WO2005005618A1 - Micro-organisme et procede pour la preparation de pravastatine - Google Patents

Micro-organisme et procede pour la preparation de pravastatine Download PDF

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WO2005005618A1
WO2005005618A1 PCT/CN2004/000699 CN2004000699W WO2005005618A1 WO 2005005618 A1 WO2005005618 A1 WO 2005005618A1 CN 2004000699 W CN2004000699 W CN 2004000699W WO 2005005618 A1 WO2005005618 A1 WO 2005005618A1
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Prior art keywords
mevastatin
pravastatin
salt
sodium
microorganism
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PCT/CN2004/000699
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English (en)
French (fr)
Inventor
Minquan Mei
Xiaoming Ji
Xiaoliang Gao
Yi Chen
Yan Li
Yong Yao
Zhonghao Zhuo
Jing Xu
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Shanghai Techwell Biopharmaceutical Co., Ltd.
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Priority to JP2006517934A priority Critical patent/JP4263741B2/ja
Priority to EP04738297A priority patent/EP1642964B1/en
Priority to DE602004019086T priority patent/DE602004019086D1/de
Publication of WO2005005618A1 publication Critical patent/WO2005005618A1/zh
Priority to US11/328,494 priority patent/US7582464B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/365—Nocardia

Definitions

  • the invention provides a novel microorganism for producing pravastatin sodium and a method for producing pravastatin sodium using the microorganism. Background technique
  • Cardiovascular disease is the leading cause of human death, and its morbidity and mortality are the highest among all diseases. Medical research has confirmed that the main pathological basis of cardiovascular disease is atherosclerosis, and hyperlipidemia is the primary factor leading to atherosclerosis. Therefore, the importance of lipid-lowering drugs in reducing the incidence of cardiovascular disease has attracted people's attention. 'Countries are researching and developing lipid-lowering drugs.
  • HMG-CoA reductase inhibitor hydroxymethylglutaryl coenzyme A reductase inhibitors
  • statins including lovastatin, simvastatin, pravastatin, fluvastatin, cerivastatin, and atorivastatin.
  • Vastatin atorvastatin
  • pravastatin has unique tissue selectivity. It selectively inhibits cholesterol synthesis in the liver and small intestine, but only slightly inhibits cholesterol synthesis in other organs. In addition, it has the advantage of low toxicity.
  • Pravastatin Sodium (formula I) production is obtained by the hydroxylation of its prodrug Mevastatin (formula Ila) or Mevastatin salt (formula lib) by microorganisms.
  • Biotransformation for the production of mevastatin sodium can be carried out by a variety of microorganisms, mainly in the following categories: multiple genera of mold (Mortierella, WO00 / 46175), Nocardia (Norcardia, US5830695), actinomycetes Madura ( Actinomadura, W096 / 40863), Streptomyces Carbopilus EP215665, Streptomyces exfoliatus W098 / 45410), Micromonospora.
  • each of the above strains has a disadvantage in the production process: Because the pravastatin precursor, mevastatin, is highly toxic to microorganisms, especially molds, it can only be maintained in the industrial production process. The low concentration and the slower conversion speed greatly increase the production cost of pravastatin microbial conversion.
  • the object of the present invention is to provide a microorganism and a method for efficiently producing pravastatin sodium.
  • a micropolyspora roseoalba is provided, and its deposit number is CGMCC 0624.
  • a method for producing pravastatin or a salt thereof comprising the following steps:
  • step (b) to maintain the concentration of mevastatin or in 0. 01-0. O5wt% 0
  • the duration of step (b) for 3-5 days In another preferred embodiment, step (b) to maintain the concentration of mevastatin or in 0. 01-0. O5wt% 0 In a further preferred embodiment, the duration of step (b) for 3-5 days.
  • step (d) is only when the concentration of mevastatin or its salt is lower than 1 mg / 1, and then the separation of pravastatin sodium is started.
  • mevastatin in steps (b) and (c) is mevastatin sodium.
  • the pravastatin salt isolated in step (d) is pravastatin sodium.
  • Fig. 1 shows a nuclear magnetic resonance spectrum of pravastatin sodium prepared by the microorganism of the present invention.
  • micropolyspora roseoalba The present invention has been completed based on this strain.
  • the micropolysporum powder TW-9918 of the present invention was stored in the General Microbial Center of the China Microbial Strain Collection and Management Committee (Beijing, China) on August 29, 2001, and the serial number was CGMCC 0624.
  • strain of the present invention or “microorganism of the present invention” refers to Micromonas powderyrum numbered CGMCC 0624, ie TW-9918.
  • the production method of the present invention is basically the same as the method for producing pravastatin sodium in the prior art except that the producing bacteria and fermentation conditions are different, such as the separation and purification process of pravastatin sodium.
  • the fermentation conditions of the strains of the present invention are similar to general micropolyspora, that is, in a medium containing a carbon source, a nitrogen source, and a trace element, at a pH of 6. 8-7. 5 (preferably pH 7. 0-7. 2). ) And 25-38 ° C (preferably 28-35 ° C).
  • Some preferred media are as follows-slanted media uses yeast malt agar (ISP2), which is composed of: malt extract 1.0%, yeast extract 0.4%, glucose 0.4%, agar 2.0 %, PH 7.0-7.2.
  • ISP2 yeast malt agar
  • the seed culture composition is: glucose 0.2-5.0%, yeast extract 0.05-5-0. 5%, peptone 0. 1-2. 0%, dipotassium phosphate 0.2-2-0. 1 %, pH 7. 0-7. 2.
  • the fermentation medium composition is: glucose 1. 0-5. 0%, yeast extract 0. 1-1. 0%, peptone 0.5-2. 0%, K 2 HP0 4 0. 01-0. 5%, MgS0 4 - 73 ⁇ 40 0. 01-0 05%, pH 7. 0-7 2...
  • TW-9918 matures on a slant at 28 ⁇ for 7-10 days.
  • the mature mycelium was taken into the seed medium, and then cultured at 28 ° C on a shaker at 210 RPM for 2-3 days.
  • the fermentation medium is inserted into 5-15% seed culture solution, and then placed in a shaker or cultured in a fermentation tank at a cultivation temperature of 28-35 ° C. 005-0. 5wt% ( ⁇ After 12-24 hours of bacterial growth, supplemented with 0.2-2. 0% glucose every day, and supplemented with mevastatin so that the concentration of mevastatin in the culture medium is maintained at 0. 005-0. 5wt% 0. 01-0. 05%).
  • the transformation culture is generally performed for 2-6 days (preferably 3-5 days), and then the supplementation of mevastatin is stopped, and the mevalastatin concentration in the culture medium is less than 1 mg / L, and the termination is terminated.
  • the fermentation broth was removed by centrifugation, the supernatant was adsorbed by a hydrophobic resin, the resin was washed with water, and pravastatin sodium was eluted with an ethanol / water mixed solution or an acetone / water mixed solution.
  • the pravastatin portion was collected and concentrated.
  • the concentrated solution was extracted with ethyl acetate, and ethanol / ethyl acetate crystallized to obtain pure pravastatin sodium.
  • the main advantages of the present invention are:
  • the strain of the present invention is highly tolerant to mevastatin sodium, and can be maintained at a higher concentration during production. The rapid conversion of mevastatin sodium resulted in a significant reduction in production costs.
  • Soil samples collected from various parts of China obtained in 10-2--10-6 diluted and then spread on a flat plate, square plate incubated at 28 ° for 7-10 days.
  • a total of about 1,500 strains were isolated for shake flask screening.
  • Three of these strains have the ability to transform mevastatin, and one of them, TW-9918, isolated from the soil of Ningbo, China, shows high tolerance to mevastatin and can be efficiently transformed into pravastatin.
  • TW-9918 After growing on Santas agar and glucose asparagine agar for 5-10 days, short chains of 3-12 spores and straight spore filaments were formed on the aerial hyphae; the hyphae within the basal membrane were separated and broken And produce a few short spore chains. The spores are oval, 0.85xl. 5um, and the surface is smooth. Training characteristics:
  • TW-9918 grown on seven media at 28 ° C for 7-15 days are as follows:
  • Cell wall component analysis The cell wall of strain TW-9918 contains mdso-DAP (Diaminopimelic acid), glycine (cell wall type IV).
  • Strain TW-9918 does not contain mycolic acid. Physiological and biochemical characteristics:
  • TW-9918 basic filaments have transverse septum and break; aerial mycelium and intracellular hyphae have short spore chains; the cell wall is type IV, and the sugar type is A.
  • Amycolic acid belongs to the genus Micropolyspora. Based on the principles of cultivation and physiological and biochemical characteristics, strain TW 9918 aerial mycelium is gray and white; the base hyphae is pale yellow with no soluble pigment; combined with physiological and biochemical characteristics, strain TW-9918 and powder white are small Polysporum similar
  • TW-9918 was used to produce pravastatin sodium.
  • the composition of the seed solution is as follows: glucose 2.0%, yeast extract 1.0%, peptone 1.0%, pH 7.2.
  • the seed solution was sterilized at 121 ° C for 20 minutes. After cooling, the TW-9918 strain was introduced and cultured on a shaker at 28 ° C for 2 days. The rotation speed of the shaker was 210 RPM.
  • the composition of the fermentation medium is: glucose 2.0%, yeast extract 0.5%, peptone 2.0%, K 2 HP0 4 0.05%, MgS0 4 ⁇ 7H 2 0 0.05%, pH 7. 2, 121 ° C for 20 minutes. 0% ⁇
  • the daily addition of glucose was 2.0%. 01%-The content of methavastatin in the solution was measured by HPLC, and the concentration was maintained at 0.01% by adding methavastatin sodium salt aqueous solution. 0. 05%. After 4 days of incubation, a total of 0.6 g (3 g / L) of mevastatin was added.
  • the culture solution was centrifuged to remove the bacterial cells.
  • the supernatant was about 180 ml, and it was adsorbed with 100 ml of HP-20 resin. After the adsorption, it was washed with 500 ml of distilled water and then eluted with 50% acetone. 2g ⁇ Partially, concentrated; the concentrate was decolorized, extracted with ethyl acetate, crystallized from ethanol / ethyl acetate to obtain pure pravastatin sodium 0.2g. Examples
  • TW-9918 was used to produce pravastatin sodium in a 5L fully automatic fermentation tank.
  • a 5L full-automatic fermentation tank was equipped with a total of 3.5 liters of fermentation medium, sterilized at 121 ° C for 20 minutes, and 300 ml of seed liquid was introduced after cooling.
  • the seed liquid culture method was the same as in Example 2.
  • the formulation of the fermentation medium is: glucose 2.0%, yeast extract 1.0%, peptone 2.0%, K 2 HP0 4 0.1%, MgS0 4 ⁇ 73 ⁇ 40 0.05%.
  • Fermentation was performed at 3CTC with aeration volume of 3L / min and stirring speed of 300-500rpm.
  • the fermentation progressed to the hour, continuous addition of methavastatin sodium salt solution and glucose solution was maintained, and the methavastatin concentration was maintained at 0.03-0. 05%, the conversion continued until the end of 96 hours, and a total of 20 g of methavastatin (5 7g / L).
  • the conversion solution was purified according to the same method as in the example, and the 1 H nuclear magnetic resonance spectrum of pravastatin sodium prepared from pravastatin sodium after purification was shown in the figure, which is the same as the pravastatin sodium standard.
  • micropolyspora roseoalba TW-9918 of the present invention has been stored at the General Microbial Center of the China Microbial Strain Collection and Management Committee (Beijing, China) on January 2001, and the serial number is CGMCC

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Description

生产普伐他汀钠的微生物和方法
技术领域
本发明提供了一种新的生产普伐他汀钠的微生物及使用该微生物生产普伐他 汀钠的方法。 背景技术
心血管疾病己是人类死的主要原因， 其发病率与死亡率都高居所有疾病之首。 医学研究证实心血管疾病的主要病理基础为动脉粥样硬化， 高血脂症是导致动脉 粥样硬化的首要因素。 因此降脂药在减少心血管疾病发病率方面的重要性己引起 人们的关注， '各国竟相研究开发降血脂药物。
在众多降血脂药物中， 八十年代初发展起来的一类化合物， 它们是羟甲基戊二 酰辅酶 A还原酶抑制剂 （ HMG- CoA reductase inhibitor ) , 由于它们降低胆固 醇的高效性， 抑制胆固醇合成的高度选择性以及低毒性， 正成为心血管药物研究 中最活跃， 发展最迅速的领域。
这类药物又被称为他汀类药物， 包括洛伐他汀（ lovastatin )， 辛伐他汀 ( simvastatin )， 普伐他汀 (pravastatin )， 氟伐他汀 ( f luvastatin )， 西立 伐他汀（ cerivastatin )， 阿伐他汀（ atorvastatin )等。
和同类药物相比，普伐他汀具有独特的组织选择性， 它选择性抑制肝脏及小肠 内的胆固醇合成， 但仅微弱抑制其它器官中的胆固醇合成， 此外它还具有低毒性 的优点。
普伐他汀钠（Pravastatin Sodium,式 I)生产是通过微生物对其前体药物美伐 他汀（Mevastatin, 式 Ila)或美伐他汀的盐（ Mevastatin salt,式 lib)羟基化而 得到。
Figure imgf000002_0001
( I ) ( Ila ) ( lib)
其中美伐他汀（Ila)的水溶性差， 因此通常通过加入碱而将式 Ila转变为美伐 他汀的盐（式 lib, 式中 R=碱金属（如 Na、 K)、 碱土金属、 和 ΝΗ4)。
生产美伐他汀钠的生物转化可以多种微生物进行， 主要为以下几类: 霉菌的多 个属（Mortierella, WO00/46175) , 诺卡氏菌（Norcardia, US5830695)， 马杜拉放 线菌 ( Actinomadura, W096/40863 )，链霉菌 (Streptomyces Carbopilus EP215665, Streptomyces exfoliatus W098/45410 )， 小单孢菌( Micromonospora)。
但上述各菌种在生产过程中都有一个缺点：由于普伐他汀的前体美伐他汀对于 微生物具有较强的毒性， 尤其是霉菌， 因而在工业化生产过程中美伐他汀仅能维 持在较低的浓度， 且转化速度也较慢， 这大大提高了普伐他汀微生物转化的生产 成本。
因此，大规模工业化生产迫切需求开发高效率生产普伐他汀钠的微生物和相应 的生产工艺。 发明内容
本发明的目的就是提供一种高效生产普伐他汀钠的微生物和方法。 在本发明的第一方面， 提供了一种粉白小多孢菌 （ Micropolyspora roseoalba )， 其保藏号是 CGMCC 0624。
在本发明的第二方面， 提供了本发明上述粉白小多孢菌的用途， 它被用于生产 普伐他汀钠。 .
在本发明的第三方面，提供了一种生产普伐他汀或其盐的方法，包括以下步骤:
(a)在 28- 35°C， pH6. 8- 7. 5条件下， 培养保藏号 CGMCC 0624的粉白小多孢菌 (Micropolyspora roseoalba)；
(b) 加入美伐他汀或其盐， 维持美伐他汀浓度在 0. 005-0. 5wt%， 持续 2- 6天; (c)停止加入美伐他汀或其盐；
(d)分离出普伐他汀或其盐。
在另一优选例中， 步骤（b)中维持美伐他汀或其盐浓度在 0. 01-0. O5wt%0 在另一优选例中， 步骤（b)的持续时间为 3-5天。
在另一优选例中， 步骤（d)是当美伐他汀或其盐的浓度低于 lmg/1后， 才开始 分离普伐他汀钠。
在另一优选例中， 步骤 (b)和（c)中美伐他汀是美伐他汀钠。
在另一优选例中， 步骤（d)中分离出的普伐他汀盐是普伐他汀钠。 附图说明
图 1显示了用本发明微生物制备的普伐他汀钠的 核磁共振波谱。 具体实施方式
本发明人经过广泛而深入的研究，通过传统的微生物筛选方法获得了一株新的 微生物 TW- 9918， 它能够耐受高浓度的美伐他汀， 并且具有极高的转化效率， 这 —菌株被命名为粉白小多孢菌 （ Micropolyspora roseoalba )。 在此菌株的基础 上完成了本发明。 本发明的粉白小多孢菌 TW- 9918已于 2001年 8月 29日保存在中国微生物菌种 保藏管理委员会普通微生物中心（中国， 北京）， 编号为 CGMCC 0624。
如本文所用， 术语 "本发明菌株"或 "本发明微生物"指编号为 CGMCC 0624 的粉白小多孢菌， 即 TW- 9918。
本发明的生产方法， 除了生产菌和发酵条件不同之外， 其他条件与现有技术中 生产普伐他汀钠的方法基本相同， 例如普伐他汀钠的分离和纯化工艺。
本发明菌株的发酵条件与一般的小多孢菌相近， 即在含碳源、氮源和微量元素 的培养基中， 在 pH6. 8-7. 5 (较佳地 pH7. 0-7. 2)和 25- 38°C (较佳地 28- 35°C )上发 酵。
一些优选的培养基如下- 斜面培养基采用酵母麦芽琼脂（ ISP2 )， 其组成为： 麦芽抽提物 1. 0%， 酵母 抽提物 0. 4%, 葡萄糖 0. 4%， 琼脂 2. 0%， pH 7. 0- 7. 2。
种子培养基组成为： 葡萄糖 0. 2-5. 0%， 酵母抽提物 0. 05-0. 5%, 蛋白胨 0. 1-2. 0%, 磷酸氢二钾 0. 02-0. 1%, pH 7. 0-7. 2。
发酵用培养基组成为： 葡萄糖 1. 0-5. 0%， 酵母抽提物 0. 1-1. 0%， 蛋白胨 0. 5-2. 0%， K2HP04 0. 01-0. 5%, MgS04 - 7¾0 0. 01-0. 05%, pH 7. 0-7. 2。
—种优选的用 TW-9918菌株转化美伐他汀为普伐他汀的生产过程如下：
TW-9918于 28Ό在斜面上培养 7-10天后即成熟。 取成熟斜面上的菌丝接入种 子培养基， 然后于 28 °C在摇床上以转速 210RPM培养 2-3天。
发酵培养基接入 5-15%种子培养液， 然后置于摇床或在发酵罐内培养， 培养温 度为 28- 35 °C。 菌体生长 12-24小时后， 每天补入 0. 2-2. 0%的葡萄糖， 并同时补 入美伐他汀使培养液中美伐他汀浓度维持在 0. 005-0. 5wt% (较佳地 0. 01-0. 05%)。 转化培养一般进行 2-6天（较佳地 3-5天)后即停止补加美伐他汀， 待培养液中美 伐他汀浓度低于 lmg/L即终止。
发酵液通过离心去除菌体， 上清液由疏水树脂吸附， 以水洗涤树脂， 普伐他汀 钠以乙醇 /水混合溶液或丙酮 /水混合溶液洗脱， 收集普伐他汀部分， 浓缩。 浓缩 液经乙酸乙酯萃取， 乙醇 /乙酸乙酯结晶得普伐他汀钠纯品。 本发明的主要优点在于： 本发明菌株对美伐他汀钠的耐受性强， 生产过程中美 伐他汀钠可维持在较高浓度。 对美伐他汀钠的转化速度快， 导致生产成本大幅下 降。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明本 发明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通 常按照常规条件， 或按照制造厂商所建议的条件。 实施例 1
普伐他汀钠生产菌株的获得和鉴定
1.微生物的分离筛选
从中国各地采集得到的土壤样本以 10- 2- 10-6稀释然后铺于平板上， 平板于 28 °〇恒温培养 7-10天。共约 1， 500个菌株被分离出来用于摇瓶筛选。其中有 3株菌 株具有美伐他汀转化能力， 其中一株从中国宁波土壤分离所得的 TW- 9918显示出 对美伐他汀的高耐受性， 并能高效转化为普伐他汀。
2.微生物的鉴定
形态特征：
TW-9918在桑塔斯琼脂和葡萄糖天冬酰胺琼脂上生长 5-10天后， 气生菌丝上 形成 3- 12个孢子的短链， 孢子丝直丝； 基内菌丝有横隔， 断裂， 并产生少量短孢 子链。 孢子椭圆形， 0. 85xl. 5um， 表面光滑。 培养特征：
TW-9918在七种培养基上 28°C生长 7-15天的特征如下：
Figure imgf000005_0001
化学分类
细胞壁组分分析- 菌株 TW- 9918的细胞壁中含有 mdso- DAP ( 二氨基庚二酸， Diaminopimelic acid )， 甘氨酸（ 细胞壁 IV型）。
全细胞糖型分析： 菌株 TW-9918水解液中含有半乳糖和阿拉伯糖（糖型 A)。
枝菌酸分析：
菌株 TW-9918不含枝菌酸。 生理生化特征:
Figure imgf000006_0001
菌种鉴定结果：
根据形态特征与细胞壁化学组分定属的原则， TW- 9918基丝有横隔、 断裂； 气 生菌丝和基内菌丝都有短的孢子链； 细胞壁为 IV型， 糖型为 A， 无枝菌酸， 属于 小多孢菌属（ Micropolyspora )。 又根据培养特征和生理生化特征定种的原则， 菌株 TW 9918气生菌丝灰白、 粉白； 基内菌丝浅黄色调， 无可溶性色素； 结合生 理生化特征， 菌株 TW-9918与粉白小多孢菌相似， 故划归为粉白小多抱菌
( Micropolyspora roseoalba )。
目前尚没有小多孢菌属的菌株用于生产普伐他汀钠的报道。 实施例 2
在本实施例中， 用 TW-9918生产普伐他汀钠。
在一个 250ml摇瓶中加入 50ml种子液。种子液的配方组成如下：葡萄糖 2. 0%， 酵母抽提物 1. 0%， 蛋白胨 1. 0%， pH 7. 2。 种子液在 121 Ό灭菌 20分钟， 冷却后 接入 TW- 9918菌种， 在摇床上 28°C培养 2天， 摇床转速 210RPM。
2天后分别取 10ml种子培养液接入含有 100ml发酵培养基的 2个 500ml摇瓶 中， 于摇床上 30°C培养， 摇床转速 290RPM。 发酵培养基组成为： 葡萄糖 2. 0%， 酵母抽提物 0. 5%，蛋白胨 2. 0%, K2HP04 0. 05%, MgS04 · 7H20 0. 05%, pH 7. 2, 121 °C灭菌 20分钟。 培养 24小时后开始补糖， 葡萄糖每天加量为 2. 0%。 溶液中的美 伐他汀含量用 HPLC检测， 通过补加美伐他汀钠盐水溶液使其浓度维持在 0. 01%- 0. 05%。 4天后培养结束， 共加入美伐他汀 0. 6g (3g/L )。
发酵终止后培养液离心去除菌体， 上清液约为 180ml， 以 100ml HP- 20树脂吸 附， 吸附完后， 以 500ml蒸熘水洗涤， 然后以 50%丙酮洗脱， 收集含普伐他汀钠 部分， 浓缩； 浓缩液经脱色， 乙酸乙酯萃取， 乙醇 /乙酸乙酯结晶制得普伐他汀钠 纯品 0. 2g。 实施例
在本实施例中用 TW- 9918在 5L全自动发酵罐生产普伐他汀钠。
在一个 5L全自动发酵罐中共装有 3. 5升发酵培养基， 于 121 Ό灭菌 20分钟， 冷却后接入 300ml 种子液， 种子液培养方法同实施例 2。 发酵培养基的配方为： 葡萄糖 2. 0%， 酵母抽提物 1. 0%，蛋白胨 2. 0%, K2HP04 0. 1%, MgS04 · 7¾0 0. 05%。
发酵在 3CTC进行， 通气量为 3L/分钟， 搅拌转速 300- 500rpm。 当发酵进行到 小时开始连续补加美伐他汀钠盐水溶液及葡萄糖溶液， 并维持美伐他汀浓度为 0. 03-0. 05%, 转化持续到 96小时结束， 共加入美伐他汀 20g ( 5. 7g/L )。
转化液按实施例 相同方法纯化， 精制后共得普伐他汀钠 制得的普伐 他汀钠的 1 H 核磁共振波谱如图 所示， 与普伐他汀钠标准品相同。 菌株保藏
本发明的粉白小多孢菌（ Micropolyspora roseoalba ) TW-9918已于 2001年 月 日保存在中国微生物菌种保藏管理委员会普通微生物中心（中国， 北京）， 编号为 CGMCC
申请人或代理人档案号 033755 f wo 国际申请号 / 0 0 0 6 9 9 关 于微生 物 保藏 的 说 明
(细则 13之二）
A.对说明书第 6 页, 第 19-21 行所述的微生物的说明<
B .保藏事项
Figure imgf000008_0001
中国微生物菌种保藏管理委员会普通微生物中心 (CGMCC) 保藏单位地址
(包括邮政编码和国名） 中国， 北京 100080
中国科学院微生物所 保藏日期 2001年 8月 29日 保藏号 CGMCC 0624
C. 补充说明 （必要时） 本栏内容有补充页 □
D. 本说明是为下列指定国作的 （如果说明不是为所有指定国而作的)
E. 补充说明 （必要时)
下列说明将随后向国际局提供 （写出说明的类别， 例如 ： "保藏的编号" ）
由受理局填写 由国际局填写
□本页已经和国际申请一起收到 □国际局收到本页日期:
受权官员 受权官员
PCT/R0/134表（1992年 7月）

Claims

权 利 要 求
1. 一禾中粉白小多抱菌 ( Micropolyspora roseoalba ) , 其特征在于， 其保藏 号是 CGMCC 0624 c
2 . 如权利要求 1所述的粉白小多孢菌的用途， 其特征在于， 用于生产普伐他 汀钠。
3 . 一种生产普伐他汀或其盐的方法， 其特征在于， 包括以下步骤：
(a)在 28- 35°C， pH6. 8- 7. 5条件下， 培养保藏号 CGMCC 0624的粉白小多孢菌 (Micropolyspora roseoalba)；
(b) 加入美伐他汀或其盐， 维持美伐他汀浓度在 0. 005-0. 5wt%, 持续 2-6天;
(c)停止加入美伐他汀或其盐；
(d)分离出普伐他汀或其盐。
4.如权利要求 3所述的方法， 其特征在于， 步骤（b)中维持美伐他汀或其盐浓 度在 0. 01-0. 05wt% o
5.如权利要求 3所述的方法， 其特征在于， 步骤（b)的持续时间为 3- 5天。
6. 如权利要求 3所述的方法， 其特征在于， 步骤（d)是当美伐他汀或其盐的浓 度低于 lmg/1后， 才开始分离普伐他汀钠。
7. 如权利要求 3所述的方法， 其特征在于， 步骤（b)和（c)中美伐他汀或其盐 是美伐他汀钠。
8. 如权利要求 3所述的方法， 其特征在于， 步骤（d)中分离出的普伐他汀盐是 普伐他汀钠。
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CN1254373A (zh) * 1997-04-10 2000-05-24 永进药品工业株式会社 新型微生物"脱叶链霉菌yj－118"及使用该菌株生产普伐他丁钠的方法
CN1399686A (zh) * 1999-07-12 2003-02-26 伊瓦克斯药品研究院有限公司 利用小单孢菌将密实菌素羟基化为普伐他汀

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DE3682557D1 (de) 1985-09-13 1992-01-02 Sankyo Co Hydroxy-ml-236b-derivate, deren herstellung und anwendung.
US5942423A (en) 1995-06-07 1999-08-24 Massachusetts Institute Of Technology Conversion of compactin to pravastatin by actinomadura
CZ291991B6 (cs) 1995-11-29 2003-07-16 Sankyo Company Limited Aktinomycetový promotor
ATE294771T1 (de) 1999-02-03 2005-05-15 Inst Drug Res Ltd Mikrobielles verfahren zur herstellung von pravastatin

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CN1254373A (zh) * 1997-04-10 2000-05-24 永进药品工业株式会社 新型微生物"脱叶链霉菌yj－118"及使用该菌株生产普伐他丁钠的方法
CN1399686A (zh) * 1999-07-12 2003-02-26 伊瓦克斯药品研究院有限公司 利用小单孢菌将密实菌素羟基化为普伐他汀

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