嗜高温sulfolobussolfataricusβ-糖苷酶(SS-βGly),具有比嗜温酶更高的稳定性和活性,具有工业人参皂苷生物转化的潜力。然而,其相对较低的人参皂苷Rd水解活性限制了药物活性次要人参皂苷化合物K(CK)的产生。在这项研究中,首先,我们使用分子对接来预测可能与人参皂苷Rd相互作用的关键酶残基。然后,基于序列比对和丙氨酸扫描诱变方法,确定了可能提高酶催化效率的关键变异位点。N264D变体酶对人参皂苷Rd的酶催化效率(kcat/Km)和底物亲和力(Km)与WT酶相比提高了60%,降低了17.9%,分别,这可能是由于变体酶和底物Rd之间的结合自由能(ΔG)降低。此外,在整个1000-nsMD模拟过程中的马尔可夫状态模型(MSM)分析表明,将N264改变为D使变体酶比野生型(WT)酶和相应的Rd复合物获得更稳定的SS-βGly构象状态。在相同的条件下,N264D酶的相对活性和CK转化率分别比WT酶高1.7和1.9倍。这项研究确定了用于人参皂苷工业生物转化的优异的嗜高温β-糖苷酶候选物。
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.