Optimization On Dehydroepiandrosterone Production Process Conditions By Microbial Conversion Method

Dehydroepiandrosterone?DHEA,3?-hydroxyandrost-5-en-17-one?is the most abundant hormone in mammals,including humans,and has a wide variety of physiological effects,including major regulatory effects upon the immune system.Two of the most striking aspects of DHEA are the steady decline in DHEA that is observed with age and a significant deficiency in DHEA in patients with several major diseases,including cancer,atherosclerosis,and Alzheimer's disease.DHEA is a representative member of the 17-ketosteroid family,members of which be transformed to pharmacologically significant steroids,such as mifepristone,drospirenone,norethindrone,abiraterone,and ulipristal.In recent years,the market demand for DHEA has increased with the continuous development of steroid industry.The effects of flask seed medium's major ingredients on the Mycobacterium sp.growth were studied.Orthogonal methodology was used to optimize the seed medium.The effect of different cosolvents on phytosterols degradation in aqueous and two phase system was studied.It was found that DHEA production was significantly increased in the presence of an organic-aqueous of two-phase system and the optimal organic phase was vegetable oil.The fermentation medium was optimized by the single factor test and the response surface experiments on the basis of above experiments.The best carbon source,nitrogen source,phosphate source and inorganic salt types were determined.The Response Surface Methodology?RSM?based on a three-factor Box-Behnken design of experiments was used to build an analytical model and finally employed as a constraint in an obvious optimization process.The optimized parameters were soybean oil 30%,corn steep liquor 2%,?NH₄?₂HPO₄ 0.005%,NaNO₃0.054%,substrate 1.5%and fermentation period of 7 d,respectively.After optimization,the concentration of DHEA increased 60.85%with compared to the former?4.13 g/L?.Moreover,the effect on fermentation result of culture conditions,such as inoculation quantity,temperature,initial pH,rating speed,liquid medium etc,was studied to obtain the optimal conditions.The condition of DHEA came to 8.03 g/L,the yield of DHEA 72.90%?mol/mol?.In comparison with the former?4.13 g/L?the concentration of DHEA increased 91.64%.Based on the above results obtained in shake-flasks,the batch biotransformation of3-methoxymethyl-phytosterols was carried out in a 30 L stirred bioreactor to enhance the production of DHEA.Batch biotransformation was carried out at 30°C with 10 L of medium in a30 L stirred bioreactor.The biotransformation medium contained 30%soybean oil and 25 g/L3-methoxymethyl-phytosterols.The pH was controlled at 8.0 with 2 mol/L NaOH and HCl during the biotransformation process.The air flow rate and agitation speed were controlled at 1.0 vvm and 300 rpm,respectively.During the biotransformation process in the bioreactor,cells grew faster and produced more DHEA due to better dissolved oxygen in the bioreactor compared with that present in shake-flasks.With an initial substrate concentration of 25 g/L,more substrate was converted by Mycobacterium sp.NRRL B-3683,and the final substrate conversion reached 96.76%in the 30 L bioreactor.At the end of the biotransformation,the DHEA concentration and yield were 16.33 g/L and 92.65%,respectively.Finally,The DHEA extraction process in the two-phase system was also studied in the article.Through a series of optimization of hydrolysis,extraction,concentration,and crystallization,DHEA products were detected by GC,IR,NMR,and MS.The purity of the product can reach more than 98%,and the technical indicators can meet the sales requirements.