新金分枝杆菌ZJUVN-08转化植物甾醇合成雄甾烯二酮的研究
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摘要
甾体类激素药物是我国医药领域的重要门类,雄甾烯二酮(AD)是甾体激素类药物不可替代的中间体。微生物选择性降解植物甾醇(PS)侧链生成AD,能替代复杂的多步化学合成法,并减轻目前由于薯蓣皂素为原料造成的资源紧缺,对合理利用我国甾体植物资源、推动制药行业的发展有着重要意义。本文对分离得到能转化植物甾醇合成AD的菌株进行性能比较,并对高产菌株进行鉴定与N+离子注入诱变选育,获得1株仅积累产物AD的菌株(新金分枝杆菌,命名为ZJUVN-08)。利用正交实验对该菌的培养基组成和培养条件进行了优化；通过几种不同的细胞通透性调节手段,提高新金分枝杆菌细胞壁的通透性,以期增加AD产量；比较了水相系统中几种不同促溶剂对植物甾醇转化的影响,并借助响应面分析方法对添加羟丙基-p-环糊精(HP-β-CD)的生长细胞转化过程进行了优化；研究了水相体系中静息细胞转化植物甾醇的最佳条件。论文的主要研究结果如下：
取山东泰安市郊几家植物油厂附近污染较为严重的土壤样品,后经富集培养和涂布平板等方法,分离得到76株能够在以植物甾醇为唯一碳源的培养基上生长的菌株,对转化产物进行薄层层析和高效液相色谱分析,发现菌株ZJUVN的转化产物产量最高,且产物AD和雄甾-1,4-二烯-3,17-二酮(ADD)的比例约为10:1。经过菌株形态观察、生理生化特征及16S rDNA序列分析方法对菌株ZJUVN进行鉴定,结果显示该菌为新金分枝杆菌(Mycobacterium neoaurum. ZJUVN)。
利用N+离子束注入技术对新金分枝杆菌ZJUVN进行诱变,通过不同注入剂量与诱变致死率的关系曲线确定N+离子束注入的最佳条件为：能量10keV,诱变剂量100×2.6×1013ions/cm2,致死率约在70%。
选择在平板筛选培养基上生长良好的单菌落,进行摇瓶发酵,得到23株积累产物AD而几乎没有副产物ADD的突变株,其中突变株ZJUVN-08的AD产量为2.29g/L,比出发菌株提高71.05%。10次传代过程中,AD产量基本保持稳定,表明该菌有较好的遗传稳定性。
利用正交实验,对突变株M. neoaurum ZJUVN-08的培养基组成进行了优化,最终确定合适培养基组成为：葡萄糖1%,蛋白胨0.6%,磷酸氢二钾0.15%,硫酸镁0.05%,硫酸亚铁0.007%。确定了最佳转化条件为：初始pH值7.0,温度30。C,10%的接种量,装液量50mL/250mL,转速为200rpm。对ZJUVN-08的转化产物利用制备型HPLC进行分离纯化,制得白色粉末结晶,通过MS,IR和NMR进行结构鉴定,结果证明纯化产物为AD。
对比了超声、杆菌肽、鱼精蛋白和甘氨酸处理对菌体生长及底物转化能力的影响。结果发现,超声功率为500W,超声时长为180s时,发酵液中AD产量为2.95g/L,比对照提高了5.8%。杆菌肽处理菌体后,几种参试浓度下,均会抑制菌体生长,且AD产量低于对照水平。10mgm鱼精蛋白处理菌体后,发酵液中AD产量最高为3.02g/L,比对照提高了8.6%。5g/L的甘氨酸有利于菌体的生长,且有利于提高菌体转化底物的能力,发酵液中AD产量最高为3.16g/L,比对照提高了13.6%。并通过药敏性实验进一步确定了甘氨酸是通过增强细胞通透性的方式提高了底物的转化能力。
探讨了水相体系中不同助溶剂对植物甾醇微生物转化的影响,确定了添加HP-β-CD的体系中植物甾醇转化效果最好；进一步采用响应面法对添加HP-β-CD的水相体系转化条件进行优化,获得最佳转化条件为诱导物浓度0.1g/L、初始pH7.0,HP-β-CD/PS摩尔比1.92:1,底物浓度8.89g/L,转化时间120h,在该条件下转化植物甾醇时,得到AD产量最大值为5.96g/L,摩尔转化率为94.69%。比未优化前有效节约了底物和HP-β-CD的用量,同时获得了较高的产量。
进一步研究了水相体系中诱变菌株M. neoaurum ZJUVN-08静息细胞转化植物甾醇的条件,确定静息细胞菌龄为72h,细胞浓度45g/L、pH为7.0的0.05mol/L Tris-HCl缓冲液中转化96h条件最佳。静息细胞重复使用4次以后,在底物浓度为8.89g/L时,摩尔转化率降为56.04%。
Steroid hormone is an important category in our pharmaceutical industry, and androstenedione is an indispensable key intermediate of steroid hormones. Androstenedione can be produced by microbial side chain cleavage of phytosterol, which is an alternative to multi-step chemical synthesis, and can alleviate the current situation caused by the raw material scarcity of diosgenin, is of great significance to full use of plant steroid resources, as well as prompting the pharmacy development. In the present study, strains with the ability to selectively degrade phtosterol were screened and their AD producing ability was tested. The strain with high AD yield was identified and mutation using low energy N+implantation was followed. As a result, a genetically stable mutant strain with AD as the sole product, designated as ZJUVN-08, was obtained. The effects of medium composition and culture conditions on phytosterol biotransformation were evaluated experimentally. Different cell wall permeabilization factors were used to enhance cell wall permeability and AD production. The effect of different cosolvents in aqueous system on the degradation of phytosterol into AD was studied. AD production was significantly increased in the presence of HP-P-CD. Then the process optimization was investigated using growing mycobacteria cells in depth. Biotransformation of phytosterol to AD using resting cell was optimized. The main results of this study were as follows:
Soil samples which were seriously polluted by vegetable oil were collected from Taian surburb, Shandong, China. After incubation in encrichment media, the enrichment broth was spread on the plates with phytosterol as the sole carbon source.76strains were isolated, and then these strains were inoculated to bioconversion media to test the ability to transform the substrate. Transformation products were analyzed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). A strain named ZJUVN was found to have the maximum production and was able to transform phytosterol to AD and androsta-1,4-diene-3,17-dione (ADD) with the ratio of10:1. The strain was identified as Mycobacterium neoaurum by morphology, biochemical identification and16S rDNA sequence analysis.
The strain was bred using low energy N+implantation method for accumulation of AD as the only product. The most suitable mutation conditions were:energy of10keV, implantation dose of100×2.6×1013ions/cm2, with the death rate of70%. Colonies appearing on the selected plates were chosen and tested for their AD production. Then23strains were selected with AD as the sole product. One of the mutated strains, designated as ZJUVN-08, produced the maximum level of AD (2.29g/L) which increased by71.05%. The strain was serially passaged for10passages and AD production was determined. The results demonstrated that M. neoaurum. ZJUVN-08has good genetic stability within10passages.
The effects of medium composition and culture conditions on AD production by strain ZJUVN-08were evaluated experimentally. The optimized medium composition was as follows:glucose1%, peptone0.6%, KH2PO40.15%, MgSO40.05%, FeSO4. The optimized fermentation conditions were as follows:initial pH value7.0; temperature30℃; inoculums volume,10%; and50mL medium in250mL shaken flask; rotate speed200rpm. The product was purified by preparation HPLC and was a white powder, then further identified as AD using NMR, MS and IR analysis.
By the study of ultrasound, bacitriacin, protamine and glycine on the strain growth and AD production, it was found that under the condition that with ultrasound power500W, treating time180s, AD yield was2.95g/L and increased by5.8%. After the treatment of different concentration of bacitriacin, the growth of strain was inhibited and AD yield was decreased under all the tested bacitriacin concentration. After the treatment of10g/L protamine, AD yield was3.02g/L and increased by8.6%. It was found that5g/L glycine was in favor of strain growth and AD production, with the AD yield of3.16g/L. Drug sensitivity assays were carried out to establish whether glycine increased AD production by increasing the cell wall permeabilization.
The effect of different cosolvents on phytosterol degradation in aqueous system was studied. It was found that AD production was significantly increased in the presence of HP-β-CD. The parameters of biotransformation process by growing mycobacteria cells were optimized using fractional factorial design and response surface methodology. The optimial process conditions were observed at0.1g/L inducer, pH7.0, and molar ratio of hydroxypropyl-β-cyclodextrin to phytosterol1.92:1,8.98g/L phytosterol and at120h of incubation time. Under these conditions, the maximum AD yield was5.96g/L and the maximum phytosterol conversion rate was94.69%, which is a cost-efficient process compared to the non-optimized condition.
The effect of phytosterol bioconversion using resting cells of strain Mneoaurum ZJUVN-08was investigated. The optimized conditons were as follows:cell age of72h; cell concentration of45g/L; Tris-HCl buffer solution pH value and concentration were7.0and0.05mol/L, respectively. After repeatedly utilizing four times, phytosterol bioconversion rate was decreased to56.04%.
取山东泰安市郊几家植物油厂附近污染较为严重的土壤样品,后经富集培养和涂布平板等方法,分离得到76株能够在以植物甾醇为唯一碳源的培养基上生长的菌株,对转化产物进行薄层层析和高效液相色谱分析,发现菌株ZJUVN的转化产物产量最高,且产物AD和雄甾-1,4-二烯-3,17-二酮(ADD)的比例约为10:1。经过菌株形态观察、生理生化特征及16S rDNA序列分析方法对菌株ZJUVN进行鉴定,结果显示该菌为新金分枝杆菌(Mycobacterium neoaurum. ZJUVN)。
利用N+离子束注入技术对新金分枝杆菌ZJUVN进行诱变,通过不同注入剂量与诱变致死率的关系曲线确定N+离子束注入的最佳条件为：能量10keV,诱变剂量100×2.6×1013ions/cm2,致死率约在70%。
选择在平板筛选培养基上生长良好的单菌落,进行摇瓶发酵,得到23株积累产物AD而几乎没有副产物ADD的突变株,其中突变株ZJUVN-08的AD产量为2.29g/L,比出发菌株提高71.05%。10次传代过程中,AD产量基本保持稳定,表明该菌有较好的遗传稳定性。
利用正交实验,对突变株M. neoaurum ZJUVN-08的培养基组成进行了优化,最终确定合适培养基组成为：葡萄糖1%,蛋白胨0.6%,磷酸氢二钾0.15%,硫酸镁0.05%,硫酸亚铁0.007%。确定了最佳转化条件为：初始pH值7.0,温度30。C,10%的接种量,装液量50mL/250mL,转速为200rpm。对ZJUVN-08的转化产物利用制备型HPLC进行分离纯化,制得白色粉末结晶,通过MS,IR和NMR进行结构鉴定,结果证明纯化产物为AD。
对比了超声、杆菌肽、鱼精蛋白和甘氨酸处理对菌体生长及底物转化能力的影响。结果发现,超声功率为500W,超声时长为180s时,发酵液中AD产量为2.95g/L,比对照提高了5.8%。杆菌肽处理菌体后,几种参试浓度下,均会抑制菌体生长,且AD产量低于对照水平。10mgm鱼精蛋白处理菌体后,发酵液中AD产量最高为3.02g/L,比对照提高了8.6%。5g/L的甘氨酸有利于菌体的生长,且有利于提高菌体转化底物的能力,发酵液中AD产量最高为3.16g/L,比对照提高了13.6%。并通过药敏性实验进一步确定了甘氨酸是通过增强细胞通透性的方式提高了底物的转化能力。
探讨了水相体系中不同助溶剂对植物甾醇微生物转化的影响,确定了添加HP-β-CD的体系中植物甾醇转化效果最好；进一步采用响应面法对添加HP-β-CD的水相体系转化条件进行优化,获得最佳转化条件为诱导物浓度0.1g/L、初始pH7.0,HP-β-CD/PS摩尔比1.92:1,底物浓度8.89g/L,转化时间120h,在该条件下转化植物甾醇时,得到AD产量最大值为5.96g/L,摩尔转化率为94.69%。比未优化前有效节约了底物和HP-β-CD的用量,同时获得了较高的产量。
进一步研究了水相体系中诱变菌株M. neoaurum ZJUVN-08静息细胞转化植物甾醇的条件,确定静息细胞菌龄为72h,细胞浓度45g/L、pH为7.0的0.05mol/L Tris-HCl缓冲液中转化96h条件最佳。静息细胞重复使用4次以后,在底物浓度为8.89g/L时,摩尔转化率降为56.04%。
Steroid hormone is an important category in our pharmaceutical industry, and androstenedione is an indispensable key intermediate of steroid hormones. Androstenedione can be produced by microbial side chain cleavage of phytosterol, which is an alternative to multi-step chemical synthesis, and can alleviate the current situation caused by the raw material scarcity of diosgenin, is of great significance to full use of plant steroid resources, as well as prompting the pharmacy development. In the present study, strains with the ability to selectively degrade phtosterol were screened and their AD producing ability was tested. The strain with high AD yield was identified and mutation using low energy N+implantation was followed. As a result, a genetically stable mutant strain with AD as the sole product, designated as ZJUVN-08, was obtained. The effects of medium composition and culture conditions on phytosterol biotransformation were evaluated experimentally. Different cell wall permeabilization factors were used to enhance cell wall permeability and AD production. The effect of different cosolvents in aqueous system on the degradation of phytosterol into AD was studied. AD production was significantly increased in the presence of HP-P-CD. Then the process optimization was investigated using growing mycobacteria cells in depth. Biotransformation of phytosterol to AD using resting cell was optimized. The main results of this study were as follows:
Soil samples which were seriously polluted by vegetable oil were collected from Taian surburb, Shandong, China. After incubation in encrichment media, the enrichment broth was spread on the plates with phytosterol as the sole carbon source.76strains were isolated, and then these strains were inoculated to bioconversion media to test the ability to transform the substrate. Transformation products were analyzed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). A strain named ZJUVN was found to have the maximum production and was able to transform phytosterol to AD and androsta-1,4-diene-3,17-dione (ADD) with the ratio of10:1. The strain was identified as Mycobacterium neoaurum by morphology, biochemical identification and16S rDNA sequence analysis.
The strain was bred using low energy N+implantation method for accumulation of AD as the only product. The most suitable mutation conditions were:energy of10keV, implantation dose of100×2.6×1013ions/cm2, with the death rate of70%. Colonies appearing on the selected plates were chosen and tested for their AD production. Then23strains were selected with AD as the sole product. One of the mutated strains, designated as ZJUVN-08, produced the maximum level of AD (2.29g/L) which increased by71.05%. The strain was serially passaged for10passages and AD production was determined. The results demonstrated that M. neoaurum. ZJUVN-08has good genetic stability within10passages.
The effects of medium composition and culture conditions on AD production by strain ZJUVN-08were evaluated experimentally. The optimized medium composition was as follows:glucose1%, peptone0.6%, KH2PO40.15%, MgSO40.05%, FeSO4. The optimized fermentation conditions were as follows:initial pH value7.0; temperature30℃; inoculums volume,10%; and50mL medium in250mL shaken flask; rotate speed200rpm. The product was purified by preparation HPLC and was a white powder, then further identified as AD using NMR, MS and IR analysis.
By the study of ultrasound, bacitriacin, protamine and glycine on the strain growth and AD production, it was found that under the condition that with ultrasound power500W, treating time180s, AD yield was2.95g/L and increased by5.8%. After the treatment of different concentration of bacitriacin, the growth of strain was inhibited and AD yield was decreased under all the tested bacitriacin concentration. After the treatment of10g/L protamine, AD yield was3.02g/L and increased by8.6%. It was found that5g/L glycine was in favor of strain growth and AD production, with the AD yield of3.16g/L. Drug sensitivity assays were carried out to establish whether glycine increased AD production by increasing the cell wall permeabilization.
The effect of different cosolvents on phytosterol degradation in aqueous system was studied. It was found that AD production was significantly increased in the presence of HP-β-CD. The parameters of biotransformation process by growing mycobacteria cells were optimized using fractional factorial design and response surface methodology. The optimial process conditions were observed at0.1g/L inducer, pH7.0, and molar ratio of hydroxypropyl-β-cyclodextrin to phytosterol1.92:1,8.98g/L phytosterol and at120h of incubation time. Under these conditions, the maximum AD yield was5.96g/L and the maximum phytosterol conversion rate was94.69%, which is a cost-efficient process compared to the non-optimized condition.
The effect of phytosterol bioconversion using resting cells of strain Mneoaurum ZJUVN-08was investigated. The optimized conditons were as follows:cell age of72h; cell concentration of45g/L; Tris-HCl buffer solution pH value and concentration were7.0and0.05mol/L, respectively. After repeatedly utilizing four times, phytosterol bioconversion rate was decreased to56.04%.
引文
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