Efficient Immobilization of ß-galactosidases using solid-binding peptides to produce galactooligosaccharides from whey

<p dir="ltr">Galactooligosaccharides (GOS) are prebiotics known for their numerous health benefits, making them valuable in the food, cosmetic, and pharmaceutical industries. Industrially, GOS are synthesized from lactose by β-galactosidase, resulting in a mixture of GOS with varying linkages, degrees of polymerization (DP), and branching. β-galactosidases involved in GOS synthesis belong to GH1, GH2, GH35, and GH42 glycoside hydrolase families, with the enzyme's origin playing a key role in determining both GOS yield and composition. Moreover, combining enzymes from different sources has been shown to enhance the diversity of GOS structures (in terms of DP), boost total GOS yield, and improve lactose conversion. The use of enzyme combinations for GOS synthesis is still largely unexplored. In particular, there is limited understanding of how different β-galactosidases combining affect the structural characteristics of GOS mixtures, in terms of DP and linkages. Moreover, the combined impact of enzyme immobilization and multi-enzyme strategies on GOS yield and composition remains unexplored. Enzyme immobilization provides several advantages, including enhanced stability, ease of separation, and reusability. For optimal efficiency, the immobilization carrier must be both cost-effective and stable, with silica and zeolite serving as exemplary supports. Additionally, dairy by-products provide a sustainable and cost-effective source for GOS synthesis.</p><p dir="ltr">This thesis explores three strategies for GOS production: (a) immobilization of β-galactosidases fused to a solid-binding peptide (SBP) linker (to give L-β-galactosidases) onto zeolite; (b) sequential and simultaneous GOS production using combinations of zeolite-immobilized L-β-galactosidases and (c) the use of cheese whey as an alternative substrate.</p><p dir="ltr">To implement these strategies, five β-galactosidases from GH1, GH2, GH35, and GH42 families were genetically fused to the SBP linker. Due to the SBP’s high affinity for silica-based materials, L-β-galactosidases were effectively immobilized onto zeolite, achieving high immobilization efficiencies. A comparative assessment of free and immobilized L-β-galactosidases was conducted, evaluating their pH and temperature optima, thermostability, kinetics, and storage stability. All immobilized enzymes retained their activity over multiple cycles, and immobilization on zeolite did not significantly alter the cation effects on L-β-galactosidases. These findings highlight the potential of SBP-mediated β-galactosidase immobilization for GOS production and lactose hydrolysis in dairy waste treatment.</p><p dir="ltr">The study further examined the structure of GOS produced through sequential and simultaneous reactions using immobilized L-β-galactosidases. GOS products were identified and quantified using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Sequential production strategies enriched GOS with DP ≥ 3, β-(1→3), and β-(1→6) linkages, improving yield while reducing galactose and glucose content. Additionally, GOS production in whey indicated that enzyme origin influenced GOS yield, with whey sometimes altering GOS composition. Immobilization on zeolite had variable effects, either enhancing or reducing GOS yield in whey, with L-β-galactosidase from <i>Bacillus circulans </i>(GH2) proving the most effective.</p><p dir="ltr">Overall, this thesis advances GOS production by developing a robust SBP-mediated immobilization platform based on zeolite, supporting efficient GOS synthesis and lactose hydrolysis in whey.</p>