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. 2023 Dec 18;24(24):17611.
doi: 10.3390/ijms242417611.

The Isolation, Identification and Immobilization Method of Three Novel Enzymes with Diosgenin-Producing Activity Derived from an Aspergillus flavus

Shirong Feng  1 , Lintao Pan  1 , Quanshun Li  1 , Yi Zhang  1 , Fangyuan Mou  1 , Zhao Liu  1 , Yuanyuan Zhang  1 , Longfei Duan  1 , Baofu Qin  1 , Zhongqiu Hu  2
Affiliations

The Isolation, Identification and Immobilization Method of Three Novel Enzymes with Diosgenin-Producing Activity Derived from an Aspergillus flavus

Shirong Feng et al. Int J Mol Sci. .

Abstract

Diosgenin is an important raw material used in the synthesis of steroid drugs, and it is widely used in the pharmaceutical industry. The traditional method of producing diosgenin is through using raw materials provided via the plant Dioscorea zingiberensis C. H. Wright (DZW), which is subsequently industrially hydrolyzed using a high quantity of hydrochloric and sulfuric acids at temperatures ranging from 70 °C to 175 °C. This process results in a significant amount of unmanageable wastewater, creates issues of severe environmental pollution and consumes high quantities of energy. As an alternative, the enzymolysis of DZW to produce diosgenin is an environmentally and friendly method with wide-ranging prospects for its application. However, there are still only a few enzymes that are suitable for production on an industrial scale. In this study, three new key enzymes, E1, E2, and E3, with a high conversion stability of diosgenin, were isolated and identified using an enzyme-linked-substrate autography strategy. HPLC-MS/MS identification showed that E1, a 134.45 kDa protein with 1019 amino acids (AAs), is a zinc-dependent protein similar to the M16 family. E2, a 97.89 kDa protein with 910 AAs, is a type of endo-β-1,3-glucanase. E3, a 51.6 kDa protein with 476 AAs, is a type of Xaa-Pro aminopeptidase. In addition, the method to immobilize these proteins was optimized, and stability was achieved. The results show that the optimal immobilization parameters are 3.5% sodium alginate, 3.45% calcium chloride concentration, 1.4 h fixed time, and pH 8.8; and the recovery rate of enzyme activity can reach 43.98%. A level of 70.3% relative enzyme activity can be obtained after employing six cycles of the optimized technology. Compared with free enzymes, immobilized enzymes have improved stability, acid and alkaline resistance and reusability, which are conducive to large-scale industrial production.

Keywords: dioscin-glycosidase; enzyme immobilization technology; identification; isolation; stability.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(a) Influence of ammonium sulfate concentration on specific enzyme activity. (b) The effect of ammonium sulfate concentration on protein content. (c) Influence of salting-out times on specific enzyme activity. (d) Influence of salting-out times on protein content. (e) Effects of different salting-out methods on specific enzyme activity. (f) Effects of different salting-out methods on protein content. (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.)
Figure 2
Figure 2
The activity verification and molecular weight analysis of the isolated DGAs. (a) Verified dioscin-glycosidases by enzyme-linked-substrate autography. (b) The SDS-PAGE results of purified E1, E2 and E3. Lane 1, 2 and 3: the enzyme solution of E1, E2 and E3 after Native-PAGE, respectively; M: protein marker.
Figure 3
Figure 3
Effect of metal ions on the production of diosgenin by solid-state fermentation. (Control: base medium.) (* p < 0.05; *** p < 0.001; **** p < 0.0001.)
Figure 4
Figure 4
Screening and optimization of enzyme protectants. (a) Effect of the addition of different protective agents on the specific activity of DGAs. (b) Effect of Tween 80 concentration on the specific activity of DGAs. (** p < 0.01; *** p < 0.001; **** p < 0.0001.)
Figure 5
Figure 5
Response surface (3D) and contour plots showing the effect and interaction of the five main factors on the recovery immobilized enzyme. (a,b) Interaction between sodium alginate concentration and CaCl2 concentrations. (c,d) Interaction between time and CaCl2 concentrations. (e,f) Interaction between CaCl2 concentrations and pH. (g,h) Interaction between sodium alginate and pH. (i,j) Interaction between sodium alginate and time. (k,l) Interaction between time and pH.
Figure 6
Figure 6
Stability of immobilized enzymes. (a) pH stability. (b) Temperature stability. (c) Time stability. (d) Stability of the number of cycles.
Figure 7
Figure 7
The scheme of this work.
See this image and copyright information in PMC

References

    1. Mohammadi M., Mashayekh T., Rashidi-Monfared S., Ebrahimi A., Abedini D. New insights into diosgenin biosynthesis pathway and its regulation in Trigonella foenum-graecum L. Phytochem. Anal. 2020;31:229–241. doi: 10.1002/pca.2887. - DOI - PubMed
    1. Zhang X., Jin M., Tadesse N., Dang J., Zhou T., Zhang H., Wang S., Guo Z., Ito Y. Dioscorea zingiberensis C. H. Wright: An overview on its traditional use, phytochemistry, pharmacology, clinical applications, quality control, and toxicity. J. Ethnopharmacol. 2018;220:283–293. doi: 10.1016/j.jep.2018.03.017. - DOI - PubMed
    1. Zhang X.X., Ito Y., Liang J.R., Liu J.L., He J., Sun W.J. Therapeutic effects of total steroid saponin extracts from the rhizome of Dioscorea zingiberensis C.H.Wright in Freund’s complete adjuvant induced arthritis in rats. Int. Immunopharmacol. 2014;23:407–416. doi: 10.1016/j.intimp.2014.07.018. - DOI - PMC - PubMed
    1. He Z., Chen H., Li G., Zhu H., Gao Y., Zhang L., Sun J. Diosgenin inhibits the migration of human breast cancer MDA-MB-231 cells by suppressing Vav2 activity. Phytomedicine. 2014;21:871–876. doi: 10.1016/j.phymed.2014.02.002. - DOI - PubMed
    1. Pang X., Huang H.Z., Zhao Y., Xiong C.-Q., Yu L.Y., Ma B.-P. Conversion of furostanol saponins into spirostanol saponins improves the yield of diosgenin from Dioscorea zingiberensis by acid hydrolysis. RSC Adv. 2015;5:4831–4837. doi: 10.1039/C4RA12709A. - DOI

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