Abstract/Summary

The human gut microbiome is an extensively researched topic due to its potential role in not only influencing the immediate integrity of the intestinal tract, but also having a systemic effect on various metabolic functions throughout the organism. Prebiotics, indigestible substances that are metabolised by gut microbiota to affect bacterial populations and metabolite production, remain of keen interest both in preventative medicine, and as therapeutic factors in a variety of disease states. The first section of the project focused on three GH2 β-galactosidases derived from the Bifidobacterium bifidum NCIMB 41171 strain. Collectively, the 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 engineer a functional chimeric enzyme with increased yield, novel GOS synthesis profile, and/or potential for patentability, detailed structure function studies were initiated. Bioinformatics tools, alongside previous literature, have been utilised to characterise β-galactosidase I (BbgI) and β-galactosidase IV (BbgIV) and evaluate their potential for bioengineering using truncation design. The enzymes were assessed for their hydrolytic and transgalactosydic activity to establish their function and potential effect in the chimera design. The β-galactosidase III (BbgIII) 33-930 truncation, previously used to produce the commercially available prebiotic BIMUNO®, has been re-evaluated in order to design an optimal catalytic core for the chimera. Finally, BbgIII-BbgI/IV chimera variants were designed and their transgalactosydic activity and GOS profile assessed against the BbgIII 33-930 enzyme. This led to the development of a functional chimeric enzyme with retained activity, as well as a GOS profile with increased prevalence of allolactose, and increased prevalence of β1→3 linked oligomers, which have been previously shown to induce a greater bifidogenic effect than β1→4 and β1→6 linked sugars. The second section of the project focused on the derivation of potential novel prebiotics, using alternative bacterial species and their ability to produce secondary metabolites that may confer a prebiotic effect with specific health benefits. Human Milk Oligosaccharides were identified as an adequate target due to their structural similarities to GOS, significantly larger structural diversity, as well as previously identified beneficial effect on the growth of Short-Chain Fatty Acid (SCFA)-producing bacteria. Six novel GH29B α-L-fucosidases derived from such producers have been examined against a previously studied Bifidobacterium bifidum homolog. Bioinformatic tools, alongside previous literature, have been utilised to characterise their tertiary structure and active site features, with the enzymes then evaluated for their ability to synthesize specific HMO products. This led to the identification of a novel enzyme target, the Akkermansia muciniphila α-L-fucosidase (AmAfcB), with further bioengineering strategies for the increase in product yield suggested. Finally, a statistical analysis of glycosyl hydrolase family sequences from prevalent SCFA and lactate-producing bacteria has been conducted in an attempt to observe trends in substrate specificity. This allowed for the identification of target carbohydrates which are likely to induce a more tailored effect through the selective stimulation of growth of butyrate and propionate producers in the human gastrointestinal tract. This work has therefore contributed to the development of novel enzymatic systems for prebiotic synthesis on an industrial scale, as well as future development of prebiotics towards a more specific beneficial effect.