Research On Study On Bioconversion Of Phytosteril Into Steroid Medicine Intermediates By Mycobacter

Sterols hormone plays an indispensable role in clinic. Androst-4-ene-3,17-dione (AD) is an middling product of Sterol hormones for which can not be substituted and have an very important regulative effect in organism,which has a very wide market prospects. We can say that almost of all hormonal medicines are made of AD. At present, every country in the world has large demand on the steroid drugs.This work has investigated the key technology to synthesize AD from Phytosterolas.Using Phytosterolas as starting material, selective side chain degradation of Phytosterolas is a main synthesis method for AD. And the main conclusions were as follows:(1)One of key factors, which hindered producing AD on industrial scale, is that the yield of AD is low. The microorganism of Mycobacterium sp.BD696 was treated by complex mutagenesis of UV,60 C o, UV+60 C o ray irradiation. And AD transformation productivity was 39.2% higher than that of its parent strain and unproduced ADD. via the fifth was also genetic stable. The experiment showed that complex mutagenesis of UV+60 C o ray irradiation was a good method of mutation breeding. The menstruation on transformation of phytosterol into AD with microorganism was established. It was to analyse with TLC firstly and selectively analyse with GC secondly.(2)A series of study on biotransformation of AD by Mycobacterium sp-uv-8 was progressed by transforming the systems of ferment. The effect of polymer-salt two-phase systems, different polymer-polymer two-phase systems on bioconversion was studied. At the same time, the effect of the general concentration of polymer-polymer on bioconversion and the rate of bioconversion in polymer-polymer two-phase systems is higher than in polymer-salt two-phase systems. when the general concentration is 20%, the rate of bioconversion is higher, and when the concentration of PEG10000 and DEX20000 are 12% and 8%, respectively, the rate of bioconversion is up to 50%; And AD transformation productivity was 5% higher than that of 10% concentration respectively. The conversion of Phytosterol to AD and ADD was influenced significantly by pH. pH 8.0～8.5 supported relatively high AD outputs, when pH was 8.5 in Phosphate, AD convertibility was 50.1%. Further accelerated when the cultivation medium was adjusted with phosphate buffer at the pH range 4.0～6.5, and when pH was at 5.5 in citric acid, AD convertibility was 15.9%.(3)The technology of obtaining AD from phytosterol by Microbial degradation in biphasic systems and the effect of concentration of substrate, surfactants and 9a-hydroxylase inhibitor on biotransformation were studied. The results showed that sunflower oil-aqueous biphasic system was the most suitable for bioconversion, and at the low concentration of substrate and 9a-hydroxylase inhibitor could increase the rate of biotransformation to some extent. Finally, the system of ferment was optimized by orthogonal experiment, and the optimized condition was sunflower oil-aqueous biphasic systems with the concentration of sunflower oil 20%, substrate 0.4%, HgCl2 0.005%. Under this condition the maximal yield of AD was 83.4%.(4)The mechanism of AD product from phytosterol by Mycobacterium SP-UC-8 was studied. The effects of various enzymatic inhibitors, accelerants, recusants on the efficiency on bioconversion rate and biomass were also investigated. Results indicated that the effect of concentration of revulsant was important and the entering of revulsant in low-concentration could increase the rate of biotrans-formation.0.15g/L revulsant was entering after incubation time 66h, enzyme active was highest and AD production achieved 2.46g/L and bioconversion rate was 84.2%, the fermenntation time decreased from 168h to 144h; The enzyme activity was stimulated by low-concentration ZnSO4. 7H2O, when concentration of ZnSO4.7H2O was 0.1g/L, the AD output was2.50g/L. The enzyme activity was inhibite by high-concentration, and when ZnSO4.7H2O was0.3g/L, the AD output had distinct decreased; low-concentration of 9a-hydroxylase inhibitor had not obviously efficiency for biomass and substrate consumption, but could increase the rate of biotransformation to a certain extent.(5) The optimal medium was obtaineded by single factor experiment,uniform design and response surface methodology, experiment showed:sunflower oil 211.6283 mL/L, molasses 58.4925 mL/L, Phytosterol 6.0119g/L, NH4NO3 3.2 g/L, (NH4)2HPO4 0.8 g/L, ZnSO4.7H2O1.0g/L, HgCl2 0.055g/L, AD production was 2.5486g/L at this condition. And repeat experiment was preferable. Depending on Central composite design, sunflower oil,molasses and Phytosterol were designed by SAS(?) Systemfor Windows 9.0 and analyzed by Response Surface Analysis method, The Response Surface and the contour were graphed. Three factors and five levels regress model was proposed.The model was provided a reasonable description for the fermentation process by F and t test. The interaction of three factors and two factors were analyzed, the influences of three factors on bioconversion were compared with. The result of influence on bioconversion was:molasses> sunflower oil>Phytosterol. Phytosterol and molasses, molasses and sunflower oil were all have interact influence, but the influences were disnotability. The interact influences of Phytosterol and sunflower oil was distinct.(6) The best fermentation condition for AD was carried out. It was obtained that AD production was effective when Mycobacterium SP-UC-8 was cultured at 30℃, stir speed 220r/min, inocu-lation volume 10%, the initial pH 8.2. The variety orderlines of pH on AD production was also studied, result indicated that Mycobacterium SP-UC-8 could also growth and work at pH 5～9. Is showed when circumstance pH was changed that microorganism could control pHin in its cell system for keeping pHin in nature point or nearly nature point, this system include in cytoplast buffer materials, acid materials, alkali materials or transportation H+ from celll in to cell out by ion pump.(7) The fermentation process of Mycobacterium sp-UV-8 was studied in batch system, and a kinetic model was proposed based on the Logistic and Leudeking-piret equations for microorganism growth, product formation and substrate consumption. Depending on the evaluated model parameters, the model appears to provide a reasonable description for the fermentation process, the average relative error was no more than 7%. The calculated results of models were compared satisfactorily with experimental data. Curve parameters which were obtained(a=0.2320,β=0.0007) by using MATLAB analyse indicated the bioconversion is growth segment associate in all batch cultures with Mycobacterium sp-UV-8. which will offer assurance for the industrial design and production of degradation of phytosterol by Mycobacterium sp-UV-8. Based on previous results, the fermentation process was enlarged 5L and 5000 L, AD yield achieved 3.1 g/L-4.2 g/L respectively.(8) This text makes reference to relevant cultural heritage and the experimentation result of the AD group. Make use of aqueous two-phase systems, separate AD from ferment. The emphasis is the separation. The quantity of table salt joined, static time and the extraction ratio was optimize and select, the result was that joining the table salt of 70 g/L, placing 8 hours and extracting the oil mutually is the best condition for condensability. The refine technics was make sure:Loss on drying =0.5%, Melting point168～173℃, Specific rotation +193°～+202°, which achieved the quality standard of AD.