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Engineered β-glycosidase from Hyperthermophilic Sulfolobus solfataricus with Improved Rd-hydrolyzing Activity for Ginsenoside Compound K Production

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Abstract

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.

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Data Availability

The data generated and analyzed during the current study are available from the corresponding author on reasonable request.

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Funding

This research was supported by the National Key R&D Program of China (2021YFC2101500, 2019YFA0905200, 2021YFC2101000) and the National Natural Science Foundation of China (21706211, 22108229). The authors would like to thank all the reviewers who participated in the review and MJEditor (http://www.mjeditor.com) for its linguistic assistance during the preparation of this manuscript.

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Authors and Affiliations

  1. Shaanxi Key Laboratory of Degradable Biomedical Materials, Shaanxi R&D Center of Biomaterials and Fermentation Engineering, Biotech. & Biomed. Research Institute, School of Chemical Engineering, Northwest University, Shaanxi, 710069, China

    Chenchen Fu, Wenfeng Shen, Weina Li, Pan Wang & Daidi Fan

  2. Beijing Bioprocess Key Laboratory, Beijing University of Chemical Technology, Beijing, 100029, People’s Republic of China

    Luo Liu

  3. Shaanxi Giant Biogene Co., Ltd, Xi’an, 710065, Shaanxi, China

    Yangfang Dong

  4. Xi’an Giant Biogene Co., Ltd, Xi’an, 710065, Shaanxi, China

    Jing He

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  1. Chenchen Fu
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  2. Wenfeng Shen
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  3. Weina Li
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  4. Pan Wang
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  5. Luo Liu
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  7. Jing He
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  8. Daidi Fan
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Contributions

CF: the acquisition of data and study conduct, and writing original draft. WL: the study conception and design, and modified the manuscript. WS: review responses and revise. PW and LL: conceptualization, validation, project administration. YD and JH: reagents or analytical tools. DF: supervision, resources.

Corresponding authors

Correspondence to Weina Li or Daidi Fan.

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Key Points

• N264D SS-βGly has improved Rd-hydrolyzing activity.

• MD simulation studies indicated that N264D mutation had no obvious effect on the enzyme structure.

• N264D mutation improved enzyme-substrate binding by 1.5 times according to binding free energy calculations.

• The engineered enzyme has a 1.9-fold higher catalytic efficiency for CK production.

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Fu, C., Shen, W., Li, W. et al. Engineered β-glycosidase from Hyperthermophilic Sulfolobus solfataricus with Improved Rd-hydrolyzing Activity for Ginsenoside Compound K Production. Appl Biochem Biotechnol (2023). https://doi.org/10.1007/s12010-023-04745-x

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