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

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-ß-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.

Identification and a phased pH control strategy of diosgenin bio-synthesized by an endogenous Bacillus licheniformis Syt1 derived from Dioscorea zingiberensis C. H. Wright.

Diosgenin is widely used as one precursor of steroidal drugs in pharmaceutical industry. Currently, there is no choice but to traditionally extract diosgenin from Dioscorea zingiberensis C. H. Wright (DZW) or other plants. In this work, an environmentally friendly approach, in which diosgenin can be bio-synthesized by the endophytic bacterium Bacillus licheniformis Syt1 isolated from DZW, is proposed. Diosgenin produced by the strain was identified by high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and Fourier transform infrared spectroscopy (FTIR). The thermal gravimetric analysis (TGA) showed that the melting point of the diosgenin product was 204 °C. The optical rotation measurement exhibited that the optical rotation was α20589 = - 126.1° ± 1.5° (chloroform, c = 1%): negative sign means that the product is left-handed, which is very important to further produce steroid hormone drugs. Cholesterol may be the intermediate product in the diosgenin biosynthesis pathway. In the batch fermentation process to produce diosgenin using the strain, pH values played an important role. A phased pH control strategy from 5.5 to 7.5 was proved to be more effective to improve production yield than any single pH control, which could get the highest diosgenin yield of 85 ± 8.6 mg L-1. The proposed method may replace phyto-chemistry extraction to produce diosgenin in the industry in the future.Key points• An endophytic Bacillus licheniformis Syt1 derived from host can produce diosgenin.• A dynamic pH industrial control strategy is better than any single pH control.• Proposed diosgenin-produced method hopefully replaces phyto-chemistry extraction.