzymes from milk sugar. Different β-galactosidase enzymes produce different GOS mixtures, with variations in composition, structures, and also in prebiotics functions. Although various β-galactosidase enzymes and the produced GOS mixtures have been studied, we still knew little about what enzyme features determine the GOS composition and structures produced. This topic therefore has been studied in detail in this PhD thesis, combining enzyme mutagenesis and detailed analysis of products formed. Also, this study has generated new types of prebiotics, which may have better functionality in infant nutrition for babies, and thus have potential industrial application.