甾体化合物微生物转化的研究
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摘要
许多甾体化合物都具有很强的生理活性,临床上广泛应用于抗炎、抗毒、抗过敏、抗休克等。甾体药物的发现及成功合成是近半个世纪以来医药工业取得的最引人注目的两大进展之一,甾体药物也成为仅次于抗生素的第二大类药物。然而利用化学法合成时,往往合成步骤多,得率低,价格昂贵。1950年Murray和Peterson利用黑根霉高效转化黄体酮为11α-羟基黄体酮,使从孕酮合成皮质酮只需要3步,这一研究成果引起了微生物学者的极大兴趣,开展了大量的微生物对甾体转化的研究工作。
本文的主要研究内容及结果如下:
1.甾体化合物转化菌株的筛选
从山东省济南市郊区及黄河附近的稻田、豆地、玉米地以及菜地采集土样,经富集培养,涂布于以植物甾醇和胆固醇为唯一碳源的平板上,分别筛选得到10株和48株菌株,将菌株接种于发酵培养基中培养72 h,对转化产物进行薄层色谱(Thin Layer Chromatography,TLC)分析,结果发现一株真菌X4和一株细菌B4分别能够将植物甾醇和胆固醇转化成几种产物。通过形态观察和18S rDNA序列分析对真菌X4进行鉴定,结果显示X4为串珠镰刀菌(Fusarium moniliformeSheld);利用菌株形态观察、BIOLOG技术和16S rDNA序列分析对细菌B4进行鉴定,结果显示B4为博德特氏菌(Bordetella sp.B4),博德特氏菌转化胆固醇尚属首次报道。
2.Fusarium moniliforme Sheld转化玉米粉和黄豆粉中的植物甾醇为AD的研究
Fusarium moniliforme Sheld能够转化植物甾醇为几种产物,利用TLC和HPLC对产物进行分析,结果显示其中一种产物的Rf(TLC)和Rt(HPLC)与标准品雄甾-4烯-3,17-二酮(Androst-4-ene-3,17-dione,AD)均一致。通过制备型TLC和HPLC对该产物进行分离纯化,利用紫外光谱(Ultraviolet spectrum,UV),红外光谱(Infrared spectrum,IR),质谱(Mass Spectrum,MS)和核磁共振(NuclearMagnetic Resonance,NMR)对该产物进行结构鉴定,结果表明该产物为AD。AD是甾体激素类药物不可替代的中间体,因此具有重要的应用价值。然而,植物甾醇在水中的溶解度非常低,导致转化率很低,寻找一种更好的植物甾醇转化体系是非常必要的。考虑到玉米粉和黄豆粉中含有丰富的植物甾醇,天然状态下的植物甾醇在水中的分散度更好,于是对Fusarium moniliforme Sheld直接转化玉米粉和黄豆粉中的植物甾醇为AD进行了研究,结果表明Fusarium moniloformeSheld能高效转化玉米粉中的植物甾醇,AD最高产率能够达到83.5%,并且省去了植物甾醇的提取过程,节省了成本;同时,利用硫磷铁显色法对玉米粉和黄豆粉中的植物甾醇进行定量,并与传统的HPLC定量方法进行比较,结果显示两种方法的定量结果吻合得比较好,而硫磷铁显色法更为快捷方便。
3.Bordetella sp.B4转化AD的研究
利用筛选得到的Bordetella sp.B4转化AD,对其转化产物进行TLC分析,结果显示Bordetella sp.B4能够将AD转化为三种主要产物,通过IR、MS、UV及NMR等技术对产物进行结构分析,三种产物分别被鉴定为雄甾-1,4-二烯-3,17-二酮(ADD),9α-羟基-雄甾-4-烯-3,17-二酮(9α-OH-AD),3-hydroxy-9,10-secoandrost-1,3,5-triene-9,17-dione(3-OH-SATD),Bordetella sp.B4转化AD此前未见报道。
对β-环糊精包合AD的条件进行了初步摸索,发现β-环糊精与AD以质量比2:1混合研磨,高温灭菌后,包合效果最好,使AD的溶解度明显增加,有利于Bordetella sp.B4转化AD的快速进行。
利用HPLC法检测转化过程中各产物浓度随时间变化情况,发现在30℃和37℃条件下,9α-OH-AD浓度在16 h就能够达到最高值,分别为0.502g/L和0.765 g/L,然而没有ADD产生;40℃和45℃的条件下都有少量的ADD产生,最高浓度分别能达到0.123g/L和0.196g/L,考虑到9α-羟化酶催化AD生成9α-OH-AD,而1,2位脱氢酶催化AD形成ADD,推测1,2位脱氢酶的最适作用温度应该高于9α-羟化酶。Bordetella sp.B4对AD的转化效率优于大多数已报道菌株,且温度对Bordetella sp.B4转化AD的影响也与已报道菌株不同。
4.Bordetella sp.B4转化胆固醇的研究
利用Bordetella sp.B4转化胆固醇,对其转化产物进行TLC分析,结果显示Bordetella sp.B4能够将胆固醇转化成一种产物,通过MS、UV及NMR等方法对产物进行分析,结果证明这个产物为胆甾酮。通过对Bordetella sp.B4发酵液中胆固醇氧化酶活性检测,发现Bordetella sp.B4能够产胞外胆固醇氧化酶。
利用正交试验对Bordetella sp.B4产酶培养基进行优化。结果显示,酵母粉对产酶的影响是最大的,随着浓度的增加,酶活迅速增加,当浓度达到20 g/L时酶活最高,为最适浓度;胆固醇浓度在3 g/L时,酶活最高,随着胆固醇浓度的增加,酶活逐渐下降,其原因可能是胆固醇浓度过高会造成底物抑制,浓度过低导致诱导作用减弱;葡萄糖浓度为7.5 g/L时,酶活最高,随着浓度提高,酶活逐渐降低,其原因可能是葡萄糖浓度过高影响菌体对胆固醇的利用;微量离子液添加体积为200 mL/L时,酶活最高。经过两轮正交试验获得最优组合为胆固醇3.0 g/L,葡萄糖7.5 g/L,酵母粉20 g/L,微量离子液200 mL/L。将Bordetella sp.B4接种于最优培养基中,胆固醇氧化酶的酶活能够达到650 U/L,较优化前提高了5倍左右。进一步对其他培养条件优化,发现优化培养基的初始pH为7.0,接种量为4%,转速为260 rpm时,酶活能够达到1700 U/L,较优化前提高了13倍。
5.Bordetella sp.B4产胆固醇氧化酶的分离纯化及酶学性质研究
利用分级硫酸铵沉淀、DEAE-cellulose离子交换色谱、Sephadex G100凝胶过滤层析对Bordetella sp.B4产胆固醇氧化酶进行分离纯化,获得电泳纯胆固醇氧化酶,SDS-PAGE显示该酶分子量约为55 kDa;胆固醇氧化酶以胆固醇为底物最适作用温度为37℃,在50℃以下都比较稳定,最适作用pH为7.0,在pH4-10的范围都比较稳定;在pH 7.0的磷酸缓冲体系中,以胆固醇为底物测定时,该酶的米氏常数K_m为5.56×10~(-4) mol/L;除了Hg~(2+)和Ag~+对该酶有抑制作用外,大部分金属离子对其酶活都有促进作用,Cu~(2+)的影响最为明显,能够将酶活提高2.19倍;辛醇/水分配系数(log P_(ow))较小的甲醇、乙醇和丙酮对该酶都有抑制作用,而log P_(ow)为-1.38的二甲基亚砜的影响却很小,该酶在log P_(ow)较大的乙酸乙酯、丁醇、氯仿、苯、二甲苯及环己胺的作用下都比较稳定,显示了很好的应用前景。
6.Bordetella sp.B4产多糖的初步分析
利用分级硫酸铵沉淀对胆固醇氧化酶进行初步分离时,有大量的多糖与胆固醇氧化酶共同析出,说明Bordetella sp.B4在产胆固醇氧化酶的同时还分泌一种多糖。十六烷基三甲基溴化铵(CTAB)能够与该多糖结合成络合物使其从发酵液中沉淀出来,将络合物解离、乙醇沉淀、干燥获得多糖粗品。利用咔唑法检测多糖粗品中葡萄糖醛酸含量,发现多糖粗品中约含40%的葡萄糖醛酸。通过DEAE-cellulose离子交换色谱对多糖进行进一步分离纯化,获得多糖精品。利用IR对多糖精品进行初步分析,结果显示此多糖具有-OH、C=O、C-N、N-H和-COOH的特征吸收峰,将此多糖的IR光谱图与透明质酸的相比较,发现二者非常相似,推测此多糖可能为透明质酸。
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 microorganisms
Soil 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.B4
Transformation 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.B4
The 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.B4
The 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 Hg~(2+),Cu~(2+) 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.B4
In 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.
本文的主要研究内容及结果如下:
1.甾体化合物转化菌株的筛选
从山东省济南市郊区及黄河附近的稻田、豆地、玉米地以及菜地采集土样,经富集培养,涂布于以植物甾醇和胆固醇为唯一碳源的平板上,分别筛选得到10株和48株菌株,将菌株接种于发酵培养基中培养72 h,对转化产物进行薄层色谱(Thin Layer Chromatography,TLC)分析,结果发现一株真菌X4和一株细菌B4分别能够将植物甾醇和胆固醇转化成几种产物。通过形态观察和18S rDNA序列分析对真菌X4进行鉴定,结果显示X4为串珠镰刀菌(Fusarium moniliformeSheld);利用菌株形态观察、BIOLOG技术和16S rDNA序列分析对细菌B4进行鉴定,结果显示B4为博德特氏菌(Bordetella sp.B4),博德特氏菌转化胆固醇尚属首次报道。
2.Fusarium moniliforme Sheld转化玉米粉和黄豆粉中的植物甾醇为AD的研究
Fusarium moniliforme Sheld能够转化植物甾醇为几种产物,利用TLC和HPLC对产物进行分析,结果显示其中一种产物的Rf(TLC)和Rt(HPLC)与标准品雄甾-4烯-3,17-二酮(Androst-4-ene-3,17-dione,AD)均一致。通过制备型TLC和HPLC对该产物进行分离纯化,利用紫外光谱(Ultraviolet spectrum,UV),红外光谱(Infrared spectrum,IR),质谱(Mass Spectrum,MS)和核磁共振(NuclearMagnetic Resonance,NMR)对该产物进行结构鉴定,结果表明该产物为AD。AD是甾体激素类药物不可替代的中间体,因此具有重要的应用价值。然而,植物甾醇在水中的溶解度非常低,导致转化率很低,寻找一种更好的植物甾醇转化体系是非常必要的。考虑到玉米粉和黄豆粉中含有丰富的植物甾醇,天然状态下的植物甾醇在水中的分散度更好,于是对Fusarium moniliforme Sheld直接转化玉米粉和黄豆粉中的植物甾醇为AD进行了研究,结果表明Fusarium moniloformeSheld能高效转化玉米粉中的植物甾醇,AD最高产率能够达到83.5%,并且省去了植物甾醇的提取过程,节省了成本;同时,利用硫磷铁显色法对玉米粉和黄豆粉中的植物甾醇进行定量,并与传统的HPLC定量方法进行比较,结果显示两种方法的定量结果吻合得比较好,而硫磷铁显色法更为快捷方便。
3.Bordetella sp.B4转化AD的研究
利用筛选得到的Bordetella sp.B4转化AD,对其转化产物进行TLC分析,结果显示Bordetella sp.B4能够将AD转化为三种主要产物,通过IR、MS、UV及NMR等技术对产物进行结构分析,三种产物分别被鉴定为雄甾-1,4-二烯-3,17-二酮(ADD),9α-羟基-雄甾-4-烯-3,17-二酮(9α-OH-AD),3-hydroxy-9,10-secoandrost-1,3,5-triene-9,17-dione(3-OH-SATD),Bordetella sp.B4转化AD此前未见报道。
对β-环糊精包合AD的条件进行了初步摸索,发现β-环糊精与AD以质量比2:1混合研磨,高温灭菌后,包合效果最好,使AD的溶解度明显增加,有利于Bordetella sp.B4转化AD的快速进行。
利用HPLC法检测转化过程中各产物浓度随时间变化情况,发现在30℃和37℃条件下,9α-OH-AD浓度在16 h就能够达到最高值,分别为0.502g/L和0.765 g/L,然而没有ADD产生;40℃和45℃的条件下都有少量的ADD产生,最高浓度分别能达到0.123g/L和0.196g/L,考虑到9α-羟化酶催化AD生成9α-OH-AD,而1,2位脱氢酶催化AD形成ADD,推测1,2位脱氢酶的最适作用温度应该高于9α-羟化酶。Bordetella sp.B4对AD的转化效率优于大多数已报道菌株,且温度对Bordetella sp.B4转化AD的影响也与已报道菌株不同。
4.Bordetella sp.B4转化胆固醇的研究
利用Bordetella sp.B4转化胆固醇,对其转化产物进行TLC分析,结果显示Bordetella sp.B4能够将胆固醇转化成一种产物,通过MS、UV及NMR等方法对产物进行分析,结果证明这个产物为胆甾酮。通过对Bordetella sp.B4发酵液中胆固醇氧化酶活性检测,发现Bordetella sp.B4能够产胞外胆固醇氧化酶。
利用正交试验对Bordetella sp.B4产酶培养基进行优化。结果显示,酵母粉对产酶的影响是最大的,随着浓度的增加,酶活迅速增加,当浓度达到20 g/L时酶活最高,为最适浓度;胆固醇浓度在3 g/L时,酶活最高,随着胆固醇浓度的增加,酶活逐渐下降,其原因可能是胆固醇浓度过高会造成底物抑制,浓度过低导致诱导作用减弱;葡萄糖浓度为7.5 g/L时,酶活最高,随着浓度提高,酶活逐渐降低,其原因可能是葡萄糖浓度过高影响菌体对胆固醇的利用;微量离子液添加体积为200 mL/L时,酶活最高。经过两轮正交试验获得最优组合为胆固醇3.0 g/L,葡萄糖7.5 g/L,酵母粉20 g/L,微量离子液200 mL/L。将Bordetella sp.B4接种于最优培养基中,胆固醇氧化酶的酶活能够达到650 U/L,较优化前提高了5倍左右。进一步对其他培养条件优化,发现优化培养基的初始pH为7.0,接种量为4%,转速为260 rpm时,酶活能够达到1700 U/L,较优化前提高了13倍。
5.Bordetella sp.B4产胆固醇氧化酶的分离纯化及酶学性质研究
利用分级硫酸铵沉淀、DEAE-cellulose离子交换色谱、Sephadex G100凝胶过滤层析对Bordetella sp.B4产胆固醇氧化酶进行分离纯化,获得电泳纯胆固醇氧化酶,SDS-PAGE显示该酶分子量约为55 kDa;胆固醇氧化酶以胆固醇为底物最适作用温度为37℃,在50℃以下都比较稳定,最适作用pH为7.0,在pH4-10的范围都比较稳定;在pH 7.0的磷酸缓冲体系中,以胆固醇为底物测定时,该酶的米氏常数K_m为5.56×10~(-4) mol/L;除了Hg~(2+)和Ag~+对该酶有抑制作用外,大部分金属离子对其酶活都有促进作用,Cu~(2+)的影响最为明显,能够将酶活提高2.19倍;辛醇/水分配系数(log P_(ow))较小的甲醇、乙醇和丙酮对该酶都有抑制作用,而log P_(ow)为-1.38的二甲基亚砜的影响却很小,该酶在log P_(ow)较大的乙酸乙酯、丁醇、氯仿、苯、二甲苯及环己胺的作用下都比较稳定,显示了很好的应用前景。
6.Bordetella sp.B4产多糖的初步分析
利用分级硫酸铵沉淀对胆固醇氧化酶进行初步分离时,有大量的多糖与胆固醇氧化酶共同析出,说明Bordetella sp.B4在产胆固醇氧化酶的同时还分泌一种多糖。十六烷基三甲基溴化铵(CTAB)能够与该多糖结合成络合物使其从发酵液中沉淀出来,将络合物解离、乙醇沉淀、干燥获得多糖粗品。利用咔唑法检测多糖粗品中葡萄糖醛酸含量,发现多糖粗品中约含40%的葡萄糖醛酸。通过DEAE-cellulose离子交换色谱对多糖进行进一步分离纯化,获得多糖精品。利用IR对多糖精品进行初步分析,结果显示此多糖具有-OH、C=O、C-N、N-H和-COOH的特征吸收峰,将此多糖的IR光谱图与透明质酸的相比较,发现二者非常相似,推测此多糖可能为透明质酸。
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 microorganisms
Soil 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.B4
Transformation 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.B4
The 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.B4
The 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 Hg~(2+),Cu~(2+) 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.B4
In 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.
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