Study Of Hydrocortisone From RSA By A Cooperative-mycelia System Of Twin Strains Of Absidia Coerulea AS 3.65 And Curvularia Lunata AS 3.4381

As the important basic steroid drug and intermediate for the synthesis of high quality steroid drugs, Hydrocortisone (HC) has been drawing eyeballs of industries and academic circles. Microbes have been used in the industry for more than semicentennial, they are the Absidia coerulea in China and Curvularia lunata in aborad. In this study, two microbes showed different features. A. coerulea displayed de-acetyl and oxidation activity; but because of the poor-specificity, it made Many by-products accumulated in the media and leaded to the difficulty for purifying the desired HC. The yield of HC was only 45% to RSA and 18% to diosgenin. C. lunata showed better specificity but de-acetyl, and the yield of HC was up to 55-60% to RS. The aim of this thesis was to seek a novel way to promote the yield of HC and the concentration of substrate, with microbes A.coerulea and C.lunata through the improvement of fermentation technology and adding substrate.The higher activity of strains A.coerulea and C.lunata were sreened with the initial strains of Absidia coerulea AS 3.65 and Curvularia lunata AS 3.4381(=ATCC 12017=NRRL 2380), and the slants were passaged 5-7 times and conserved in 4℃. Through comparison of two microbes in the stage of screening, the fermentation feature was to match to the described above. These two microbes were combined to form a cooperative system. RSA (solved in 4% ethanol) being the substrate, three batches which were sequential bioconversion with microbes cultivated respectively, synchronal one with microbes cultivated respectively and mixed one with microbes cultivated together were given, and the results showed that the best one was the sequential bioconversion. Adding C.lunata time, the amount of mycelia and load of substrate, these parameters were optimized. And the inoculum size for two microbes was 5% (v/v) in second stage. The mycelium of A.coerulea had been added to the PBS containing substrate RSA for biotransforming 20h firstly, mycelium of C. lunata was added to construct the cooperative-biocatalyst system. And as the concentration of substrate being 2 and 1g/L, the cooperative-biocatalyst could be reused up to 3 and 6 times respectively. Compare to the transformation of A.coerulea or C.lunata, by-products of 14α-hydroxylation and others produced in single-conversion could be inhibited in this cooperative conversion media effectively. In the level of shake flask, the yield of HC could be up to 81.6% (detected by HPLC).Organic solvent used to transformation of steroid reported in some literatures usually, which was to be inspected in this thesis to increase the total substrate load. In the study of screening solvent, Glycerin investigated the stability of mycelia infirmly was the best choice to the single and cooperative transformation. The system of Tween80/Gly/RSA (1.0/3.0/1.0, v/v/w) for substrate added was given by optimization of Tween80 added and its pre-processing of grinding, ultrasonic waves (UW) and mixed method. This substrate system used to the cooperative conversion could make the load be up to 5g/L, and 104h, RSA could be roundly converted to HC. The scale up experiments in the level of shake flask presents 2-3 Runs utilization of cooperative catalysts and hydrocortisone average yield of 52% with initial RSA substrate concentration of 3g/L. Yield with this improved way was increased by 7-5% from 45-47% with the style of RSA solved in ethanol commonly used to the industrial level nowadays. So the system established was suitable to improve technology of RSA to HC, and it showed that the valuable reference to promote the level of steroid HC manufacture.The substrate system described above, i.e. Tween80/Gly/RSA (1.0/4.0/1.0, v/v/w), was used to the single C.lunata transformation, which gave the interesting thing of high de-acetyl activity to RSA and high yield of HC. Yield of 53.3% could be obtained in 5L fermenter with the load of 2g/L. It's thus clear that better de-acetyl activity of C. lunata to RSA could be demonstrated in suitable bioreactor. This way omited the chemical step of RSA to RS, further more, energy sources be saved and be gentle environment. In one words, this study of the novel biocatalysis and bioprocessing system developed would possess the strong potential for industrial manufacture of HC.