Pathways of biotransformation of zingiberen newsaponin from Dioscorea zingiberensis C. H. Wright to diosgenin

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Highlights

  • A new steroidal saponin-β-glucosidase from A. flavus was purified and identified.

  • Catalytic characteristics of the new enzyme were studied.

  • Pathways of converting zingiberen newsaponin to diosgenin were firstly elucidated.

Abstract

A new steroidal saponin-β-glucosidase from Aspergillus flavus that specifically hydrolyzed the terminal β-d-glucosyl group at the C-3 position of zingiberen newsaponin, deltonin and trillin from Dioscorea zingiberensis C. H. Wright (DZW) was purified, and characterized. The optimal temperature and pH for the new steroidal saponin-β-glucosidase was 50 °C and pH 5.0. The steroidal saponin-β-glucosidase was stable at 30–60 °C, and retained more than 80% activity. Further, the purified protein was analyzed by ESI-Q-TOF proteomic analyzer. The results indicated that this enzyme is a β-glucosidase of the type glycosidase hydrolase 3 (GH3). Using a combination of the steroidal saponin-β-glucosidase and steroidal saponin-α-1,2-rhamnosidase from Curvularia lunata obtained previously in our lab, the saponins zingieren newsaponin and deltonin could be converted to diosgenin. The pathways of converting zingiberen newsaponin and deltonin into diosgenin by the two key enzymes were elucidated in this study.

Graphical abstract

A new steroidal saponin-β-glucosidase from A. flavus was purified and identified. Pathways of biotransformation of zingiberen newsaopin to diosgenin with the two key enzymes, named steroidal saponin-α-1,2-rhamnosidase and the steroidal saponin-β-glucosidase, were elucidated firstly.

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Introduction

Diosgenin (25R-spirost-en-3β-ol), an aglycon of variety of steroidal saponins in plants, is an important precursor for synthesizing steroidal hormones and steroidal contraceptives [1]. Recently, there are many reports on the significant pharmacological attributes of diosgenin, such as antagonistic effect on cardiovascular diseases [2], anticancer activity, anti-skin aging and anti-hepatitis C virus [3], [4], [5]. Diosgenin mainly exists in the plants rhizomes as steroidal saponins which are its glycosides with different sugar groups [6]. In China, Dioscorea zingiberensis C. H. Wright (DZW) is the preferred species as the resources of its raw material. In industry, diosgenin is produced by strong acid treatment of the DZW rhizomes. However, a great deal of wastewater generated during the production process leads to the serious environmental problems [7]. With the development of biotechnology, mild, safe and environmentally friendly methods are being applied to biotransformation of more and more natural products. In recent years, enzymatic productions of diosgenin from DZW have been reported. Some saponin glycosidases were extracted and purified from microorganisms, plants and livers. The dioscin-α-l-rhamnosidase from pig liver [8] hydrolyzes terminal 1,2-linked rhamnosyl and 1,4-linked rhamnosyl from dioscin to 3-O-β-D-Glc-diosgenin. The dioscin-glycosidase from Absidia sp.d38 [9] gradually hydrolyzes terminal 1,2 linked rhamnosyl, 1,4 linked rhamnosyl and 3-O-β-d-glucosyl from dioscin to diosgenin as final product. In our lab, related reports have been published. Feng [10], [11] found that a glucoamylase from Curvularia lunata has steroidal saponin–rhamnosidase activity. The enzyme can hydrolyze the terminal α-1,2-rhamnosyl residue at the C-3 position of dioscin. Very recently, Lei [12] purified a β-glucosidase from Aspergillus fumigates that can hydrolyze DZW spirostanosides, such as trillin, diosgenin-diglucoside, dioscin, deltonin and gracillin, to produce diosgenin. However, the pathway of the transformation process had not been elucidated. Dong [13] presumes the pathways of biotransformation of DZW by Aspergillus oryzae, but do not purify the key enzymes to confirm the pathways.

In this study, we purified and identified a β-glucosidase from Aspergillus flavus that could hydrolyze terminal 1,4-linked glucosyl residue from zingiberen newsaponin and terminal 1,3-linked glucosyl residue from deltonin to prosapogenin A of dioscin. Using a combination of the β-glucosidase and steroidal saponin-α-1,2-rhamnosidase from C. lunata obtained previously in our lab, the saponins zingiberen newsaponin and deltonin could be converted into diosgenin step by step, so that the pathways of biotransformation of zingiberen newsaponin into diosgenin by the two key enzymes were elucidated for the first time.

Section snippets

Materials

Zingiberen newsaponin, deltonin, prosapogenin A of dioscin, trillin and diosgenin were prepared by our laboratory. A. oryzae 3.4437, Aspergillus niger 3.7390, A. flavus 3.2792, Penicillium purpurogenum 3.5160 and Mucor wutungkiao 3.0025 were purchased from Institute of Microbiology Chinese Academy of Sciences. Middle molecular weight marker proteins for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were obtained from Fermentas (Amercia). Coomassie brilliant blue R-250 was

Screening of fungi for transforming zingiberen newsaponin and deltonin

Five fungi A. oryzae (Ao), A. niger (An), A. flavus (Af), P. purpurogenum (Pp) and M. wutungkiao (Mw) were selected, and their culture broths were used for transforming zingiberen newsaponin and deltonin. According to the TLC analytical results, the culture broth of A. flavus showed the best performance on hydrolyzing zingiberen newsaponin to deltonin and prosapogenin A of dioscin, and hydrolyzing deltonin to prosapogenin A of dioscin. A. oryzae showed weak activity on hydrolyzing zingiberen

Conclusions

The combination of steroidal saponin-β-glucosidase and steroidal saponin-α-1,2-rhamnosidase was used for conversion of zingiberen newsaponin to diosgenin and the yield was more than 80%. The two enzymes were also used for hydrolysis of total saponins from DZW to diosgenin, and reached around 90% yield (data not shown).

The present study elucidates firstly the key enzymes of every step in bioconversion of two main steroidal saponins from DZW, namely zingiberen newsaponin and deltonin, to

Acknowledgements

We are grateful to Shuo Cheng of the Beijing Proteome Research Center for ESI-Q-TOF measurements. This work was financially supported by the Major Program of Municipal Natural Science Foundation of Beijing (No. 7090001), the National Natural Science Foundation of China (No. 81202417), and National Infrastructure of Microbial Resource (No. NIMR-2012-3).

References (13)

  • R. Saunders et al.

    Enzyme Microb. Technol.

    (1986)
  • G.H. Gong et al.

    Chem. Biol. Interact.

    (2010)
  • Y. Tada et al.

    Steroids

    (2009)
  • J.B. Zhu et al.

    J. Pharm. Biomed. Anal.

    (2010)
  • W. Huang et al.

    Bioresour. Technol.

    (2008)
  • B. Feng et al.

    Tetrahedron

    (2007)
There are more references available in the full text version of this article.

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1

These two authors contributed equally to this study.

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