Abstract/Summary

The human gut microbiome is a complex ecosystem which includes predominantly bacteria, but also protozoa, archaea, eukaryotes, and viruses, that live symbiotically within various sites of the human body. This living microbiome plays a crucial role in human health. However, the gut microbiome equilibrium is delicate, and different strategies to modulate bacterial populations have been proposed. One approach involves the use of prebiotics and probiotics, as supplements, able to alter the microbial composition in the gut and increase its beneficial effects on the host. This project focuses on two Lactobacillus strains already used as a probiotic for their beneficial effects on lipid metabolism. Recent sequencing of specific Lactobacillus plantarum 2830 and Lactobacillus rhamnosus GR1 has revealed potential novel enzymes responsible for beneficial effects on human health. Specifically, this project focuses on three β-galactosidases from L. plantarum (belonging to GH2 and GH42 protein families) and one β-galactosidase from L. rhamnosus (Bgal_LR) belonging to the GH35 protein family. Collectively, the GH family enzymes are responsible for galactooligosaccharide (GOS) synthesis, which can provide prebiotic effects to the host and, consequently, lower cholesterol levels and modulate the gut microflora. In an effort to elucidate the functional role of these lactobacilli enzymes for the production of new GOS products as potential prebiotics, detailed structure-function studies were initiated for these newly identified β-galactosidases. Bioinformatics tools have been utilized to identify important functional and structural domains for these enzymes and X-ray crystallography has been used for their structural characterisation. Subsequently, protein engineering was used to identify specific mutations, for each enzyme of interest, to improve the production of their corresponding novel GOS mixtures. The results obtained from the solved structure of Bgal_LR and the assessment of its corresponding activity profile revealed key residues for successful GOS production. The structure-based protein engineering approach, utilised in this work, highlighted the importance of residues Glu128 and Tyr295 for, respectively, hydrolysis and transgalactosylation. Thus, targeting these and other residues in the active site could further improve the production of a novel GOS mixture. This work has contributed to our understanding of the detailed mechanisms of action of specific lactobacilli enzymes and for the first time has shown that these enzymes can be used in the production of novel GOS.