WO2020074009A1 - 一种青霉及其生产烟曲霉素的方法 - Google Patents

一种青霉及其生产烟曲霉素的方法 Download PDF

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WO2020074009A1
WO2020074009A1 PCT/CN2019/110893 CN2019110893W WO2020074009A1 WO 2020074009 A1 WO2020074009 A1 WO 2020074009A1 CN 2019110893 W CN2019110893 W CN 2019110893W WO 2020074009 A1 WO2020074009 A1 WO 2020074009A1
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penicillium
powder
fumagillin
yeast extract
sulfate
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PCT/CN2019/110893
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French (fr)
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张延青
钱娉婷
邓爱文
姜南
张杭锋
李建宋
张辉
应灵萍
王继栋
滕云
郑玲辉
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浙江海正药业股份有限公司
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    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • 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
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/80—Penicillium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/336—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having three-membered rings, e.g. oxirane, fumagillin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N1/14—Fungi; Culture media therefor
    • 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
    • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
    • C12P17/162—Heterorings having oxygen atoms as the only ring heteroatoms, e.g. Lasalocid

Definitions

  • the invention relates to the technical field of microbial engineering, in particular to a new penicillium and its application in the preparation of fumagillin.
  • Fumagillin (Formula I) was originally used to kill microsporidia parasites of honeybees, but was later found to be useful for the treatment of microsporidiosis and / or cryptosporidiosis caused by human intestinal infections. The disease is quite fatal for immunodeficiency patients.
  • fumagillin and its analogues have anti-angiogenic activity, which can be linked to human methionine aminopeptidase-2 ( MetAP-2) binds, thereby blocking the formation of blood vessels. Therefore, fumagillin and its analogs are widely used as angiogenesis inhibitors for the treatment of cancer.
  • scabrosum FCB 353 has been cultured and optimized to produce fumagillin
  • the potency can reach 110mg / L. It can be seen that the fumagillin-producing bacteria used in the prior art are relatively primitive and have low fermentation levels, so it is necessary to find a new and efficient fumagillin-producing bacteria.
  • One of the objects of the present invention is to provide a new fumagillin-producing strain, which is Penicillium (Penicillium sp.) HS-NF-684Z, and its deposit number is CGMCC No. 14144.
  • the microbial strain of Penicillium HS-NF-684Z of the present invention was deposited on July 24, 2017 at the General Microbiology Center of the China Microbial Culture Collection and Management Committee (Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Chinese Academy of Sciences Institute of Microbiology), the deposit number is CGMCC No. 14144, the classification name is Penicillium (Penicillium sp.), And it is registered in the book to prove survival.
  • the object of the present invention is also to provide an application of penicillium HS-NF-684Z (CGMCC NO.14144) in the preparation of fumagillin or a pharmaceutical composition containing fumagillin.
  • the present invention also provides a method for preparing fumagillin.
  • the method includes using Penicillium HS-NF-684Z (CGMCC No. 14144) in a nutrient medium containing an assimitable carbon source and / or nitrogen source. Steps to carry out aerobic fermentation.
  • the assimilable carbon source is selected from starch, dextrin, glucose, sucrose, lactose, maltose, industrial molasses, glycerin, sorbitol, mannitol, or a combination of the above substances, preferably starch, pastesperm, glucose, sucrose.
  • the aforementioned assimitable nitrogen source is selected from the group consisting of yeast extract powder, yeast powder, yeast extract, soybean cake powder, cottonseed cake powder, peanut cake powder, gluten powder, corn steep powder, soybean meal, peptone,
  • yeast extract powder yeast powder, yeast extract, soybean cake powder, cottonseed cake powder, peanut cake powder, gluten powder, corn steep powder, soybean meal, peptone,
  • One of urea and ammonium salt or a combination of the above is preferably soybean cake powder, yeast extract, yeast extract powder, and yeast powder.
  • the above nutrient medium further includes an inorganic salt selected from trisodium citrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, zinc sulfate, Copper sulfate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride, manganese sulfate, or a combination of the foregoing, preferably calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, lemon Trisodium acid.
  • an inorganic salt selected from trisodium citrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, zinc sulfate, Copper sulfate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride, manganese sulfate
  • the nutrient medium contains sucrose 40-1000g / L, dextrin 20-1000g / L, glycerol 10-80g / L, yeast extract powder 1-15g / L, yeast powder 5-20g / L, yeast extract 5-20g / L, magnesium sulfate 1-10g / L, potassium dihydrogen sulfate 1-5g / L, calcium carbonate 0-50g / L.
  • the temperature of the aerobic fermentation is 20-30 ° C, preferably 23-28 ° C; the culture medium pH is 5.0-8.0, preferably 5.0-7.0; the cultivation time is 24-226 hours, preferably 72- 168 hours; oxygen flux is 0.1-2.0vvm, preferably 0.5-2.0vvm.
  • the Penicillium HS-NF-684Z is inoculated into the nutrient medium by seed liquid for the fermentation culture; wherein, the seed liquid is the Penicillium HS-NF-684Z (CGMCC No. 14144) obtained by seed culture in a seed medium; the conditions of the seed culture are: the temperature of the seed culture is 20 ° C-30 ° C, preferably 23 ° C-28 ° C; the pH of the medium is 5.0-8.0 , Preferably 5.0-7.0; cultivation time is 24-80 hours, preferably 24-60 hours.
  • the seed liquid is the Penicillium HS-NF-684Z (CGMCC No. 14144) obtained by seed culture in a seed medium
  • the conditions of the seed culture are: the temperature of the seed culture is 20 ° C-30 ° C, preferably 23 ° C-28 ° C; the pH of the medium is 5.0-8.0 , Preferably 5.0-7.0; cultivation time is 24-80 hours, preferably 24-60 hours.
  • the seed medium contains glucose 5-20g / L, starch 5-20g / L, soybean cake powder 5-20g / L, yeast extract powder 1-10g / L, calcium carbonate 1 -20g / L, magnesium sulfate 1-10g / L, potassium dihydrogen sulfate 1-10g / L.
  • the fumagillin of the present invention was tested by HPLC under the following conditions:
  • Mobile phase A 0.1% (volume percentage) aqueous acetic acid solution
  • Mobile phase B 0.1% (volume percent) acetic acid in acetonitrile
  • Penicillium HS-NF-684Z CGMCC NO.14144
  • the main biological characteristics of Penicillium HS-NF-684Z (CGMCC NO.14144) of the present invention are: the spores are round, the spore surface is blue-green, the colony color is yellow, the reverse side is yellowish brown, and the surface is radial Grooved, velvety, 20-22mm in diameter.
  • Penicillium (Penicillium sp.) HS-NF-684Z (CGMCC NO.14144) is a brand-new strain of fumagillin-producing bacteria, and its ability to produce fumagillin is better than other strains in the prior art. With a substantial increase, the potency can be as high as 1000mg / L, which is conducive to industrial production.
  • the fungal DNA extraction kit used was purchased from Beijing Sanbo Yuanzhi Biotechnology Co., Ltd .;
  • Penicillium HS-NF-684Z (CGMCC No. 14144) is a strain of Penicillium isolated from the soil of a hillside in Qingcheng Mountain, Chengdu City, Sichuan province, China.
  • the culture characteristics of the strain use CA medium (Czapek Agar) at 25 °C for 12 days, CYA medium (Czapek Yeast Autolysate Agar) at 5 °C / 25 °C / 37 °C for 7 days, and G25N medium (25 % Glycerol Gitrate Agar) cultured at 25 °C for 7 days, observe the morphology, color and pigment of mycelium.
  • the culture characteristics of the strains are shown in Table 1.
  • Using PDA, SDA, MEA, and MEPG four kinds of culture media, after 5 to 7 days of cultivation at 25 °C, observe the color and pigment of mycelium.
  • the culture characteristics of the strains are shown in Table 2.
  • ISP9 is used as the basic medium, and the concentrations of potassium nitrate and ammonium sulfate are both 0.1%, as shown in Table 3.
  • PDA medium is used for oxidase and catalase test, pH test and temperature test.
  • the oxidase and catalase test results are shown in Table 5, the pH test results are shown in Table 6, and the temperature test results are shown in Table 7.
  • Carbon source growth situation Carbon source growth situation Inorganic nitrogen source growth situation D-glucose 4 Salicin 3 Ammonium sulfate + D-raffinose 3 D-lactose 3 Potassium nitrate +
  • Degradants Degradation concentration result* Degradants Degradation concentration result Adenine 0.5% 4, + Casein 1.0% 4,- Guanine 0.5% 4,- Tyrosine 1.0% 4,- Xanthine 0.4% 4,- Tween-40 1.0% 4,- Xylan 0.4% 4,- Tween-60 1.0% 4,- Hypoxanthine 0.4% 4, + Tween-80 1.0% 4,-
  • Example 3 18s rDNA sequence analysis and strain identification
  • the measured 18s rDNA sequence (SEQ ID NO: 3) was compared with the homologous sequence BLAST alignment with the related species and gene sequences in the GenBank database after proofreading to determine the taxonomic status of the strain.
  • the HS-NF-684Z (CGMCC No. 14144) 18s rDNA sequence was BLAST aligned with related sequences in the GenBank database. The results are shown in Table 10 (only strains with high homology are listed in the table).
  • the 18s rDNA sequence alignment results of the strain HS-NF-684Z show that the strain HS-NF-684Z (CGMCC No. 14144) and Penicillium (Penicillium sp.) Have very high homology, The highest reached 99.4%, combined with the morphological and cultural characteristics of the strain HS-NF-684Z (CGMCC No. 14144), it was found that the strain and Penicillium (Penicilliurn sp.) Classification characteristics are very similar, so the strain HS-NF-684Z (CGMCC No. 14144) was identified as Penicillium (Penicillium sp.).
  • Penicillium HS-NF-684Z CGMCC No. 14144
  • JCFrisvad et al. (Persoonia, vol. 14, no. 2, 177-182, 1990) disclosed that Penicillium scabrosum was cultured on CYA medium at 25 ° C for 7 days. The colony diameter was 26-32 mm, and there were few radial grooves. Exudate appeared; while Penicillium HS-NF-684Z (CGMCC No. 14144) of the present invention was cultured on CYA medium at 25 ° C for 7 days, with a colony diameter of 22 mm, obvious radial grooves, and little exudate in the central area.
  • the strain HS-NF-684Z (CGMCC No. 14144) of the present invention belongs to the genus Penicillium ( Penicillium sp.), And it is different from the known fumagillin-producing bacteria, so the penicillium HS-NF-684Z (CGMCC No. 14144) of the present invention is a brand new strain.
  • the slant medium uses potato glucose agar medium (g / L): potato 200g / L, glucose 20g / L, agar 20g / L, distilled water 1000mL, natural pH.
  • High-pressure steam sterilization at 121 ° C for 20min wait for the medium to cool to 50-60 ° C and place the slope, inoculate a ring of spores with a sterile inoculation ring on the ultra-clean workbench, coat evenly and place it at 25 ° C in the dark to cultivate 3- 5 days.
  • the volume of the bottled liquid is 30mL / bottle, sterilized by high-pressure steam at 121 °C for 20min.
  • the spore inoculation volume was 10 5 -10 6 cfu / mL, the cultivation temperature was 25 ⁇ 1 ° C., 250 rpm, and the shaker was shaken for 24 hours.
  • the volume of the shake bottle is 20mL / bottle, which is sterilized by high-pressure steam at 121 °C for 20min.
  • the seed liquid inoculation amount is 5% (volume percentage)
  • the cultivation temperature is 25 ⁇ 1 ° C., 250 rpm
  • shaking culture is carried out for 168 hours with a shaker to obtain a fumagillin fermentation broth.
  • the fermentation unit is 1000mg / L.
  • step (2) in Example 4 for the ratio of seed culture medium Put 9L of seed culture medium in the 15L seed tank (see step (2) in Example 4 for the ratio of seed culture medium), and add 0.1% (volume percentage) of the enemy as defoaming agent, steam sterilize at 121 °C, After 20min, after cooling to 25 °C, 30mL shake flask seed solution was connected, the cultivation temperature was 25 ⁇ 1 °C, the stirring speed was 100-600rpm, the oxygen flux was 0.5-2.0vvm, and the cultivation was for 24h.
  • the cultivation temperature is 25 ⁇ 1 ° C
  • the stirring speed is 100-600rpm
  • the oxygen flux is 0.5-2.0vvm
  • the cultivation is 120 hours to obtain the fumagillin fermentation broth.
  • the fermentation unit was 1010mg / L.
  • the wet solid was dissolved in 200 ml of a mixed solvent of dichloromethane and methanol (the volume ratio of dichloromethane and methanol was 1: 1), and filtered to obtain 220 ml of filtrate.
  • the filtrate was at a temperature of -10 ° C, a pressure of -0.08 Mpa, and a stirring rate After stirring at 100 rpm, 66 ml of methanol was added to the filtrate after 1 h, and then 66 ml of methanol was added every 1 h. After 8 h, filtration was performed to obtain 41.1 g of solid powder.
  • Example 6 The solid powder obtained in Example 6 was analyzed by MS to determine that its molecular weight was 458.55. Through 1 H NMR and 13 C NMR analysis, it was determined to be fumagillin, the structure is as follows:
  • Solubility easily soluble in methyl tert-butyl ether, methylene chloride, insoluble in water

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Abstract

一种新的青霉(Penicillium sp.)HS-NF-684Z，保藏编号为：CGMCC No.14144；同时公开了通过培养它制备烟曲霉素或者含有烟曲霉素的药物组合物的方法。

Description

一种青霉及其生产烟曲霉素的方法 技术领域
本发明涉及微生物工程技术领域，具体涉及一种新的青霉及其在制备烟曲霉素中的应用。
背景技术
烟曲霉素(式I)最初被用来杀死蜜蜂的微孢子虫寄生虫，后来发现可以用于治疗人肠道感染引起的微孢子虫病和(或)隐孢子虫病，而这种疾病对于免疫缺陷患者是相当致命的。近年来，随着对烟曲霉素及其类似物研究的深入，有报道表明烟曲霉素及其类似物具有抗血管生成的活性，可以通过与人甲硫氨酸氨肽酶-2(MetAP-2)结合，从而阻断血管的形成。因此，烟曲霉素及其类似物被广泛用于治疗癌症的血管生成抑制剂。
Figure PCTCN2019110893-appb-000001
现有技术中，F.R.Hanson等报道了烟曲霉素最初是从烟曲霉Aspergillus fumigatus的发酵培养物中分离得到的天然产物，并在随后的研究中公开了生产工艺(Eble and Hanson,1951；McCowen et al.,1951)；J.C.Frisvad(Persoonia，vol.14，no.2，177-182，1990)等公开了首次从一种青霉菌Penicillium scabrosum的发酵培养物中发现烟曲霉素，并对该菌株的形态进行了详细的描述；Z.Barboráková等(Journal of Microbiology,Biotechnology and Food Sciences，2012:1(4)466-477)公开了P.scabrosum FCB 353经过培养优化，产烟曲霉素的效价可以达到110mg/L。由此可见，现有技术中所使用的烟曲霉素生产菌比较原始，发 酵水平低，因此寻找一种新的高效的烟曲霉素生产菌很有必要。
发明内容
本发明的目的之一在于提供一种新的烟曲霉素生产菌，该菌为青霉(Penicillium sp.)HS-NF-684Z，其保藏编号为CGMCC No.14144。
本发明青霉HS-NF-684Z的微生物菌种于2017年07月24日保藏于中国微生物菌种保藏管理委员会普通微生物中心(地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所)，保藏编号为CGMCC No.14144，分类名为青霉(Penicillium sp.)，并登记在册，证明存活。
本发明的目的还在于提供了一种青霉HS-NF-684Z(CGMCC NO.14144)在制备烟曲霉素或者含有烟曲霉素的药物组合物的应用。
本发明还提供了一种烟曲霉素的制备方法，该方法包括采用青霉HS-NF-684Z(CGMCC No.14144)在含有可同化的碳源和/或氮源的营养培养基里，进行有氧发酵的步骤。
在优选的实施方案中，上述可同化的碳源选自淀粉、糊精、葡萄糖、蔗糖、乳糖、麦芽糖、工业糖蜜、甘油、山梨醇、甘露醇之一或者上述物质的组合，优选淀粉、糊精、葡萄糖、蔗糖。
在优选的实施方案中，上述可同化的氮源选自酵母抽提粉、酵母粉、酵母膏、黄豆饼粉、棉籽饼粉、花生饼粉、麸质粉、玉米浆干粉、豆粕、蛋白胨、尿素、铵盐之一或者上述物质的组合，优选黄豆饼粉、酵母膏、酵母抽提粉、酵母粉。
在优选的实施方案中，上述营养培养基还包括无机盐，所述无机盐选自柠檬酸三钠、磷酸二氢钾、磷酸氢二钾、硫酸铵、碳酸钙、硫酸亚铁、硫酸锌、硫酸铜、氯化钠、氯化钾、氯化钙、硫酸镁、氯化铁、硫酸锰之一或上述物质的组合，优选碳酸钙、磷酸二氢钾、磷酸氢二钾、硫酸镁、柠檬酸三钠。
在优选的实施方案中，所述营养培养基含有蔗糖40-1000g/L、糊精20-1000g/L、甘油10-80g/L、酵母抽提粉1-15g/L、酵母粉5-20g/L、酵母膏5-20g/L、硫酸镁1-10g/L、硫酸二氢钾1-5g/L、碳酸钙0-50g/L。
在优选的实施方案中，所述有氧发酵的温度为20-30℃，优选23-28℃；培养基pH为5.0-8.0，优选5.0-7.0；培养时间为24-226小时，优选72-168小时；通氧量为0.1-2.0vvm，优选0.5-2.0vvm。
在优选的实施方案中，所述青霉HS-NF-684Z是通过种子液接种至所述营养培养基中进行所述发酵培养的；其中，所述种子液是将青霉HS-NF-684Z(CGMCC No.14144)在种子培养基里进行种子培养得到的；所述种子培养的条件为：种子培养的温度为20℃-30℃，优选23℃-28℃；培养基pH为5.0-8.0，优选5.0-7.0；培养时间为24-80小时，优选24-60小时。
在优选的实施方案中，所述的种子培养基含有葡萄糖5-20g/L、淀粉5-20g/L、黄豆饼粉5-20g/L、酵母抽提粉1-10g/L、碳酸钙1-20g/L，硫酸镁1-10g/L，硫酸二氢钾1-10g/L。
本发明烟曲霉素通过以下条件进行HPLC检测：
色谱柱：Accchrom C18(4.6×250mm，5μm)
柱温：25℃；
进样体积：5μL；
流速：1mL/min；
检测波长：350nm；
分析时间：40min；
流动相：
流动相A：0.1％(体积百分比)乙酸水溶液；
流动相B：0.1％(体积百分比)乙酸乙腈溶液；
时间(min) 流动相A(％) 流动相B(％)
00:00 65 35
20:00 10 90
30:00 10 90
34:01 65 35
36:00 65 35
本发明青霉(Penicillium sp.)HS-NF-684Z(CGMCC NO.14144)的主要生物学特征是：孢子呈圆形，孢子面呈蓝绿色，菌落颜色为黄色，反面黄褐色，表面呈现放射状沟纹，绒状，直径20-22mm。
本发明青霉(Penicillium sp.)HS-NF-684Z(CGMCC NO.14144)为一株全新的烟曲霉素产生菌，且其生产烟曲霉素的能力比现有技术中其他菌种有了大幅度的提高，效价可高达1000mg/L，有利于实现工业化生产。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料，试剂等，如无特殊说明，均可从商业途径得到。
其中所用真菌DNA提取试剂盒购自北京三博远志生物技术有限责任公司；
PCR产物纯化回收所用的SanPrep柱式PCR产物纯化试剂盒购自生工生物工程(上海)股份有限公司。
以下结合具体实施例，对本发明作进一步说明。应理解，以下实施例仅用于说明本发明而非限定本发明的范围。
实施例1：菌株来源
青霉HS-NF-684Z(CGMCC No.14144)是一株从中国四川省成都市青城山某山坡的土壤中分离得到的青霉。
在青城山区域土壤进行交叉采样，随机取5个采样点，每个点取土壤样品10g，放入锥形瓶中，混合均匀后取样品10g，加入到一个装入90mL无菌水的锥形瓶中(瓶中有一个磁力搅拌器)，漩涡搅拌30分钟，使其充分混匀制成悬浊液，即为10 -1菌悬液。用稀释涂布平板法将上述悬浊液与无菌水按照体积比1：9稀释成10 -2，10 -3，10 -4，10 -5浓度，取不同稀释倍数菌悬液0.1mL，涂布于察氏培养基(Czapek–Dox Medium)平板中，用无菌涂布棒在培养基表面轻轻涂布，室温下静置30分钟后置于25℃恒温培养箱。待菌落长出后，观察记录菌落颜色、透明度、菌落表面、边缘形态。最终挑取1000株菌株接种于察氏培养基制成斜面，并进行发酵验证。用接种环挑取斜面培养的菌体一环，分别接种于含有30mL 种子培养基的250mL锥形瓶中，于25℃条件下震荡培养1天后，再吸取1mL移种于含有20mL发酵培养基的250mL锥形瓶中，于25℃条件下震荡培养2天后，经HPLC检测所得发酵液中烟曲霉素的含量，挑选出最高产菌株即青霉HS-NF-684Z(CGMCC No.14144)。
种子培养基配方(g/L)：葡萄糖10g/L，淀粉10g/L，黄豆饼粉10g/L，酵母抽提粉5g/L，碳酸钙15g/L，硫酸镁1.5g/L，硫酸二氢钾1g/L，加水定容至1000mL，pH 6.0±0.1。
发酵培养基配方(g/L)：蔗糖40g/L，糊精40g/L，甘油30g/L，酵母抽提粉5g/L，酵母粉10g/L，酵母膏10g/L，碳酸钙30g/L，加水定容至1000mL，pH 6.0±0.1。
实施例2：菌种鉴定
参照《中国真菌志》、《普通真菌学》、《常见细菌系统鉴定手册》、《分子克隆实验指南》及《中国药典》(2015版)等书中的有关内容进行实验；颜色判断参照RAL K7色卡系列的颜色进行对照。
1、菌种编号：HS-NF-684Z(CGMCC No.14144)。
2、菌株的培养学特征：采用CA培养基(Czapek Agar)25℃培养12天，采用CYA培养基(Czapek Yeast Autolysate Agar)5℃/25℃/37℃培养7天，采用G25N培养基(25％Glycerol Gitrate Agar)25℃培养7天，观察菌丝体的形态、颜色和色素情况，菌株的培养特征见表1。采用PDA、SDA、MEA和MEPG 4种培养基，25℃培养5～7天后，观察菌丝体的颜色及色素情况，菌株的培养特征见表2。
3、生理生化试验：除温度实验外，均为25℃培养5～7天。
a)碳源的利用：采用ISP9作为基础培养基，各种碳源的终浓度均为1.0％，见表3。
b)无机氮源的利用：采用ISP9作为基础培养基，硝酸钾和硫酸铵的浓度均为0.1％，见表3。
c)降解试验和NaCl耐受实验采用基础培养基为GYEA(pH6.8)，各种降解 物的浓度及降解试验结果见表4；NaCl耐受实验结果见表8。
d)氧化酶和过氧化氢酶试验、pH试验和温度试验均采用PDA培养基。氧化酶和过氧化氢酶试验结果见表5，pH试验结果见表6，温度试验结果见表7。
e)M.R、V-P和脲酶实验采用《常见细菌系统鉴定手册》方法，其结果见表5。
实验结果
1、菌株的培养学特征：HS-NF-684Z(CGMCC No.14144)的培养特征见表1和表2。
表1 菌株HS-NF-684Z(CGMCC No.14144)在CA/CYA/G25N培养基上的培养特征
Figure PCTCN2019110893-appb-000002
表2 菌株HS-NF-684Z(CGMCC No.14144)在PDA、SDA、MEA和MEPG 4种培养基上的培养特征
Figure PCTCN2019110893-appb-000003
2、生理生化特征：见表3-表8。
表3 菌株HS-NF-684Z(CGMCC No.14144)的碳源和氮源的利用情况
碳源 生长情况 碳源 生长情况 无机氮源 生长情况
D-葡萄糖 4 水杨苷 3 硫酸铵 +
D-棉子糖 3 D-乳糖 3 硝酸钾 +
D-木糖 2 半乳糖 4    
D-山梨醇 4 肌醇 4    
L-阿拉伯糖 2 甘露醇 4    
甘油 4 甘氨酸 2    
麦芽糖 4 木聚糖 4    
D-果糖 4 菊粉 2    
D-蔗糖 4        
表4 菌株HS-NF-684Z(CGMCC No.14144)的降解试验结果
降解物 降解物浓度 结果* 降解物 降解物浓度 结果
腺嘌呤 0.5％ 4，+ 酪蛋白 1.0％ 4，-
鸟嘌呤 0.5％ 4，- 酪氨酸 1.0％ 4，-
黄嘌呤 0.4％ 4，- Tween-40 1.0％ 4，-
木聚糖 0.4％ 4，- Tween-60 1.0％ 4，-
次黄嘌呤 0.4％ 4，+ Tween-80 1.0％ 4，-
表5 菌株HS-NF-684Z(CGMCC No.14144)主要的生理生化特征
试验项目 结果 试验项目 结果 试验项目 结果
明胶液化 + 牛奶胨化 - 纤维素利用 -
淀粉水解 - 硝酸盐还原 - 过氧化氢酶 -
牛奶凝固 + 硫化氢产生 -    
V.P实验 - M.R实验 -    
表6 菌株HS-NF-684Z(CGMCC No.14144)生长的pH试验
pH 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5
生长情况 3 3 3 4 4 4 4 4 4
表7 菌株HS-NF-684Z(CGMCC No.14144)生长的温度试验
温度(℃) 7 14 28 37 45
生长情况 2 3 4 0 0
表8 菌株HS-NF-684Z(CGMCC No.14144)对NaCl的耐受性
NaCl浓度 1％ 4％ 7％ 10％
菌株生长情况 4 4 3 2
*注：表1-8中：0，无生长；1，生长很弱；2，能生长，有少量孢子；3，生长良好，有大量孢子；4，生长最好，有丰富孢子；+，阳性；-，阴性。
实施例3：18s rDNA序列分析及菌种鉴定
参照《分子克隆实验指南》书中的有关内容进行实验。收集菌株的新鲜菌体，然后采用真菌DNA提取试剂盒提取菌体总DNA，采用通用引物NS1(SEQ ID NO:1)/NS8(SEQ ID NO:2)进行18S rDNA序列扩增，扩增体系和PCR反应程序如表9所示，PCR产物检测采用0.8％琼脂糖凝胶电泳，PCR产物纯化回收采用SanPrep柱式PCR纯化产物试剂盒，纯化后的PCR产物直接送南京金斯瑞生物科技有限公司进行序列测定。
表9 PCR扩增体系和反应程序
Figure PCTCN2019110893-appb-000004
所测的18s rDNA序列(SEQ ID NO:3)经过校对后与GenBank数据库中相关种、属序列进行同源序列BLAST比对，以确定该菌株的分类地位。
菌株HS-NF-684Z(CGMCC No.14144)18s rDNA序列与GenBank数据库中相关序列进行BLAST比对，结果见表10(表中只列出同源性较高的菌株)。
表10 菌株HS-NF-684Z和相关菌株的同源性结果
Figure PCTCN2019110893-appb-000005
菌株HS-NF-684Z(CGMCC No.14144)的18s rDNA序列比对结果显示：菌株HS-NF-684Z(CGMCC No.14144)和青霉属(Penicillium sp.)有非常高的同 源性，最高的达到99.4％，结合菌株HS-NF-684Z(CGMCC No.14144)的形态学和培养学特征实验结果，发现该菌株和青霉(Penicilliurn sp.)分类的特征非常相似，因此将该菌株HS-NF-684Z(CGMCC No.14144)鉴定为青霉属(Penicillium sp.)。
本发明青霉HS-NF-684Z(CGMCC No.14144)和其他烟曲霉素产生菌的比较如下：
J.C.Frisvad等(Persoonia，vol.14，no.2，177-182，1990)公开了Penicillium scabrosum在CYA培养基上25℃培养7天，菌落直径26-32mm，很少放射状沟纹，中心区域常出现渗出液；而本发明青霉HS-NF-684Z(CGMCC No.14144)在CYA培养基上25℃培养7天，菌落直径22mm，明显放射状沟纹，中心区域少有渗出液。
F.R.Hanson等(Journal of Bacteriology，1949，58(4)：527-529)和Wen Yang等(Current Microbiology，2003，46(1)：0024-0027)公开的烟曲霉素生产菌均为烟曲霉Aspergillus fumigatus；而本发明中的菌株HS-NF-684Z(CGMCC No.14144)经过18s rDNA序列比对，鉴定为青霉菌属Penicillium sp.。
Z.Barboráková等(Journal of Microbiology，Biotechnology and Food Sciences，2012：1(4)：466-477)公开了烟曲霉素生产菌P.scabrosum FCB 353产烟曲霉素的效价为110mg/L；而本发明青霉HS-NF-684Z(CGMCC No.14144)产烟曲霉素的效价高达1000mg/L。
结合前述本发明的菌株HS-NF-684Z(CGMCC No.14144)的形态学、培养特征和DNA序列鉴定结果可知，本发明的菌株HS-NF-684Z(CGMCC No.14144)属于青霉属(Penicillium sp.)，且它不同于已知的烟曲霉素产生菌，因此本发明青霉HS-NF-684Z(CGMCC No.14144)是一株全新的菌种。
实施例4：摇瓶发酵制备烟曲霉素
(1)斜面菌落的制备与培养
斜面培养基采用马铃薯葡萄糖琼脂培养基(g/L)：马铃薯200g/L，葡萄糖20g/L，琼脂20g/L，蒸馏水1000mL，自然pH。
高压蒸汽灭菌121℃，20min，待培养基冷却至50-60℃摆放斜面，在超净工作台使用无菌接种环接种一环孢子，涂布均匀后置于25℃避光培养3-5天。
(2)种子培养基的制备与培养
种子培养基配方(g/L)：葡萄糖10g/L，淀粉10g/L，黄豆饼粉10g/L，酵母抽提粉5g/L，碳酸钙15g/L，硫酸镁1.5g/L，硫酸二氢钾1g/L，加水定容至1000mL，pH 6.0±0.1。
摇瓶装液量为30mL/瓶，高压蒸汽121℃灭菌，20min。孢子接种量为10 5-10 6c.f.u/mL，培养温度为25±1℃，250rpm，摇床振荡培养24小时。
(3)发酵培养基的制备与培养
发酵培养基配方(g/L)：蔗糖80g/L，糊精20g/L，甘油40g/L，酵母抽提粉5g/L，酵母粉10g/L，酵母膏10g/L，硫酸镁1.5g/L，硫酸二氢钾1g/L，碳酸钙30g/L，加水定容至1000mL，pH 6.0±0.1。
摇瓶装液量为20mL/瓶，高压蒸汽121℃灭菌，20min。种子液接种量为5％(体积百分比)，培养温度为25±1℃，250rpm，摇床振荡培养168小时得烟曲霉素发酵液。经HPLC检测，发酵单位为1000mg/L。
实施例5：发酵罐制备烟曲霉素
(1)种子罐种子液的制备与培养
在15L种子罐中投入9L的种子培养基(种子培养基配比见实施例4中步骤(2))，并添加0.1％(体积百分比)泡敌作为消泡剂)，蒸汽121℃灭菌，20min，待冷却至25℃后，接入30mL摇瓶种子液，培养温度为25±1℃，搅拌转速100-600rpm，通氧量0.5-2.0vvm，培养24h。
(2)发酵罐发酵液的制备与培养
在50L发酵罐中投入30L的发酵培养基(发酵培养基配比见实施例4中步骤(3)，并添加0.05％(体积百分比)泡敌作为消泡剂)，蒸汽121℃灭菌，20min，待冷却至25℃后，移入3L种子罐种子液，培养温度为25±1℃，搅拌转速100-600rpm，通氧量0.5-2.0vvm，培养120小时得烟曲霉素发酵液。经HPLC检测，发酵单位为1010mg/L。
实施例6：提取制备烟曲霉素
取实施例5的烟曲霉素发酵液(烟曲霉素效价为1010mg/L)30L，加入45L甲基叔丁基醚(MTBE)，搅拌2h，静置8h，分液，收集MTBE萃取相42L，将MTBE或ETBE萃取相浓缩至1.1L，浓缩温度为30℃，然后降温至0℃，搅拌8h，过滤得到50.0g湿固体。然后，将湿固体用二氯甲烷与甲醇的混合溶剂200ml(二氯甲烷与甲醇的体积比为1：1)溶解，过滤得220ml滤液，滤液在温度-10℃，压力-0.08Mpa，搅拌速率100rmp条件下搅拌，1h后向滤液中加入66ml的甲醇，之后每1h加入66ml的甲醇，8h后过滤得到固体粉末41.1g。
实施例7：烟曲霉素的鉴定
(1)将实施例6所得固体粉末经过MS分析确定其分子量为458.55。通过 1H NMR和 13C NMR分析，确定其为烟曲霉素，结构如下：
Figure PCTCN2019110893-appb-000006
(2)烟曲霉素的理化性质如下：
性状：白色至淡黄色无定型粉末状
溶解性：易溶于甲基叔丁基醚、二氯甲烷，不溶于水
分子式：C 26H 34O 7
烟曲霉素在CDCl 3中的核磁数据(氢谱，400MHz；碳谱，100MHz)的具体结果见表11。
表11 烟曲霉素在CDCl 3中的核磁数据
Figure PCTCN2019110893-appb-000007
Figure PCTCN2019110893-appb-000008

Claims (10)

  1. 一种青霉(Penicillium sp.)HS-NF-684Z，其保藏编号为CGMCC No.14144。
  2. 根据权利要求1所述的青霉HS-NF-684Z在制备烟曲霉素或者含有烟曲霉素的药物组合物的应用。
  3. 一种烟曲霉素的制备方法，其特征在于：包括采用如权利要求1所述的青霉HS-NF-684Z在含有可同化的碳源和/或氮源的营养培养基里，进行有氧发酵的步骤。
  4. 根据权利要求3所述的方法，其特征在于：所述可同化的碳源选自淀粉、糊精、葡萄糖、蔗糖、乳糖、麦芽糖、工业糖蜜、甘油、山梨醇、甘露醇之一或者上述物质的组合，优选淀粉、糊精、葡萄糖、蔗糖。
  5. 根据权利要求3所述的方法，其特征在于：所述可同化的氮源选自酵母抽提粉、酵母粉、酵母膏、黄豆饼粉、棉籽饼粉、花生饼粉、麸质粉、玉米浆干粉、豆粕、蛋白胨、尿素、铵盐之一或者上述物质的组合，优选黄豆饼粉、酵母膏、酵母抽提粉、酵母粉。
  6. 根据权利要求3-5任一项所述的方法，其特征在于：所述营养培养基还包括无机盐，所述无机盐选自柠檬酸三钠、磷酸二氢钾、磷酸氢二钾、硫酸铵、碳酸钙、硫酸亚铁、硫酸锌、硫酸铜、氯化钠、氯化钾、氯化钙、硫酸镁、氯化铁、硫酸锰之一或上述物质的组合，优选碳酸钙、磷酸二氢钾、磷酸氢二钾、硫酸镁、柠檬酸三钠。
  7. 根据权利要求3-6任一项所述的方法，其特征在于：所述营养培养基含有蔗糖40-1000g/L、糊精20-1000g/L、甘油10-80g/L、酵母抽提粉1-15g/L、酵母粉5-20g/L、酵母膏5-20g/L、硫酸镁1-10g/L、硫酸二氢钾1-5g/L、碳酸钙0-50g/L。
  8. 根据权利要求3-7任一项所述的方法，其特征在于：所述有氧发酵的温度为20-30℃，优选23-28℃；培养基pH为5.0-8.0，优选5.0-7.0；培养时间为24-226小时，优选72-168小时；通氧量为0.1-2.0vvm，优选0.5-2.0vvm。
  9. 根据权利要求3-8任一项所述的方法，其特征在于：所述青霉HS-NF-684Z是通过种子液接种至所述营养培养基中进行所述发酵培养的；
    其中，所述种子液是将权利要求1所述的青霉HS-NF-684Z在种子培养基里进行种子培养得到的；所述种子培养的条件为：种子培养的温度为20℃-30℃，优选23℃-28℃；培养基pH为5.0-8.0，优选5.0-7.0；培养时间为24-80小时，优选24-60小时。
  10. 根据权利要求9所述的方法，其特征在于：所述的种子培养基含有葡萄糖5-20g/L、淀粉5-20g/L、黄豆饼粉5-20g/L、酵母抽提粉1-10g/L、碳酸钙1-20g/L，硫酸镁1-10g/L，硫酸二氢钾1-10g/L。
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