Development And Research Of Novel Environmental Process For The 9-OH-AD Preparation Through Phytosterols Bio-conversion

9-OH-AD(9ɑ-hydroxyandrost-4-ene-3,17-dione)is an important intermediate of steroid drugs.As the presence of 9-hydroxyl in the chemical structure,a halogen atom can be introduced to the site of C9.9-OH-AD can be used for the synthesis of several high-efficient glucocorticoids with the halogen(fluorine,chlorine),so it is attracted a lot of attention by the people.Recently,researches about the one-step biotransformation preparation of 9-OH-AD from phytosterols have been reported at home and abroad.However,the concentration of the obtained conversed product is still relatively low,the production strain and transformation preparation conditions of9-OH-AD for industrial production are required for the further exploration.Specially,phytosterols are generally dissolved in the oil or other lipid reagents to perform the transformation preparation of 9-OH-AD among the common researches.A large number of organic reagents are needed for the products extraction due to the presence of oil,which make the complex extract process and unfriendly environment,so the paper focuses on the study of the new environmental oil-free process substituting the oil-using process to explore the preparation of 9-OH-AD transformed from phytosterols,and the main researches were carried out as follows:1.Firstly,to obtain the high-efficient strain,which could adapt to the oil-free process,the conditions of the protoplasts preparation from the original strain Mycobacterium sp.have been investigated in detail.The results showed that the best osmotic stabilizer for protoplast preparation of Mycobacterium sp.was Na Cl,and the optimum concentration of Na Cl was 0.5 mol/L.Under that condition,3.10×10~8/m L protoplasts were achieved.Subsequently,we explored the growth and UV mutagenesis conditions of the Mycobacterium sp..The results demonstrated that the logarithmic growth phase of Mycobacterium sp.was from 48 h to 96 h and the UV irradiation time was 65 s.Based on the above conditions,143 mutant strains were obtained.Then we selected 63 mutant strains for the preliminary screening and 11effective mutant strains were determined.Finally,a high-yield strain named Mycobacterium sp.MS23,which could transform phytosterols into 9-OH-AD was finally obtained.Compared with the original strain,the 9-OH-AD yield of Mycobacterium sp.MS23 increased by 84.70%.Through genetic stability assay,the9-OH-AD yields of Mycobacterium sp.MS23 for three generations were 37.32 mg/L,41.64 mg/L and 38.77 mg/L,respectively.Obviously,the strain had a good genetic stability and could be used for further experimental researches.2.Secondly,the one-step preparation conditions of 9-OH-AD transformed from phytosterols were further optimized in the oil-free process with the high 9-OH-AD yield strain of Mycobacterium sp.MS23.The single-factor optimal experiments were firstly carried out to optimize the transformation preparation conditions and medium including pH and the concentration of corn syrup,sodium nitrate,and diammonium hydrogen phosphate.The results revealed that the opitimal preparation conditions were pH 9.0,corn syrup 30 g/L,sodium nitrate 5.5 g/L,diammonium hydrogen phosphate 0.6 g/L,respectively.Then the preparation transformation conditions of9-OH-AD were further optimized through the Box-Benhnken design and the results were analyzed by Design Expert 8.0.6 software for the quadratic regression analysis.Among the analysis,the accumulation of 9-OH-AD was regarded as the response value.Finally the results showed that the optimal values of corn syrup,sodium nitrate,diammonium hydrogen phosphate and pH were 32.88 g/L,4.83 g/L,0.68 g/L and8.73,respectively.Under the optimal conditions,the model predicted value of9-OH-AD concentration was 55.53 mg/L and the validated value was 56.68 mg/L.Between that the relative error was 2.07%.3.Finally,to further improve the accumulation of 9-OH-AD transformed from phytosterols in the oil-free process,this chapter emphasized to investigate the effects of the 3-ketosteroid-Δ1-dehydrogenase(KstD)inhibitors on the preparation of9-OH-AD in the transformation preparation medium due to that the 9-OH-AD accumulation was severely affected by the presence of KstD in Mycobacterium sp..KstD could make the intermediate metabolite androstendione(AD)transformed from phytosterols converted to 1,4-androstadien-3,17-dione(ADD),which leads to the less accumulation of 9-OH-AD.Firstly seventeen KstD inhibitors with different degrees of inhibition on KstD,including glycyrrhetinic acid,emodin,8-hydroxyquinoline,4-hydroxyindole,boldenone undecylenate and others were assayed and ultimately the boldenone undecylenate was determined the relatively best inhibitor that made the9-OH-AD concentration reached 72.95 mg/L,which increased by 66.78%compared with the control group.Then the optimal supplement time and work concentration of boldenone undecylenate were determined as 72 h and 15 m M,respectively.Under the optimal conditions,the 9-OH-AD concentration reached 170.47 mg/L with the supplement of boldenone undecylenate,which increased by 2.7 times compared with the control group of 46.07 mg/L.At the same time,the enzyme activities of KstD at different times were assayed with the results that the enzyme activity of KstD approached the highest level(60.20 U/g)at 72 h.It further illustrated that boldenone undecylenate could cause the inhibition on the KstD effectively.At last,the preparation of 9-OH-AD transformed from phytosterols with the supplement of boldenone undecylenate in oil-free process was performed in 5-L fermentor for the preliminary study.It was found that the accumulation of 9-OH-AD reached 172.33mg/L at 132 h,which was 1.1%higher than that of 170.47 mg/L in shake flask.It further illustrated that KstD could be inhibited with the supplement of boldenone undecylenate.The research further provides theoretical support for the higher production of 9-OH-AD for industrialization.In summary,the preliminary researches on the one-step biotransformation preparation in the oil-free process of 9-OH-AD from phytosterols were carried out in this paper.We firstly obtained a mutant strain named Mycobacterium MS23 with a higher yield of 9-OH-AD compared with the original strain through protoplasts mutagenesis.Then the preparation conditions of 9-OH-AD transformed from phytosterols by Mycobacterium MS23 were optimized.Finally it was firstly found that the boldenone undecylenate could inhibit the enzyme activity of KstD,which effectively improve the biotransformation preparation of 9-OH-AD from phytosterols.This research could provide theoretical foundation and experimental data support for the development of new environmental preparation of 9-OH-AD transformed from phytosterols.