Protein engineering and in silico approaches to enhance bacterial lactase activity: A global perspective

This study explores several strategies to develop new lactases with enhanced properties: (1) modification of TmLacS, (2) random mutagenesis, and (3) in-silico approaches for the discovery of novel lactases. Initial efforts focused on introducing a loop into TmLacS, a heat-resistant lactase, resulting in the TmLacS_SL variant, which exhibited a marked reduction in its maximum catalytic rate. Random shuffling mutagenesis was then applied to generate variants of both TmLacS and TmLacS_SL. This approach yielded more active enzymes: the TmLacS mutant TmLac_3H6 displayed a significant increase in total activity, while the TmLacS_SL mutant TmLacS_SL_3A11 showed up to a fivefold improvement compared to TmLacS_SL. In parallel, an in-silico strategy was employed to identify novel β-galactosidases with potential lactase activity. This included bioinformatics screening, phylogenetic analysis to refine the candidate list, and the selection of sequences from thermoresistant organisms with similarities to the desired β-galactosidases. Through this process, the initial pool of over 100 sequences was narrowed to four promising proteins: ThStLac, PsTheLac, CalHydLac, and TeLac. Further characterization revealed TeLac as the most efficient enzyme. • Structural modifications were made to mimic the catalytic site of Kluyveromyces lactis lactase. • DNA shuffling was used to generate mutants with improved enzymatic activity. • In Silico Approach allowed identification of five promising enzymes (TeMaLac, ThStLac, PsTheLac, CalHydLac, and TeLac. • Both the 3H6 mutant and TeLac enzymes are suitable for producing lactose-free products and GOS.