Fine-Tuning the Active-Site Microenvironment of β-Galactosidase to Enhance the Synthesis Ability of Galactooligosaccharides while Minimizing the Impairment to Transglycosylation Activity

Most reported mutations at -1 subsites of β-galactosidases that improved galactooligosaccharides (GOS) synthesis ability significantly reduced total activity (e.g., <10%), likely due to the introduction of significant disturbances within the active-site microenvironment. In this study, a fine-tuning strategy encompassing aromatic residue interchange as well as substitution among T, S, and A was proposed and subsequently evaluated in Bgal1-3 and three commercial β-galactosidases. For each of them, 2-4 positive mutants were acquired with residual activities of 30-357%. When 40% (w/v) lactose was employed as a substrate, their GOS yields were 1.2-18% higher than those of wild types. Moreover, the best mutants produced greater amounts of GOS in skim milk (2.9-11.8 g/L higher) at a lactose conversion rate of 90%. Ultimately, a mutation set (∼14 mutations) was designed for the convenience of using this approach in other glycoside hydrolases. This fine-tuning strategy may hold great potential for promoting the enzymatic synthesis of valuable carbohydrate-containing compounds.