Bioconversion Of Dehydroepiandrosterone By Colletotrichum Lini Resting Cells And Purification Of Products

3β,7α,15α-trihydroxy-5-androsten-17-one(7α,15α-di OH-DHEA) is an important drug intermediate for the synthesis of drospirenone. Drospirenone is the major composition of the oral contraceptives Yasmin. 7α,15α-di OH-DHEA can be obtained by bioconversion of dehydroepiandrosterone(DHEA). But the concentraction of substrate and conversion efficiency are still low using the traditional transformation process, which affected the economic of industrial process. This work was focused on the bioconversion of DHEA into 7α,15α-di OH-DHEA by Colletotrichum lini ST-1 resting cells and purification of products. The main results are as follows.(1) The resting cells bioconversion of DHEA was investigated by Colletotrichum lini ST-1, and the optimized conversion conditions in 250 m L shake flashs were as follows: cell concentration of 12 g·L-1, p H value of 6.5, sodium phosphate buffer concentration of 0.2 M, liquid volume of 30 m L, rotational speed of 220 r min-1 and temperature of 30℃. Under above conditions, the product molar yield was 38.5%, with substrate concentration of 10 g·L-1, which was 21.0% higher than that of control.(2) Methods of substrate griding and co-solvents were tried to enhance the bioconversion efficiency of DHEA. The molar yield of 7α,15α-di OH-DHEA was increased to 72.0% with 10 g·L-1 ground substrate and 2%(w/v) Tween 80. The mechanism of the enhancement caused by Tween 80 was also studied. It was found that adding with 2% Tween 80 could improve the substrate dispersion and change the permeability of cell membrane. Based on above work, batch-conversion with resting cells was investigated. With a total substrate concentration of 18 g·L-1, the molar yield of 7α,15α-di OH-DHEA reached 60.5% through two-batch bioconversion of 78 h.(3) The isolation and purification process of products was optimized. Firstly, the pretreatment process of transforming liquor and extraction of products were optimized, and the recovery yield of crude extraction was 95%. Secondly, the crude extraction of products was isolated through column chromatography and the product of 7α,15α-di OH-DHEA was obtained successfully. The separated product was crystallized by ethyl acetate and the purity of the product reached 98%. Based on all above process, the product recovery efficiency was 91.3%. In addition, the structure of the product was proved to be 7α,15α-di OH-DHEA by high performance liquid chromatography, mass spectra, infrared and nuclear magnetic resonance analysis.