Studies On The Microbial Transformation Of Steroids

Many steroids have very strong physiological activity,which were widely used for anti-inflammation,antitoxicity,anti-allergy and anti-shock,etc.Discovery and successful synthesis of steroid drugs was one of the most conspicuous developments in pharmaceutical industry for over half a century,and steroid drugs were the second largest category of drugs only lower than antibiotics.However,chemical synthesis of steroid drugs was usually tedious,unefficient and expensive.In 1950,Murray and Peterson discovered that progesterone could be transformed effectively to 11α-hydroxy--progesterone by Rhizopus nigricans,which shortened the process from progesterone to corticosterone to only three steps.This research achievement attracted lots of microbiologists to focus their attentions on microbial transformation of steroids.The main results of this paper are as follows:1.Screening of steroids transformation microorganismsSoil samples were collected from the paddyfields,bean fields,corn fields and vegetable gardens in Jinan suburb and the riverside of the Yellow River,after incubation in enrichment media,the enriched broth was spread on the plates with phytosterol or cholesterol as sole carbon source.Ten strains and 48 strains were isolated respectively.These strains were inoculated to bioconversion media and grown for 72h.Transformation products were analyzed by thin layer chromatography(TLC).A fungus named X4 was able to transform phytosterol and a bacterium named B4 can convert cholesterol into several products.The strain X4 was identified as Fusarium moniliforme Sheld by morphological traits and 18S rDNA sequence analysis,and the strain B4 was identified as Bordetella sp.B4 by morphological characteristics,BIOLOG and 16S rDNA sequence analysis. Transformation of cholesterol by Bordetella sp.was first reported.2.Microbial transformation of Phytosterol in corn flour and soybean flour to androst-4-ene-3,17-dione by Fusarium moniliforme Sheld. Phytosterol could be transformed to several products by Fusarium moniliforme Sheld.After analysis of products by TLC and HPLC,the results indicated that one of products showed identical Rf value(0.31) and Retention time(7.3min) with that of standard AD.The product was isolated by preparative TLC and HPLC,and characterized as AD by ultraviolet spectrum(UV),infrared spectrum(IR),mass spectrum(MS),and nuclear magnetic resonance(NMR).AD,as an important intermediate for the synthesis of many steroid medicines,is widely applied in the transformation of steroid compounds and mainly produced from phytosterol. However,the low solubility of phytosterol in aqueous media is one of the most important causes of low bioconversion rate.Therefore,it is necessary to find a better bioconversion system of phytosterol.Considering that corn flour and soybean flour are rich in phytosterol and the phytosterol therein could be better dispersed in aqueous media,phytosterols in corn flour and soybean flour were directly transformed to AD by Fusarium moniliforme Sheld.The results revealed that corn flour could be transformed to AD very well,and maximum of AD yield could reach 83.5%.The transformation procedure was simplified and the cost was greatly cut down. Meanwhile,the quantification of phytosterol in corn flour and soybean flour was performed by Sulfate-phosphate-ferric(SPF) method.The measured value by SPF method matched reasonably well with that by HPLC,which indicated the validity of SPF method.Determination of phytosterol content by SPF method was more rapid.3.Microbial transformation of androst-4-ene-3,17-dione by Bordetella sp.B4Transformation of AD by Bordetella sp.B4 was investigated.The metabolites of AD by strain B4 was detected by TLC,and the results revealed that three main metabolites were produced.Three metabolites were purified by preparative TLC and HPLC,and identified as androst-1,4-diene-3,17-dione(ADD),9α-hydroxy-androst -4-ene-3,17-dione(9α-OH-AD),and 3-hydroxy-9,10-secoandrost-1,3,5-triene-9, 17-dione(3-OH-SATD) by IR,MS,UV and NMR.Transformation of AD by Bordetella sp.has not been reported before.β-cyclodextrin inclusion was used to increase the solubility of cholesterol.When cholesterol andβ-cyclodextrin were mixed by the mass ratio of 1:2 and ground for 5min,after high temperature sterilization,inclusion effect was best and solubility of AD was increased obviously,which was good to transformation of AD by Bordetella sp.B4.In the process of AD degradation,concentrations of AD,ADD and 9α-OH-AD were determined by HPLC.During biotransformation of AD at 30 and 37℃, 9α-OH-AD was first produced and reached peak production(0.502 g/L at 30℃and 0.765 g/L at 37℃) at 16h,and no ADD appeared;However,a little ADD(0.123 g/L at 40℃and 0.196 g/L at 45℃) was produced by the strain at 40℃and 45℃. Considering that the transformation of AD to ADD was catalyzed by 1, 2-dehydrogenase,this suggest that 1,2-dehydrogenase probably requires higher temperatures for activity presence than 9-OH-hydroxylase.Transformation efficiency of AD by the strain B4 was higher than that by most microorganisms reported,and effect of temperature on transformation was different from that in the previous reports.4.Microbial transformation of cholesterol by Bordetella sp.B4The metabolite of cholesterol by Bordetella sp.B4 was detected by TLC and identified as cholest-4-en-3-one by MS,UV and NMR.Most of cholesterol oxidase (COD) produced by Bordetella sp.B4 are extracellular enzyme that were secreted to fermentation broth.The COD-producing medium was optimized by orthogonal test.The results of orthogonal experiments suggested that effect of yeast extract on production of enzyme was most obvious.The activity of COD increased rapidly with the increase of concentration of yeast extract,and was highest when yeast extract concentration reached 20g/L;The activity of COD was highest when cholesterol concentration was 3 g/L,and decreased with the increase of cholesterol concentration.Substrate cholesterol,at an appropriate concentration,could be the inducer of COD activity,but the inhibitor of COD activity at high concentration;The appropriate concentration of glucose was 3 g/L.High concentration of glucose might inhibit the utilization of cholesterol by the strain and low concentration might inhibit the growth of the strain; The optimum volume of trace ionic liquid was 200 mL/L.Yeast extract and trace ionic liquid would accelerate the growth of the strain.The preliminarily optimized medium contained:cholesterol 3.0g/L,glucose 7.5g/L,yeast extract 20.0 g/L,trace ionic liquid 200mL/L.The activity of COD produced in the above optimized medium could exceed 650 U/L.The strain grew exuberantly at pH 5.4～10.0,especially at pH 7.0,at which the biomass reached the maximum and the yield of COD simultaneously achieved the highest.Rotation speed affected activity of COD markedly,with the increase of rotation speed,the yield of COD noticeably increased and the production period of COD was obviously shortened,which rests with the fact that the genus of Bordetella is obligate aerobe.The results of optimization of cultivation conditions demonstrated that the activity of extracellular cholesterol oxidase from Bordetella sp. B4 could reach 1700 U/L in 24h at pH 7.0,260 rpm and 37℃.5.Purification and characterization of COD from Bordetella sp.B4The COD from Bordetella sp.B4 was purified using ammonium sulfate fractional precipitation,DEAE-cellulose anion exchange chromatography and Sephadex G100 gel filtration chromatography,the enzymed was finally purified to homogeneity confirmed by SDS-PAGE and its molecular mass was 55kDa.Using cholesterol as substrate,COD exhibited optimum activity around pH 7.0,and was quite stable at a pH range of pH 4.0 10.0;The optimum temperature for the enzyme was 37℃,and the enzyme was stable below 50℃;The K_m value of the enzyme for cholesterol was estimated to be 5.56×10^-4 M by the method of Lineweaver and Burk;The activity of the COD was enhanced in the presence of most metal ions except Ag⁺ and Hg²⁺,Cu²⁺ enhanced the enzyme activity by 2.19 fold;The COD was almost inactivated by methanol,ethanol,and acetone.However,the enzyme was stable in the presence of ethyl acetate,butanol,chloroform,benzene,xylene and cyclohexane.Noticeably, COD was stable in the presence of dimethylsulfoxide with low log P_ow,which is an advantageous trait in future industrial application.6.The preliminary analysis of polysaccharide from Bordetella sp.B4In the meantime of COD production,a polysaccharide was produced by Bordetella sp.B4 and they can be precipitated with 60%ammonium sulphate.The polysaccharide could be complex precipitate by binding cetyl trimethyl ammonium bromide(CTAB).Then,the crude polysaccharide was obtained by complex-dissociation,ethanol precipitation and drying.The glucuronic acid content of dried precipitate was determined to be about 40%by the method of carbazole. The polysaccharide was further purified by DEAE-cellulose anion exchange chromatography eluted with 0.5M NaCl.The fractions with polysaccharide were collected together and deproteinized by organic solvent containing n-butanol and chloroform(1:4,v/v).After centrifugation at 10000 rpm for 10min,the supernatant was precipitated by adding triple ethanol.The precipitate was dried by the vacuum drying oven.The infrared(IR) spectra revealed that the polysaccharide contained -OH,C=O,C-N,N-H and -COOH,and was almost identical with the authentic hyaluronic acid(HA).Therefore,it was concluded that the polysaccharide was HA.