Multidimensional Engineering of β-Glucosidase Cel1A for Enhanced Sophorose Synthesis

Abstract Sophorose is a potent cellulase inducer, but its enzymatic synthesis from glucose is limited by native β-glucosidases. Cel1A, a GH1 β-glucosidase from Trichoderma reesei, was engineered using a structure-, energy-, and sequence-guided semirational strategy combining alanine scanning, MM/PBSA analysis, UniKP prediction, saturation mutagenesis, and combinatorial screening, yielding T299G/K240H. This variant exhibited a 10.29-fold apparent higher catalytic efficiency (kcat/Km) relative to wild-type Cel1A, resulting in 12.24 ± 0.67 g L–1 sophorose in 12 h at 37 °C, corresponding to 1.02 ± 0.06 g L–1 h–1. Channel analysis, CaverDock calculations, and molecular dynamics simulations suggested that the mutations favored glucose entry and binding, weakened sophorose binding, and reduced conformational fluctuations. K240 substitutions modulated hydrolysis, transglycosylation, isomerization, and synthesis. These findings identify an improved Cel1A variant and provide a computational interpretation of its enhanced sophorose-synthesizing activity.