Abstract

The telomerase activator cycloastragenol (CA) is regarded as a potential anti-aging drug with promising applications in the food and medical industry. However, one remaining challenge is the low efficiency of CA production. Herein, we developed an enzyme-based approach by applying two enzymes (β-xylosidase: Xyl-T; β-glucosidase: Bgcm) for efficient CA production. Both key glycosidases, mined by activity tracking or homology sequence screening, were successfully over-expressed and showed prominent enzymatic activity profiles, including widely pH stability (Xyl-T: pH 3.0–8.0; Bgcm: pH 4.0–10.0), high catalytic efficiency (k_cat/K_m: 0.096 mM^-1s⁻¹ (Xyl-T) and 3.08 mM^-1s⁻¹ (Bgcm)), and mesophilic optimum catalytic temperature (50 °C). Besides, the putative catalytic residues (Xyl-T: Asp311/Glu 521; Bgcm: Asp311/Glu 521) and the potential substrate-binding mechanism of Xyl-T and Bgcm were predicted by comprehensive computational analysis, providing valuable insight into the hydrolysis of substrates at the molecular level. Notably, a rationally designed two-step reaction process was introduced to improve the CA yield and increased up to 96.5% in the gram-scale production, providing a potential alternative for the industrial CA bio-production. In essence, the explored enzymes, the developed enzyme-based approach, and the obtained knowledge from catalytic mechanisms empower researchers to further engineer the CA production and might be applied for other chemicals synthesis.

Key points

• A β-xylosidase and a β-glucosidase were mined to hydrolyze ASI into CA.

• The two recombinant glycosidases showed prominent catalytic profiles.

• Two-step enzymatic catalysis for CA production from ASI was developed.

中文翻译：

---

高效生产抗衰老药物环雌三醇：两种糖苷酶的酶解作用

摘要

端粒酶激活剂环黄芪醇（CA）被认为是一种潜在的抗衰老药物，在食品和医疗行业具有广阔的应用前景。但是，仍然存在的挑战是CA生产效率低下。本文中，我们通过应用两种酶（β-木糖苷酶：Xyl-T；β-葡萄糖苷酶：Bgcm）开发了一种基于酶的方法，以有效地生产CA。通过活性追踪或同源序列筛选得到的两个关键糖苷酶均成功表达，并显示出突出的酶活性特征，包括广泛的pH稳定性（Xyl-T：pH 3.0-8.0； Bgcm：pH 4.0-10.0），高催化效率（k _cat / K _m：0.096 mM ^-1 s ^-1（Xyl-T）和3.08 mM ^-1 s ^-1（Bgcm））和中温最佳催化温度（50°C）。此外，通过全面的计算分析预测了推定的催化残基（Xyl-T：Asp311 / Glu 521； Bgcm：Asp311 / Glu 521）以及潜在的Xyl-T和Bgcm底物结合机理，为深入研究水解提供了有价值的见解。分子水平的底物。值得注意的是，引入了合理设计的两步反应过程以提高CA产量，并在克级生产中提高至96.5％，为工业CA生物生产提供了潜在的替代方法。本质上，探索的酶，已开发的基于酶的方法以及从催化机理中获得的知识使研究人员能够进一步设计CA的生产，并可能用于其他化学合成。

关键点

•开采β-木糖苷酶和β-葡萄糖苷酶以将ASI水解为CA。

•两种重组糖苷酶显示出突出的催化特性。

•开发了用于从ASI生产CA的两步酶催化法。