title: The bioengineering of β-galactosidase enzymes from the Bifidobacterium bifidum NCIMB 41171 strain to increase GOS production yield and evaluation of novel targets for the discovery of next generation prebiotics
Creators: name: Gawrzak, Anatol
Publisher: name: University of Reading
Year: 2024

description: 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.