雄甾-4-烯-3，17-二酮的11α羟化及人参皂苷Rd微生物转化研究
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摘要
微生物转化反应具有专一性强、污染少和产率高的优点,羟化反应是甾体微生物转化反应中最重要的反应,微生物能在甾体母核的任何位置进行羟化反应,为甾体药物的合成提供关键中间体,而化学方法除了较易在C17引入羟基外,在其它位置都很难引入。雄甾-4-烯-3,17-二酮(AD)是制备孕激素、皮质激素、利尿剂、雄激素和避孕药等甾体药物的重要中间体,在其甾体母核引入11-羟基能增强甾体药物的抗炎活性。雄甾-4-烯-3,17-二酮(AD)的11α羟基衍生物是制备依普利酮(Eplerenone,商品名Inspra,第一个获准上市的选择性醛固酮受体阻断剂)的关键中间体。
本研究从不同来源的霉菌中筛选得到能在雄甾-4-烯-3,17-二酮上引入11α羟基的菌株Metarhizium anisopliae M28,并通过紫外诱变获得一株高转化活性的突变株Metarhizium anisopliae M28-203。本研究对突变株Metarhizium anisopliaeM28-203进行代谢调控研究,从营养代谢调控和转化条件控制两个方面着手,提高突变株转化效率。培养基正交试验结果表明该突变株培养基最佳配方为葡萄糖3%、玉米浆2%、蚕蛹粉0.5%、硫酸铵0.25%、磷酸氢二钾0.1%、硫酸镁0.05%、硫酸亚铁0.002%。转化条件控制试验表明,该突变株在28-32℃、pH6.0-6.5、菌龄48-60h、250 mL摇瓶装液为30ml和40ml、接种量5%和转化时间72h时能取得最佳转化效果。在底物投料量0.2%(w/v)时,采用4%(v/v)无水乙醇溶解底物或加入0.75 mg/mL Tween 80均有利于羟化反应,在以上优化条件下转化率分别达到62.5%和66.8%。
甾体的水不溶性造成投料浓度低(仅0.1%-0.2%,w/v),转化效率不高。由于有机溶剂和表面活性剂对菌体的毒性作用,采用无水乙醇和Tween80不能解决投料量的问题。β-环糊精(CD)是一种外部亲水内部疏水的笼形分子,能与疏水性分子形成包合物,增加其水溶性。本研究考察了β-环糊精(CD)对甾体底物在水中的溶解度及菌种羟化反应的影响,结果表明β-环糊精能显著提高底物AD在发酵培养基中的溶解度,增溶效果优于有机溶剂。在底物投料浓度0.2%(w/v)时,与4%无水乙醇投料方式相比,8 g/L CD能使AD转化率提高约10%,达到73.2%,并且使转化时间缩短至60h左右。说明CD的增溶作用能使底物迅速转移到菌体内部与酶接触,从而提高甾体微生物转化效率。在CD与AD的最佳摩尔比1:1的条件下,能使投料浓度提高至0.3%(w/v)。本研究采用ROESY对CD-AD包合物结构进行鉴定,结果表明甾体母核深入到环糊精笼形分子空腔内部,形成稳定的包合物。
本研究利用原生质体、无细胞抽提液和酶抑制剂等生物合成机制研究方法考察了Metarhizium anisopliae M28-203的羟化酶及羟化反应机理。原生质体的羟化反应实验表明,该菌的羟化酶属于胞内诱导酶,真菌细胞壁是甾体微生物转化的一个很重要的速率限制因子。原生质体的一氧化碳差示光谱显示在450nm处有最大吸收峰,结合无细胞抽提液的酶抑制试验表明Metarhizium anisopliaeM28-203的11α-羟化酶是一种细胞色素P450依赖的氧化还原酶系统,包括细胞色素P450和NADPH依赖的细胞色素C还原酶。不同亚细胞组分的羟化反应活性检测表明,该羟化酶的两个组成部分细胞色素P450和NADPH依赖的细胞色素C还原酶分别处于不同的亚细胞部位,前者位于微粒体上,后者位于去除微粒体的上清液中,分别起到结合底物和传递电子的作用。
本研究获得了雄甾-4-烯-3,17-二酮的11α-羟化高转化菌种Metarhizium anisopliae M28-203,为制备依普利酮及其他甾体药物的关键中间体11α-羟基雄甾-4-烯-3,17-二酮提供了一个新的微生物转化方法。β-环糊精与雄甾-4-烯-3,17-二酮形成包合物的方法为解决困扰甾体工业化生产的主要问题——甾体的水不溶性提供了有益的参考。对Metarhizium anisopliae M28-203的11α羟化酶和羟化反应机理的探索,为进一步分离纯化和研究该微生物甾体羟化酶系奠定基础。
人参皂苷Rd是原人参二醇型稀有人参皂苷,具有多种药理活性,在野生人参中含量极低。本研究的前期工作是从野山参土样中分离筛选获得了高转化活性菌株Paecilomyces bainier sp.229能转化二醇型人参皂苷Rb1制备稀有人参皂苷compound K,并利用UV-LiCl联合诱变对亲代菌株进行改造,运用高浓度人参皂苷定向筛选的方法,得到一株能耐受高浓度底物,并且能专一性转化人参总皂苷中Rb1到人参皂苷Rd的突变株Paecilomyces bainier sp.229-7。前期研究在摇瓶发酵条件下对Paecilomyces bainier sp.229-7的发酵培养基和转化条件进行了优化。
本研究在前期研究的基础上,对突变株Paecilomyces bainier sp.229-7转化人参总皂苷为人参皂苷Rd的10L发酵罐制备进行研究,进一步证实突变株Paecilomyces bainier sp.229-7能专一性的将三七茎叶总皂苷中的Rb1转化为Rd,并且具有高的底物耐受性。10L发酵罐装液量为6L,投料浓度2%(w/v)时,通过控制溶氧、转速、pH和温度等,克分子转化率可以达到88.9%。
本研究首次采用大孔树脂对发酵液中的人参皂苷Rd进行分离纯化,通过大孔树脂HP20粗分、脱水及大孔树脂H41精制后,转化产物人参皂苷Rd纯度可以达到93%左右,回收率55%以上。利用大孔树脂从发酵液中分离纯化人参皂苷Rd方法的建立将有利于其工业化生产。
Paecilomyces bainier sp.229-7与已报道的其他具有专一性转化能力的菌种比较,具有底物耐受性好、投料浓度高和转化时间短的优点,且作用的底物为三七茎叶总皂苷而非纯化的人参皂苷Rb1,因此Paecilomyces bainier sp.229-7更具有工业化生产潜力。
Microbial transformation has the advantages of high selectivity, low pollution and high efficiency. Hydroxylation is the most widespread type of steroid bioconversion which can be used to build intermediates for further chemical synthesis by offering access to otherwise inaccessible sites of the steroid molecule. Androst-4-ene-3,17-dione (AD) is an important intermediate of many steroid drugs such as corticosteroids, androgens, diuretics and anabolic agents. The oxygen group in position C11 is regarded as essential for anti-inflammatory action. The 11α-hydroxylation of androst-4-ene-3,17-dione leads to an intermediate in the production of Eplerenone (Trade name INSPRA), a selective aldosterone receptor blocking agent (SARA) which was developed by Pfizer and Pharmacia Company.
Metarhizium anisopliae M28, which has the ability of introducing 11α-hydroxyl to its precursor androst-4-ene-3,17-dione (AD), was screened from various filamentous fungi. A mutant strain Metarhizium anisopliae M28-203 with high transformation efficiency was obtained by UV-LiCl mutation. The optimal fermentation culture determined by orthogonal test contains glucose 3%, corn steep 2%, silkworm chrysalis powder 0.5%, (NH4)2SO4 0.25%, K2HPO4 0.1%, MgSO4 0.05% and FeSO4 0.002%. The single factor experiments for temperature, pH, fermentation time, inoculum amount, substrate addition method and substrate concentration indicated that the optimal transformation condition was 28℃, pH6.0-6.5,5% inoculum amount,48-60 h mycelium age and 72 h transformation time. The conversion rate declined with the increase of substrate concentration because of the water-insolubility of steroid. Both 4% ethanol and 0.75 mg/mL Tween 80 were helpful for substrate solubility and then improve transformation efficiency. Conversion rate arrived above 65% under the optimal culture conditions when substrate concentration was 2 mg/mL (0.2%, w/v).
Special problems such as low substrate concentration and transformation rate in steroid bioconversion originate from, in most cases, nearly total water-insolubility of the steroid substrates and difficulty to be transported inside the cell for biotransformation to take place. These can not be solved by organic reagents and surfactants because of their toxicity.β-cyclodextrin possesses a cage-like supramolecular structure with a hydrophobic cavity and hydrophilic outside and is able to form inclusion complexes with a wide variety of hydrophobic guest molecules and then improve their solubility. The effect ofβ-cyclodextrin on water-solubility of steroid substrate and hydroxylation by Metarhizium anisopliae M28-203 was investigated in this study. The results indicate that P-cyclodextrin could improve the solubility of androst-4-ene-3,17-dione in fermentation medium efficiently. Compared with 4% ethanol,8 g/L P-cyclodextrin could increase the conversion rate by 10% when the substrate concentration was 2 mg/mL(0.3%, w/v), furthermore the transformation time was shortened to 60 h. These suggest that the solubilization ofβ-cyclodextrin can facilitate the transportation of substrate inside the cell and then improve steroid bioconversion efficiency. Moreoverβ-cyclodextrin can improve substrate concentration to 3 mg/mL (0.3%, w/v) with the optimal molar ratio ofβ-cyclodextrin:AD of 1:1. Rotating frame overhauser effect spectroscopy (ROESY) was applied in structure determination ofβ-cyclodextrin:AD inclusion complex. The data indicate that the steroid skeletal is immersed in theβ-cyclodextrin cavity and stable inclusion complex is formed.
Hydroxylase and hydroxylation mechanism of Metarhizium anisopliae M28-203 were investigated by protoplasts, cell-free extract and enzyme inhibitors. Hydroxylation by protoplasts of the strain indicated that its hydroxylase is endoenzyme which could be induced by steroid substrate and cell wall of this fungus is an important rate limitation factor. Both carbon monoxide difference spectrum of protoplasts, which revealed a maximum absorbance at 450 nm, and enzyme inhibition test of cell-free extract indicated that the steroid hydroxylase of Metarhizium anisopliae M28-203 belongs to cytochrome P450-dependent oxidoreductase system which includes cytochrome P450 and NADPH dependent cytochrome C reductase. The results on hydroxylation of androst-4-ene-3,17-dione (AD) by different subcellular fractions of the cell homogenate of Metarhizium anisopliae M28-203 suggested that two compositions of hydroxylase are distributed in different subcellular fractions and cytochrome P450 is presented in microsome.
A high-yielding strain Metarhizium anisopliae M28-203 for lla-hydroxylation of androst-4-ene-3,17-dione (AD) was obtained, which provides a new biotransformation method for production of intermediate for Eplerenone and other steroid drugs. The inclusion complex of P-cyclodextrin and AD provides a benefit explore to improve substrate concentration. The investigation for hydroxylation mechanism of Metarhizium anisopliae M28-203 lays the groundwork on further purification of hydroxylase.
Ginsenoside Rd is a rare protopanaxadiol ginsenoside which has various medicinal functions. Unfortunately, the content of ginsenoside Rd in wild ginseng is so low (less than 0.4%) that isolation of Rd from natural products is extremely difficult and costly. We have previously reported on a filamentous fungus Paecilomyces bainier 229 that effectively transforms ginsenoside Rbl to ginsenoside compound K (CK). A mutant Paecilomyces bainier 229-7 was obtained after UV-LiCl mutation and then selection on high concentration of ginsenosides substrate from the parent strain Paecilomyces bainier 229. Paecilomyces bainier 229-7 can transform ginsenoside Rbl to Rd with high selectivity and substrate tolerance and culture conditions required for biotransformation of ginsenoside Rbl to Rd under high substrate concentration had been optimized in 250 mL flask.
In this study, Paecilomyces bainier 229-7 was applied in 10-L fermenter and the results further proved its high specificity and substrate tolerance. The mutant produces ginsenoside Rd from ginsenoside Rbl with as high as 88.9% bioconversion rate under optimized culture conditions in 10-L fermenter when supplied with 20 mg/mL of saponin from Panax notoginseng leaves (SPNL) by control of dissolved O2, agitation rate, pH and temperature。
The isolation and purification of ginsenoside Rd from fermentation medium with macroporous resin was first reported. After macroporous resin HP20 crude separation, HP20 dehydration and H41 refine, the chromatographic purity and recovery of ginsenoside Rd arrived above 93% and 55%, respectively.
Compared with other reported strains with high selectivity, Paecilomyces bainier 229-7 has the advantages of high substrate tolerance, high substrate concentration and short biotransformation time. Furthermore, the substrate used is SPNL extracted from stems and leaves of P. notoginseng (Burk.) F.H. Chen (Araliaceae), a Chinese herb often known by its common name, Tienchi ginseng, which is cheaper then pure ginsenoside Rbl. These results suggest that Paecilomyces bainier 229-7 could be useful for the preparation of ginsenoside Rd in the pharmaceutical industry.
本研究从不同来源的霉菌中筛选得到能在雄甾-4-烯-3,17-二酮上引入11α羟基的菌株Metarhizium anisopliae M28,并通过紫外诱变获得一株高转化活性的突变株Metarhizium anisopliae M28-203。本研究对突变株Metarhizium anisopliaeM28-203进行代谢调控研究,从营养代谢调控和转化条件控制两个方面着手,提高突变株转化效率。培养基正交试验结果表明该突变株培养基最佳配方为葡萄糖3%、玉米浆2%、蚕蛹粉0.5%、硫酸铵0.25%、磷酸氢二钾0.1%、硫酸镁0.05%、硫酸亚铁0.002%。转化条件控制试验表明,该突变株在28-32℃、pH6.0-6.5、菌龄48-60h、250 mL摇瓶装液为30ml和40ml、接种量5%和转化时间72h时能取得最佳转化效果。在底物投料量0.2%(w/v)时,采用4%(v/v)无水乙醇溶解底物或加入0.75 mg/mL Tween 80均有利于羟化反应,在以上优化条件下转化率分别达到62.5%和66.8%。
甾体的水不溶性造成投料浓度低(仅0.1%-0.2%,w/v),转化效率不高。由于有机溶剂和表面活性剂对菌体的毒性作用,采用无水乙醇和Tween80不能解决投料量的问题。β-环糊精(CD)是一种外部亲水内部疏水的笼形分子,能与疏水性分子形成包合物,增加其水溶性。本研究考察了β-环糊精(CD)对甾体底物在水中的溶解度及菌种羟化反应的影响,结果表明β-环糊精能显著提高底物AD在发酵培养基中的溶解度,增溶效果优于有机溶剂。在底物投料浓度0.2%(w/v)时,与4%无水乙醇投料方式相比,8 g/L CD能使AD转化率提高约10%,达到73.2%,并且使转化时间缩短至60h左右。说明CD的增溶作用能使底物迅速转移到菌体内部与酶接触,从而提高甾体微生物转化效率。在CD与AD的最佳摩尔比1:1的条件下,能使投料浓度提高至0.3%(w/v)。本研究采用ROESY对CD-AD包合物结构进行鉴定,结果表明甾体母核深入到环糊精笼形分子空腔内部,形成稳定的包合物。
本研究利用原生质体、无细胞抽提液和酶抑制剂等生物合成机制研究方法考察了Metarhizium anisopliae M28-203的羟化酶及羟化反应机理。原生质体的羟化反应实验表明,该菌的羟化酶属于胞内诱导酶,真菌细胞壁是甾体微生物转化的一个很重要的速率限制因子。原生质体的一氧化碳差示光谱显示在450nm处有最大吸收峰,结合无细胞抽提液的酶抑制试验表明Metarhizium anisopliaeM28-203的11α-羟化酶是一种细胞色素P450依赖的氧化还原酶系统,包括细胞色素P450和NADPH依赖的细胞色素C还原酶。不同亚细胞组分的羟化反应活性检测表明,该羟化酶的两个组成部分细胞色素P450和NADPH依赖的细胞色素C还原酶分别处于不同的亚细胞部位,前者位于微粒体上,后者位于去除微粒体的上清液中,分别起到结合底物和传递电子的作用。
本研究获得了雄甾-4-烯-3,17-二酮的11α-羟化高转化菌种Metarhizium anisopliae M28-203,为制备依普利酮及其他甾体药物的关键中间体11α-羟基雄甾-4-烯-3,17-二酮提供了一个新的微生物转化方法。β-环糊精与雄甾-4-烯-3,17-二酮形成包合物的方法为解决困扰甾体工业化生产的主要问题——甾体的水不溶性提供了有益的参考。对Metarhizium anisopliae M28-203的11α羟化酶和羟化反应机理的探索,为进一步分离纯化和研究该微生物甾体羟化酶系奠定基础。
人参皂苷Rd是原人参二醇型稀有人参皂苷,具有多种药理活性,在野生人参中含量极低。本研究的前期工作是从野山参土样中分离筛选获得了高转化活性菌株Paecilomyces bainier sp.229能转化二醇型人参皂苷Rb1制备稀有人参皂苷compound K,并利用UV-LiCl联合诱变对亲代菌株进行改造,运用高浓度人参皂苷定向筛选的方法,得到一株能耐受高浓度底物,并且能专一性转化人参总皂苷中Rb1到人参皂苷Rd的突变株Paecilomyces bainier sp.229-7。前期研究在摇瓶发酵条件下对Paecilomyces bainier sp.229-7的发酵培养基和转化条件进行了优化。
本研究在前期研究的基础上,对突变株Paecilomyces bainier sp.229-7转化人参总皂苷为人参皂苷Rd的10L发酵罐制备进行研究,进一步证实突变株Paecilomyces bainier sp.229-7能专一性的将三七茎叶总皂苷中的Rb1转化为Rd,并且具有高的底物耐受性。10L发酵罐装液量为6L,投料浓度2%(w/v)时,通过控制溶氧、转速、pH和温度等,克分子转化率可以达到88.9%。
本研究首次采用大孔树脂对发酵液中的人参皂苷Rd进行分离纯化,通过大孔树脂HP20粗分、脱水及大孔树脂H41精制后,转化产物人参皂苷Rd纯度可以达到93%左右,回收率55%以上。利用大孔树脂从发酵液中分离纯化人参皂苷Rd方法的建立将有利于其工业化生产。
Paecilomyces bainier sp.229-7与已报道的其他具有专一性转化能力的菌种比较,具有底物耐受性好、投料浓度高和转化时间短的优点,且作用的底物为三七茎叶总皂苷而非纯化的人参皂苷Rb1,因此Paecilomyces bainier sp.229-7更具有工业化生产潜力。
Microbial transformation has the advantages of high selectivity, low pollution and high efficiency. Hydroxylation is the most widespread type of steroid bioconversion which can be used to build intermediates for further chemical synthesis by offering access to otherwise inaccessible sites of the steroid molecule. Androst-4-ene-3,17-dione (AD) is an important intermediate of many steroid drugs such as corticosteroids, androgens, diuretics and anabolic agents. The oxygen group in position C11 is regarded as essential for anti-inflammatory action. The 11α-hydroxylation of androst-4-ene-3,17-dione leads to an intermediate in the production of Eplerenone (Trade name INSPRA), a selective aldosterone receptor blocking agent (SARA) which was developed by Pfizer and Pharmacia Company.
Metarhizium anisopliae M28, which has the ability of introducing 11α-hydroxyl to its precursor androst-4-ene-3,17-dione (AD), was screened from various filamentous fungi. A mutant strain Metarhizium anisopliae M28-203 with high transformation efficiency was obtained by UV-LiCl mutation. The optimal fermentation culture determined by orthogonal test contains glucose 3%, corn steep 2%, silkworm chrysalis powder 0.5%, (NH4)2SO4 0.25%, K2HPO4 0.1%, MgSO4 0.05% and FeSO4 0.002%. The single factor experiments for temperature, pH, fermentation time, inoculum amount, substrate addition method and substrate concentration indicated that the optimal transformation condition was 28℃, pH6.0-6.5,5% inoculum amount,48-60 h mycelium age and 72 h transformation time. The conversion rate declined with the increase of substrate concentration because of the water-insolubility of steroid. Both 4% ethanol and 0.75 mg/mL Tween 80 were helpful for substrate solubility and then improve transformation efficiency. Conversion rate arrived above 65% under the optimal culture conditions when substrate concentration was 2 mg/mL (0.2%, w/v).
Special problems such as low substrate concentration and transformation rate in steroid bioconversion originate from, in most cases, nearly total water-insolubility of the steroid substrates and difficulty to be transported inside the cell for biotransformation to take place. These can not be solved by organic reagents and surfactants because of their toxicity.β-cyclodextrin possesses a cage-like supramolecular structure with a hydrophobic cavity and hydrophilic outside and is able to form inclusion complexes with a wide variety of hydrophobic guest molecules and then improve their solubility. The effect ofβ-cyclodextrin on water-solubility of steroid substrate and hydroxylation by Metarhizium anisopliae M28-203 was investigated in this study. The results indicate that P-cyclodextrin could improve the solubility of androst-4-ene-3,17-dione in fermentation medium efficiently. Compared with 4% ethanol,8 g/L P-cyclodextrin could increase the conversion rate by 10% when the substrate concentration was 2 mg/mL(0.3%, w/v), furthermore the transformation time was shortened to 60 h. These suggest that the solubilization ofβ-cyclodextrin can facilitate the transportation of substrate inside the cell and then improve steroid bioconversion efficiency. Moreoverβ-cyclodextrin can improve substrate concentration to 3 mg/mL (0.3%, w/v) with the optimal molar ratio ofβ-cyclodextrin:AD of 1:1. Rotating frame overhauser effect spectroscopy (ROESY) was applied in structure determination ofβ-cyclodextrin:AD inclusion complex. The data indicate that the steroid skeletal is immersed in theβ-cyclodextrin cavity and stable inclusion complex is formed.
Hydroxylase and hydroxylation mechanism of Metarhizium anisopliae M28-203 were investigated by protoplasts, cell-free extract and enzyme inhibitors. Hydroxylation by protoplasts of the strain indicated that its hydroxylase is endoenzyme which could be induced by steroid substrate and cell wall of this fungus is an important rate limitation factor. Both carbon monoxide difference spectrum of protoplasts, which revealed a maximum absorbance at 450 nm, and enzyme inhibition test of cell-free extract indicated that the steroid hydroxylase of Metarhizium anisopliae M28-203 belongs to cytochrome P450-dependent oxidoreductase system which includes cytochrome P450 and NADPH dependent cytochrome C reductase. The results on hydroxylation of androst-4-ene-3,17-dione (AD) by different subcellular fractions of the cell homogenate of Metarhizium anisopliae M28-203 suggested that two compositions of hydroxylase are distributed in different subcellular fractions and cytochrome P450 is presented in microsome.
A high-yielding strain Metarhizium anisopliae M28-203 for lla-hydroxylation of androst-4-ene-3,17-dione (AD) was obtained, which provides a new biotransformation method for production of intermediate for Eplerenone and other steroid drugs. The inclusion complex of P-cyclodextrin and AD provides a benefit explore to improve substrate concentration. The investigation for hydroxylation mechanism of Metarhizium anisopliae M28-203 lays the groundwork on further purification of hydroxylase.
Ginsenoside Rd is a rare protopanaxadiol ginsenoside which has various medicinal functions. Unfortunately, the content of ginsenoside Rd in wild ginseng is so low (less than 0.4%) that isolation of Rd from natural products is extremely difficult and costly. We have previously reported on a filamentous fungus Paecilomyces bainier 229 that effectively transforms ginsenoside Rbl to ginsenoside compound K (CK). A mutant Paecilomyces bainier 229-7 was obtained after UV-LiCl mutation and then selection on high concentration of ginsenosides substrate from the parent strain Paecilomyces bainier 229. Paecilomyces bainier 229-7 can transform ginsenoside Rbl to Rd with high selectivity and substrate tolerance and culture conditions required for biotransformation of ginsenoside Rbl to Rd under high substrate concentration had been optimized in 250 mL flask.
In this study, Paecilomyces bainier 229-7 was applied in 10-L fermenter and the results further proved its high specificity and substrate tolerance. The mutant produces ginsenoside Rd from ginsenoside Rbl with as high as 88.9% bioconversion rate under optimized culture conditions in 10-L fermenter when supplied with 20 mg/mL of saponin from Panax notoginseng leaves (SPNL) by control of dissolved O2, agitation rate, pH and temperature。
The isolation and purification of ginsenoside Rd from fermentation medium with macroporous resin was first reported. After macroporous resin HP20 crude separation, HP20 dehydration and H41 refine, the chromatographic purity and recovery of ginsenoside Rd arrived above 93% and 55%, respectively.
Compared with other reported strains with high selectivity, Paecilomyces bainier 229-7 has the advantages of high substrate tolerance, high substrate concentration and short biotransformation time. Furthermore, the substrate used is SPNL extracted from stems and leaves of P. notoginseng (Burk.) F.H. Chen (Araliaceae), a Chinese herb often known by its common name, Tienchi ginseng, which is cheaper then pure ginsenoside Rbl. These results suggest that Paecilomyces bainier 229-7 could be useful for the preparation of ginsenoside Rd in the pharmaceutical industry.
引文
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