Efficient Mutagenesis Strategy Based on Nonpolar Amino Acids Scanning for the Improvement of Transglycosylation Ability of β-Galactosidases

A commercial β-galactosidase from Aspergillus oryzae was genetically modified through semirational design to enhance its transglycosylation ability for galactooligosaccharides (GOSs) production. By disrupting hydrogen bonds, altering the hydrophobicity and enlarging the catalytic pocket, 12 single-point mutants and a combinatorial mutant (M3) with enhanced transgalactosylation abilities were obtained. Mutant M3 was successfully expressed in Aspergillus niger, and a β-galactosidase production of 228.2 U/mL was achieved. M3 efficiently catalyzed the synthesis of GOSs, with a high yield of 62.3% (w/w), which was comparable to that of the highest value for GOS production (63.3%, w/w) ever reported. Structural analysis revealed that weak enzyme-galactose interaction and high hydrophobicity of the catalytic pocket may contribute to the enhancement of transgalactosylation ability of AoBgal35A. Thus, a mutagenesis strategy named nonpolar amino acids scanning was constructed on the basis of adjusting enzyme-galactose interaction as well as the hydrophobicity of the catalytic pocket. To validate the strategy, 3 β-galactosidases were further modified and the GOS yields of 2 were improved by 30-40%. This study may provide an excellent catalyst for commercial GOS production as well as a rapid strategy for the modification of β-galactosidases.