Study On The Conversion Of Phytosterol To 9α-OH-AD By Mycobacterium Sp.YH

Steroid drugs are currently one of the most important drugs in the world,and it is the second largest class of drugs after antibiotics.There is a great demand at home and abroad,and it has high economic value and broad market prospects.9 a-hydroxy-4-androstene-3,17-Dione(9-hydroxy androstenedione or 9a-OH-AD)can be used to synthesize a variety of glucocorticoid drugs with high anti-inflammatory and anti-sensitivity,which is the key raw material for the synthesis of steroid drugsAt present,steroid active pharmaceutical ingredient(API)intermediates including 9a-OH-AD are mainly obtained by microbial transformation.Microbial transformation of phytosterol to produce 9a-OH-AD has the advantages of less pollution,short cycle and less by-products.However,due to the poor water solubility of phytosterols,the amount of substrate is small and the conversion rate of the product is not highIn this study,Mycobacterium sp.YH was used as the experimental strain and phytosterol as the substrate.Through the optimization of culture medium,fermentation conditions,substrate dosage and oil-water ratio,the whole fermentation system was studied in 15L fermentor and scaled up in 50L fermentor.The corresponding optimum fermentation conditions were obtained.At present,this process has been applied to the actual production of 20t fermentors and 75%fermentation volume,the annual output of 9 a-OH-AD reaches 200 tons.The main results are as follows1.The optimum medium composition was as follows:corn steep powder 18g/L,NaNO3 5g/L,concentrated phospholipid lg/L,(NH4)2HPO4 0.7g/L,glycerol 1g/L,FeSO4·7H2O 0.05g/L,ZnSO4·7H2O 0.1g/L,MgSO4·7H2O 0.15g/L.Under these conditions,the conversion time of 9a-OH-AD was the shortest and the conversion rate was the highest2.The best two-phase fermentation system:soybean oil 15%,phytosterol 5%.Compared to the original technology,the feeding amount of substrate phytosterol was increased by 2.5 times,and the amount of vegetable oil was reduced by 3%3.The optimum fermentation conditions of 50L fermentor:stirring speed 600r/min,ventilation rate 0.5vvm,the amount of inoculation 10%,fermentation volume 70%,the pH of before sterilization 7.0,the fermentation temperature 32℃.Under these conditions,the transformation time of 9a-OH-AD was the shortest4.The optimum fermentation conditions of 20t fermentor are as follows:fermentation volume 70%,stirring speed 180r/min,aeration rate 0.5vvm,inoculum 10%,pH7.0,fermentation temperature 32℃,soybean oil 15%and phytosterol 5%,after conversed for 88h under the above conditions,conversion of 9-hydroxyandrostenedione 81.3 8 ± 1.56%.Under the industrial condition optimized by this research,the microbial transformation of phytosterol to produce 9 a-OH-AD has the advantages of large substrate dosage,low oil consumption,short conversion time and high conversion rate.