Pathways of biotransformation of zingiberen newsaponin from Dioscorea zingiberensis C. H. Wright to diosgenin
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.
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)
- et al.
Enzyme Microb. Technol.
(1986) - et al.
Chem. Biol. Interact.
(2010) - et al.
Steroids
(2009) - et al.
J. Pharm. Biomed. Anal.
(2010) - et al.
Bioresour. Technol.
(2008) - et al.
Tetrahedron
(2007)
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- 1
These two authors contributed equally to this study.