The Isolation, Identification and Immobilization Method of Three Novel Enzymes with Diosgenin-Producing Activity Derived from an <i>Aspergillus flavus</i>
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 <i>Dioscorea zingiberensis</i> C. H. Wright (<i>D...
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MDPI AG
2023-12-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/24/17611 |
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| author | Shirong Feng Lintao Pan Quanshun Li Yi Zhang Fangyuan Mou Zhao Liu Yuanyuan Zhang Longfei Duan Baofu Qin Zhongqiu Hu |
| author_facet | Shirong Feng Lintao Pan Quanshun Li Yi Zhang Fangyuan Mou Zhao Liu Yuanyuan Zhang Longfei Duan Baofu Qin Zhongqiu Hu |
| author_sort | Shirong Feng |
| collection | DOAJ |
| description | 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 <i>Dioscorea zingiberensis</i> C. H. Wright (<i>DZW</i>), 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 <i>DZW</i> 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. |
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| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-08T20:41:18Z |
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| spelling | doaj.art-e92b38a861a7455985b9d49a0dc6caea2023-12-22T14:15:21ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-12-0124241761110.3390/ijms242417611The Isolation, Identification and Immobilization Method of Three Novel Enzymes with Diosgenin-Producing Activity Derived from an <i>Aspergillus flavus</i>Shirong Feng0Lintao Pan1Quanshun Li2Yi Zhang3Fangyuan Mou4Zhao Liu5Yuanyuan Zhang6Longfei Duan7Baofu Qin8Zhongqiu Hu9College of Life Sciences, Northwest A&F University, Xianyang 712100, ChinaCollege of Life Sciences, Northwest A&F University, Xianyang 712100, ChinaCollege of Life Sciences, Northwest A&F University, Xianyang 712100, ChinaCollege of Life Sciences, Northwest A&F University, Xianyang 712100, ChinaCollege of Life Sciences, Northwest A&F University, Xianyang 712100, ChinaCollege of Life Sciences, Northwest A&F University, Xianyang 712100, ChinaCollege of Life Sciences, Northwest A&F University, Xianyang 712100, ChinaCollege of Life Sciences, Northwest A&F University, Xianyang 712100, ChinaCollege of Life Sciences, Northwest A&F University, Xianyang 712100, ChinaCollege of Food Science and Engineering, Northwest A&F University, Xianyang 712100, ChinaDiosgenin 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 <i>Dioscorea zingiberensis</i> C. H. Wright (<i>DZW</i>), 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 <i>DZW</i> 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.https://www.mdpi.com/1422-0067/24/24/17611dioscin-glycosidaseisolationidentificationenzyme immobilization technologystability |
| spellingShingle | Shirong Feng Lintao Pan Quanshun Li Yi Zhang Fangyuan Mou Zhao Liu Yuanyuan Zhang Longfei Duan Baofu Qin Zhongqiu Hu The Isolation, Identification and Immobilization Method of Three Novel Enzymes with Diosgenin-Producing Activity Derived from an <i>Aspergillus flavus</i> International Journal of Molecular Sciences dioscin-glycosidase isolation identification enzyme immobilization technology stability |
| title | The Isolation, Identification and Immobilization Method of Three Novel Enzymes with Diosgenin-Producing Activity Derived from an <i>Aspergillus flavus</i> |
| title_full | The Isolation, Identification and Immobilization Method of Three Novel Enzymes with Diosgenin-Producing Activity Derived from an <i>Aspergillus flavus</i> |
| title_fullStr | The Isolation, Identification and Immobilization Method of Three Novel Enzymes with Diosgenin-Producing Activity Derived from an <i>Aspergillus flavus</i> |
| title_full_unstemmed | The Isolation, Identification and Immobilization Method of Three Novel Enzymes with Diosgenin-Producing Activity Derived from an <i>Aspergillus flavus</i> |
| title_short | The Isolation, Identification and Immobilization Method of Three Novel Enzymes with Diosgenin-Producing Activity Derived from an <i>Aspergillus flavus</i> |
| title_sort | isolation identification and immobilization method of three novel enzymes with diosgenin producing activity derived from an i aspergillus flavus i |
| topic | dioscin-glycosidase isolation identification enzyme immobilization technology stability |
| url | https://www.mdpi.com/1422-0067/24/24/17611 |
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