Enhancing ginsenoside biotransformation by engineering a thermophilic enzyme: Disulfide bond-induced...

The thermophilic β-glycosidase from Sulfolobus solfataricus (SS-Bgl) has great potential for producing rare ginsenoside CK from ginseng stems and leaves, which are rich in main ginsenosides. To enhance its thermal stability, we designed disulfide bonds based on MD simulations and the Disulfide by Design 2.0 (DbD2) tool. Unexpectedly, introducing disulfide bonds in flexible loops (mutants P95C/R178C and F98C/R178C) significantly increased catalytic activity but decreased stability. The CK productivity from ginsenosides Rb1, Rb2, and Rd of the mutant P95C/R178C increased by 83.51%, 10.90%, and 13.83%, respectively. Correspondingly, the F98C/R178C mutant exhibited increases of 105.79%, 43.80%, and 32.94% under the same conditions. Further MD simulations revealed that the disulfide bond induced conformational changes, shortening the distance between the catalytic residue Glu206 and the substrate to facilitate hydrolysis, while increasing flexibility in distal regions and reducing stability. This study reveals a unique activity-stability trade-off, providing both promising industrial catalysts and insights for glycosidase design.