Recombinant bacterial β-galactosidases for lactose bioconversion and galacto-oligosaccharide production: a comparative overview

The increasing demand for lactose-free dairy products, sustainable whey valorization and high-value prebiotic ingredients has intensified the need for efficient bacterial β -galactosidases capable of both lactose hydrolysis and galacto-oligosaccharide (GOS) synthesis. Although numerous bacterial β -galactosidases have been identified and recombinantly produced, information on their structural characteristics, catalytic properties, recombinant production efficiency and performance under application-relevant conditions remains scattered across individual studies, complicating their comparative evaluation for specific biotechnological applications. This review presents a comprehensive comparative overview of recombinant bacterial β -galactosidases with particular emphasis on their potential for lactose bioconversion and GOS production. Current knowledge on enzyme diversity, glycoside hydrolase families, structural organization, molecular determinants of catalysis, recombinant expression systems, purification strategies, hydrolytic activity and transglycosylation capacity is critically evaluated together with recent advances in protein engineering, computational enzyme design and commercially available β -galactosidase preparations. Comparative analysis of published studies indicates that catalytic efficiency alone is insufficient to assess the potential suitability of β -galactosidases for industrial applications. Enzyme performance is influenced by the combined effects of molecular architecture, catalytic properties, recombinant production efficiency, thermostability, product inhibition and transglycosylation capacity. However, direct comparisons remain limited by differences in experimental conditions and by the relatively small number of studies conducted in real dairy matrices or under industrially relevant process conditions. Accordingly, this review compares the major biochemical, molecular, recombinant production and process-related factors that should be considered when evaluating β -galactosidases for lactose hydrolysis, GOS production and large-scale bioprocessing. Particular attention is given to the current limitations in translating laboratory-scale enzyme characterization into industrial application, including limited validation in complex dairy matrices, regulatory considerations, scalable catalyst configurations and downstream processing requirements. Current limitations of commercial β -galactosidase preparations and future opportunities offered by protein engineering, artificial intelligence-assisted enzyme discovery and structure-guided design are also discussed. Overall, this review provides a comparative perspective on recombinant bacterial β -galactosidases and identifies the principal factors influencing their potential application in lactose bioconversion and GOS production, while highlighting the need for further validation under application-relevant and industrial-scale conditions.