Improving Galactooligosaccharide Synthesis Efficiency of β‑Galactosidase Bgal1‑3 by Reshaping the Active Site with an Intelligent Hydrophobic Amino Acid Scanning

There are ongoing interests in improving the galactooligosaccharide (GOS) synthesis efficiency of β-galactosidase by protein engineering. In this study, an intelligent double-hydrophobic amino acid scanning strategy was proposed and employed to target nine residues forming the glycon-binding site (−1 subsite) of β-galactosidase Bgal1-3. Two mutants C510V and H512I with significantly improved GOS synthesis efficiency were obtained. When 40% (w/v) lactose was used as a substrate, Bgal1-3 reached a maximum GOS yield of 45.3% at 16 h, while the mutants reached higher yields in a much shorter time (59.1% at 10 h for C510V, 51.5% at 2 h for H512I). When skim milk was treated with these enzymes, more GOS was produced (19.9 g/L for C510V, 12.7 g/L for H512I) than that for Bgal1-3 (10.3 g/L) at a lactose conversion of 90%. These results validated hydrophobicity scanning as an efficient method to engineer β-galactosidases into promising catalysts for the preparation of GOS and GOS-enriched milk.