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 (kcat/Km: 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.
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Acknowledgment
We thank the instrument support from Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology, Beijing, China.
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This study was funded by the National Key Research Development Program [Grant No. 2016YFD0400601].
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Yuan Q and Wang W conceived and designed research. Cheng L and Zhang H conducted experiments and computational analysis. Cheng L analyzed data and wrote the manuscript. Wang W and Cui H improved the manuscript. All authors read and approved the manuscript.
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Cheng, L., Zhang, H., Cui, H. et al. Efficient production of the anti-aging drug Cycloastragenol: insight from two Glycosidases by enzyme mining. Appl Microbiol Biotechnol 104, 9991–10004 (2020). https://doi.org/10.1007/s00253-020-10966-5
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DOI: https://doi.org/10.1007/s00253-020-10966-5