去氢表雄酮的生物法羟基化修饰研究
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摘要
1α-羟基去氢表雄酮(1α-hydroxy dehydroepiandrosterone )是用于合成活性维生素D3及其类似物的一个重要的中间体。活性维生素D3作用于多种靶组织,生理作用也多种多样,广泛用于治疗骨质疏松、甲状旁腺亢进(肾衰)、牛皮癣、癌症以及免疫疾病等。在由去氢表雄酮半化学合成活性维生素D3类药物的过程中,去氢表雄酮(dehydroepiandrosterone, DHEA)的1α-羟基化是首要步骤,也是最关键的步骤之一。
本文采用微生物转化的方法对去氢表雄酮进行1α-羟基化修饰。
首先采用富集培养技术和梯度底物浓度定向筛选技术从土壤中筛选出能转化去氢表雄酮为1α-羟基去氢表雄酮的菌株。通过形态学及分子生物学手段对该菌株进行了鉴定,确定该菌为青霉属的斜卧青霉。对转化产物的结构进行确认,证实了转化产物为1α-羟基化去氢表雄酮。
通过紫外诱变等手段对筛选出的菌株进行诱变处理,通过3轮紫外诱变得到了一株转化率较高且遗传较稳定的菌株。
对ph-13的发酵和转化条件的进行了研究,结果表明种子培养基的最适碳氮源分别为葡萄糖、酵母浸粉;转化培养基的最佳碳氮源分别为蔗糖和硝酸钠。孢子粉接到种子培养基中的最佳浓度为5×10~7个/mL。
对ph-13转化去氢表雄酮1α-羟基化工艺优化后,得到了最佳转化培养基配方:蔗糖为4.75 g/L,硝酸钠为3.69 g/L,硫酸镁为0.3 g/L,硫酸锰为0.0001 g/L,硫酸亚铁为0.0001 g/L。在优化后的培养基中目标产物1α-羟基去氢表雄酮转化率达到18.83%。
最后,研究了1α-羟基化去氢表雄酮的分离纯化方法。转化结束后,抽滤,固液分离,用二氯甲烷对菌体和发酵液进行萃取。用硅胶层析、LH-20凝胶层析、PTLC、结晶/重结晶等手段对二氯甲烷萃取物进行分离,得到了白色的针状1α-羟基化去氢表雄酮,同时得到另一种白的粉末状转化产物,经鉴定为7α-羟基去氢表雄酮。
1α-Hydroxy-Dehydroepiandrosterone is an important drug intermediate which is used for the chemical synthesis of active vitamin D3 and its analogues. The vitamin D3 which has diverse biological mechanisms could act on variant target tissue. So it is wildly used in treating of osteoporosis, parathyroid(ARF), psoriasis, cancer and immune diseases. The moost critical step in semi-synthesis of vitamin D3 by dehydroepiandro- sterone is the 1α-hydroxylation in the first step.
This paper reported the 1α-hydroxylation of dehydroepiandrosterone by the micro- bial transformation.
Strains which could hydroxyl the dehydroepiandrosterone at 1αsite were directly screened by enriched cultivation with substrate concentration gradient, and one strain was screened out. Morphological characteristics and the phylogenetic analysis of 18S rDNA indicated that it was a typical Penicillium decumbens. Identification of the product in Subsequent biotransformation experiments confirmed that this strain had the bio-transform activity.
Three cycles of UV mutagenesis was introduced and one mutant named ph-13 was screened out for its higher transforming ratio and stable genetic characteristics.
Variant conditions of fermentation and transformation of ph-13 were tested and result showed that the optimal carbon and nitrogen sources for seed medium were glucose and yeast extract, while the favorite ones in transforming medium were sucrose and sodium nitrate. Best inoculating concentration was 5×10~7个/mL.
After optimizing conditions of the 1α-hydroxylation of dehydroepiandrosterone we got the best conversion medium which contained the components below: sucrose 4.75 g/L, sodium nitrate was 3.69 g/L, magnesium sulfate was 0.3g/L, manganese sulfate to 0.0001 g/L, ferrous sulfate 0.0001 g/L. In this optimum medium,the transformation rate of 1α-OH-DHEA was 18.83 %.
Subsequently we optimized the separation and purification or 1α-hydroxyled dehy- Droepiandrosterone. The broth was filtrated afer conversion, and then the supernatant was extracted with methylene chloride. The condensate was separated by silica gel chr- matography, LH-20 gel chromatography, PTLC, crystallization-ecrystallization and fin- ally got two compounds. a white needle-like crystal which was 1α-hydroxyled dehydro- epiandrosterone and another white powder-like product which was identified as 7α-hyd- roxy dehydroepiandrosterone.
本文采用微生物转化的方法对去氢表雄酮进行1α-羟基化修饰。
首先采用富集培养技术和梯度底物浓度定向筛选技术从土壤中筛选出能转化去氢表雄酮为1α-羟基去氢表雄酮的菌株。通过形态学及分子生物学手段对该菌株进行了鉴定,确定该菌为青霉属的斜卧青霉。对转化产物的结构进行确认,证实了转化产物为1α-羟基化去氢表雄酮。
通过紫外诱变等手段对筛选出的菌株进行诱变处理,通过3轮紫外诱变得到了一株转化率较高且遗传较稳定的菌株。
对ph-13的发酵和转化条件的进行了研究,结果表明种子培养基的最适碳氮源分别为葡萄糖、酵母浸粉;转化培养基的最佳碳氮源分别为蔗糖和硝酸钠。孢子粉接到种子培养基中的最佳浓度为5×10~7个/mL。
对ph-13转化去氢表雄酮1α-羟基化工艺优化后,得到了最佳转化培养基配方:蔗糖为4.75 g/L,硝酸钠为3.69 g/L,硫酸镁为0.3 g/L,硫酸锰为0.0001 g/L,硫酸亚铁为0.0001 g/L。在优化后的培养基中目标产物1α-羟基去氢表雄酮转化率达到18.83%。
最后,研究了1α-羟基化去氢表雄酮的分离纯化方法。转化结束后,抽滤,固液分离,用二氯甲烷对菌体和发酵液进行萃取。用硅胶层析、LH-20凝胶层析、PTLC、结晶/重结晶等手段对二氯甲烷萃取物进行分离,得到了白色的针状1α-羟基化去氢表雄酮,同时得到另一种白的粉末状转化产物,经鉴定为7α-羟基去氢表雄酮。
1α-Hydroxy-Dehydroepiandrosterone is an important drug intermediate which is used for the chemical synthesis of active vitamin D3 and its analogues. The vitamin D3 which has diverse biological mechanisms could act on variant target tissue. So it is wildly used in treating of osteoporosis, parathyroid(ARF), psoriasis, cancer and immune diseases. The moost critical step in semi-synthesis of vitamin D3 by dehydroepiandro- sterone is the 1α-hydroxylation in the first step.
This paper reported the 1α-hydroxylation of dehydroepiandrosterone by the micro- bial transformation.
Strains which could hydroxyl the dehydroepiandrosterone at 1αsite were directly screened by enriched cultivation with substrate concentration gradient, and one strain was screened out. Morphological characteristics and the phylogenetic analysis of 18S rDNA indicated that it was a typical Penicillium decumbens. Identification of the product in Subsequent biotransformation experiments confirmed that this strain had the bio-transform activity.
Three cycles of UV mutagenesis was introduced and one mutant named ph-13 was screened out for its higher transforming ratio and stable genetic characteristics.
Variant conditions of fermentation and transformation of ph-13 were tested and result showed that the optimal carbon and nitrogen sources for seed medium were glucose and yeast extract, while the favorite ones in transforming medium were sucrose and sodium nitrate. Best inoculating concentration was 5×10~7个/mL.
After optimizing conditions of the 1α-hydroxylation of dehydroepiandrosterone we got the best conversion medium which contained the components below: sucrose 4.75 g/L, sodium nitrate was 3.69 g/L, magnesium sulfate was 0.3g/L, manganese sulfate to 0.0001 g/L, ferrous sulfate 0.0001 g/L. In this optimum medium,the transformation rate of 1α-OH-DHEA was 18.83 %.
Subsequently we optimized the separation and purification or 1α-hydroxyled dehy- Droepiandrosterone. The broth was filtrated afer conversion, and then the supernatant was extracted with methylene chloride. The condensate was separated by silica gel chr- matography, LH-20 gel chromatography, PTLC, crystallization-ecrystallization and fin- ally got two compounds. a white needle-like crystal which was 1α-hydroxyled dehydro- epiandrosterone and another white powder-like product which was identified as 7α-hyd- roxy dehydroepiandrosterone.
引文
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