Hyperthermophilic Sulfolobus solfataricus β-glycosidase (SS-βGly), with higher stability and activity than mesophilic enzymes, has potential for industrial ginsenosides biotransformation. However, its relatively low ginsenoside Rd-hydrolyzing activity limits the production of pharmaceutically active minor ginsenoside compound K (CK). In this study, first, we used molecular docking to predict the key enzyme residues that may hypothetically interact with ginsenoside Rd. Then, based on sequence alignment and alanine scanning mutagenesis approach, key variant sites were identified that might improve the enzyme catalytic efficiency. The enzyme catalytic efficiency (kcat/Km) and substrate affinity (Km) of the N264D variant enzyme for ginsenoside Rd increased by 60% and decreased by 17.9% compared with WT enzyme, respectively, which may be due to a decrease in the binding free energy (∆G) between the variant enzyme and substrate Rd. In addition, Markov state models (MSM) analysis during the whole 1000-ns MD simulations indicated that altering N264 to D made the variant enzyme achieve a more stable SS-βGly conformational state than the wild-type (WT) enzyme and corresponding Rd complex. Under identical conditions, the relative activities and the CK conversion rates of the N264D enzyme were 1.7 and 1.9 folds higher than those of the WT enzyme. This study identified an excellent hyperthermophilic β-glycosidase candidate for industrial biotransformation of ginsenosides.
Keywords: Alanine scanning mutagenesis; Ginsenoside Rd hydrolysis; Ginsenosides biotransformation; Ginsenosides compound K; Glycosidase enzyme designing.
© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.