WO2020119828A1 - 一株克劳氏芽孢杆菌及使用其生产四氢嘧啶的方法 - Google Patents

一株克劳氏芽孢杆菌及使用其生产四氢嘧啶的方法 Download PDF

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WO2020119828A1
WO2020119828A1 PCT/CN2019/128701 CN2019128701W WO2020119828A1 WO 2020119828 A1 WO2020119828 A1 WO 2020119828A1 CN 2019128701 W CN2019128701 W CN 2019128701W WO 2020119828 A1 WO2020119828 A1 WO 2020119828A1
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bacillus clausii
tetrahydropyrimidine
medium
inorganic salt
strain
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PCT/CN2019/128701
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French (fr)
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谭剑
邓莉川
于淼
王靖
王小艳
殷红
田芳
王灿
许克家
江俊杰
李榕榕
陈博
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北京百奥茵诺生物科技有限公司
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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
    • C12N1/20—Bacteria; Culture media therefor
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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
    • C12N1/20—Bacteria; Culture media therefor
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus

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  • the present invention belongs to the field of microbiology. Specifically, the present invention relates to a strain of Bacillus clausii producing tetrahydropyrimidine, as well as applications and methods for producing tetrahydropyrimidine using the strain.
  • Tetrahydropyrimidine (Ectoine) is a compatible solute produced by many salt-tolerant and halophilic microorganisms in cells to maintain osmotic pressure balance. It can be used to stimulate cells under extreme conditions such as high temperature, freezing, radiation, and drying. , Proteins, cell membranes and nucleic acids, etc. provide protection.
  • tetrahydropyrimidine has a certain effect on neurological diseases such as Alzheimer's disease and Parkinson's disease, and the latest research found that tetrahydropyrimidine can improve the skin's regenerative ability and delay skin aging. Therefore, tetrahydropyrimidine will have broad application prospects in industries such as fine chemicals and biomedicine.
  • the production method of tetrahydropyrimidine is mainly obtained by high-density fermentation of halophilic microorganisms (especially Halomonas).
  • this process is produced by a method called "bacterial milking method/lactation method", that is, culturing bacteria under high osmotic pressure, then releasing solute by low osmotic shock, and then re-hyperosmotic culturing the bacteria, low Osmotic shock releases the solute, which is cycled up to 8-9 times in sequence to obtain the product.
  • This method has higher requirements for the stability of the reactor material, and this discontinuous production process and high concentration of salt increase the difficulty of the downstream purification process.
  • Bacillus clausii is a probiotic strain, which not only can meet the production with high safety requirements, but also has a mature fermentation process, simple fermentation and cultivation requirements, and can also meet the industrial application conditions of simple fermentation operation and low cost, but the current Bacillus clausii is mostly used in the industrial production of proteases, and there have been no reports about its use in the industrial production of tetrahydropyrimidine.
  • the object of the present invention is to provide a strain for producing tetrahydropyrimidine with high biological safety, high yield of tetrahydropyrimidine, simple fermentation operation and low cultivation cost, and a method for producing tetrahydropyrimidine by fermentation to overcome the prior art Deficiencies.
  • the present invention provides a strain of Bacillus clausii ET01 (Bacillus clausii ET01) producing tetrahydropyrimidine, and the deposit number is CGMCC NO.16310.
  • the strain described in the present invention can be grown in a liquid medium with an inorganic salt concentration of 0-100 g/L, and can produce tetrahydropyrimidine at a low salt concentration (less than 100 g/L and greater than 0 g/L, such as 50 g/L). (Up to 25g/L).
  • the present invention provides the application of the Bacillus clausii ET01 described in the first aspect in the production of tetrahydropyrimidine.
  • the present invention provides a method for producing tetrahydropyrimidine, the method comprising:
  • the Bacillus clausii ET01 of the first aspect is subjected to aerobic fermentation in a Bacillus clausii culture medium with an inorganic salt concentration of less than 100 g/L to obtain tetrahydropyrimidine.
  • the beneficial effects of the present invention are as follows: Compared with other tetrahydropyrimidine-producing strains in the prior art (for example, Halomonas in CN103451137A), the Bacillus clausii ET01 of the present invention has a higher ability to synthesize tetrahydropyrimidine ( 25g/L), which can be fermented and produced well under low salt concentration (less than 100g/L) culture conditions, reducing the risk of corrosion of fermentation equipment and environmental pollution caused by excessive salinity of other halophilic strains in fermentation production; After the aerobic fermentation of Bacillus claus ET01 invented, the fermentation fermentation by-products and other impurities are low in content, and no hydroxyl pyrimidine is produced. The purity of tetrahydropyrimidine obtained after simple separation and purification can reach more than 98%, which reduces Separation and purification costs have good industrial application prospects.
  • Figure 1 is Bacillus clausii ET01 observed under a 100x microscope.
  • the present inventors used a gradient dilution method to isolate and screen a high-hydrogen pyrimidine-producing bacterium from the mud of the offshore salt farm in Weihai. After cultivating the strain on LB solid medium (10g/LNaCl) at 38°C for 24 hours, it formed milky white colonies with irregular edges and protrusions on the surface; when viewed under a microscope at a magnification of 100 times, the bacterial cells were elongated ( As shown in Figure 1); Gram-positive staining is positive, Gram-positive bacteria.
  • Bacillus clausii ET01 Bacillus clausii ET01
  • the strain was deposited in the General Microbiology Center (CGMCC) of China Microbial Culture Collection Management Committee (Beijing No. 1, Beichen West Road, Chaoyang District, Beijing). The deposit date is August 17, 2018, and the taxonomic name is Bacillus clausii (Bacillus clausii), the deposit number is CGMCC NO.16310.
  • CGMCC General Microbiology Center
  • Bacillus clausii culture medium refers to a medium well known to those skilled in the art that can be used to cultivate Bacillus clausii, such as a nutrient medium containing a carbon source, a nitrogen source, and inorganic salts (such as LB medium or nutrient broth medium).
  • the formulation of the medium may be a formulation known in the art, for example, the commonly used LB medium formulation is: 0.8-1wt% peptone, 0.5-0.8wt% yeast powder, 1-1.5wt% sodium chloride, pH It is 6.8-7.0; the formula of nutrient broth medium is 0.8-1wt% peptone, 0.3-0.5wt% beef extract, 0.5-0.8wt% sodium chloride, and the pH is 7.2-7.6.
  • those skilled in the art can select and adjust the specific carbon source, nitrogen source, and inorganic salts and their respective contents in the culture medium according to needs, especially the types and concentrations of inorganic salts, so as to be suitable for the present invention
  • C. clausii is cultured and used, for example, the sodium chloride concentration in the LB medium is adjusted to 5 wt% for the production of tetrahydropyrimidine.
  • the term "carbon source” refers to nutrients that provide microorganisms with carbon elements required for growth and reproduction.
  • Examples of the carbon source include but are not limited to glucose, dextrin, sucrose, monosodium glutamate, molasses, glycerin, lactic acid, maltodextrin, and maltose.
  • the "carbon source” is preferably glucose.
  • the term "nitrogen source” refers to nutrients that provide microorganisms with nitrogen elements required for growth and reproduction.
  • the nitrogen source include, but are not limited to, yeast extract, yeast extract, tryptone, monosodium glutamate, ammonium chloride, ammonium sulfate, soy peptone, soybean meal, corn meal, peanut cake, cottonseed meal.
  • the "nitrogen source” is preferably yeast extract, yeast extract, tryptone, ammonium chloride or ammonium sulfate, or any combination thereof.
  • inorganic salt refers to mineral salts in inorganic compounds, such as sodium salt, potassium salt, calcium salt, phosphate, sulfate, and the like.
  • examples of the inorganic salts include but are not limited to: sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, iron sulfate heptahydrate, calcium chloride dihydrate, potassium chloride, sodium sulfate, hydrogen phosphate Disodium.
  • seed medium refers to a medium used for activating strains
  • the seed medium may be a common medium conventionally used in the art, such as LB medium or nutrient broth medium.
  • a person skilled in the art can select specific components and respective contents in the culture medium according to needs, so as to be suitable for the activation of Bacillus clausii of the present invention, especially to select and adjust the types and concentrations of inorganic salts.
  • the present invention provides a strain of Bacillus clausii ET01 (Bacillus clausii ET01) producing tetrahydropyrimidine, and the deposit number is CGMCC NO.16310.
  • the strain described in the present invention is grown in a liquid medium with an inorganic salt concentration of 0-100 g/L, and produces high tetrahydropyrimidine at a low salt concentration (less than 100 g/L and greater than 0 g/L).
  • the Bacillus clausii ET01 can be grown in a liquid medium with an inorganic salt concentration of 0-100 g/L, preferably 30-70 g/L, and more preferably 50 g/L.
  • the present invention also provides the application of the Bacillus clausii ET01 of the present invention in the production of tetrahydropyrimidine.
  • the present invention provides a method for producing tetrahydropyrimidine, the method comprising: applying Bacillus clausii ET01 of the present invention in a Bacillus clausii culture medium with an inorganic salt concentration of less than 100 g/L Carry out aerobic fermentation to obtain tetrahydropyrimidine.
  • the Bacillus clausii culture medium is a liquid medium for cultivating Bacillus clausii in the art, for example, a nutrient medium containing a carbon source, a nitrogen source, and inorganic salts, such as LB culture Base or nutrient broth medium.
  • a nutrient medium containing a carbon source, a nitrogen source, and inorganic salts, such as LB culture Base or nutrient broth medium.
  • the inorganic salt is selected from sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, iron sulfate heptahydrate, calcium chloride dihydrate, potassium chloride, sodium sulfate, Disodium hydrogen phosphate, or any combination thereof.
  • the inorganic salt is sodium chloride.
  • the salt concentration of the inorganic salt is less than 100 g/L and greater than 0 g/L, preferably 30-70 g/L, more preferably 50 g/L.
  • the carbon source is selected from glucose, dextrin, sucrose, monosodium glutamate, molasses, glycerin, lactic acid, maltodextrin, maltose, or any combination thereof.
  • the nitrogen source is selected from yeast extract, yeast extract, tryptone, monosodium glutamate, ammonium chloride, ammonium sulfate, soy peptone, soybean meal, corn meal, peanut cake, cottonseed meal, or they Any combination.
  • the nitrogen source is selected from yeast extract, yeast extract, tryptone, ammonium chloride or ammonium sulfate, or any combination thereof.
  • the Bacillus clausii medium with an inorganic salt concentration of less than 100 g/L is an LB medium with a NaCl concentration of less than 100 g/L.
  • aerobic fermentation may be carried out using aerobic fermentation equipment or methods commonly used in the art.
  • a person skilled in the art can select suitable fermentation equipment and fermentation conditions according to actual fermentation conditions.
  • the fermentation conditions are 28-38°C, preferably 37°C, 150-220rpm, preferably 200rpm, for 12-24h. It should be noted that any optimization and adjustment of aerobic fermentation conditions by those skilled in the art on the basis of the present invention are also within the scope of the present invention.
  • the method for producing tetrahydropyrimidine includes the Bacillus clausii ET01 according to the present invention having a sodium chloride concentration of less than 100g/L, preferably 30-70g/L, more preferably 50g/L LB medium or nutrient broth medium, cultured at 28-38 °C, preferably 37 °C, 150-220rpm, preferably 200rpm for 12-24h.
  • the tetrahydropyrimidine is obtained by separating and/or purifying the fermentation broth of aerobic fermentation (also referred to as "aerobic fermentation broth").
  • the tetrahydropyrimidine in the fermentation broth can be separated and/or purified using equipment or methods commonly used in the art for separation and/or purification, such as centrifugation, filtration, extraction, and the like.
  • the tetrahydropyrimidine is obtained by an extraction method, for example, the tetrahydropyrimidine in the aerobic fermentation broth is extracted with 80% ethanol.
  • the extraction of tetrahydropyrimidine in the aerobic fermentation broth with 80% ethanol includes the following steps: the aerobic fermentation broth is allowed to stand for 12-24h with an equal volume of 80% ethanol aqueous solution, and centrifuged , Take the supernatant to obtain tetrahydropyrimidine. Before adding the 80% ethanol aqueous solution, the fermentation broth can also be centrifuged to separate the bacterial cells and/or bacterial liquid for extraction.
  • the method further comprises activating the Bacillus clausii.
  • the activation includes: inoculating the Bacillus clausii ET01 in a seed culture medium for shaking culture, and cultivating at a temperature of 28-38°C and a rotation speed of 150-220rpm for 12- 24h.
  • the seed medium is LB medium (0.8-1wt% peptone, 0.5-0.8wt% yeast powder, 1-1.5wt% sodium chloride; pH 6.8-7.0) or nutrient broth Medium (0.8-1wt% peptone, 0.3-0.5wt% beef extract, 0.5-0.8wt% sodium chloride; pH 7.2-7.6).
  • the purity of the tetrahydropyrimidine prepared by the method of the present invention is above 98%.
  • LB medium tryptone 10g/L; yeast extract powder 5g/L; sodium chloride 0-100g/L; pH 7.0, 121°C sterilization for 15min.
  • LB solid medium tryptone 10g/L; yeast extract 5g/L; agar 15g/L; sodium chloride 20-100g/L; pH 7.0, 121°C sterilization for 15min.
  • Bacterial morphology identification was performed on this strain. After cultivating the strain on LB solid medium of 10g/L NaCl at 38°C for 24 hours, it formed milky white colonies with irregular edges and protrusions on the surface; under 100 times magnification, the microbial cells were long rod-shaped (as shown in the figure (1)) Gram positive staining is Gram-positive bacteria.
  • the strain was identified and named Bacillus clausii ET01 (Bacillus clausii ET01) according to the morphology of the cells and 16S rDNA sequencing comparison, and it was deposited with the deposit number CGMCC NO.16310.
  • Bacillus clausii ET01 stored in a glycerol tube was inoculated into 60 mL of 10 g/L NaCl LB medium in an amount of 2 v/v%, and cultured and activated on a shaker at 37° C. and 200 rpm overnight.
  • the absorbance value (OD 600 ) of the fermentation broth at 600 nm wavelength was measured using a spectrophotometer at 2.3.
  • the activated Bacillus clausii ET01 was inoculated into 60mL LB medium containing 0g/L, 30g/L, 50g/L, 70g/L and 100g/L NaCl in an amount of 1v/v% and shaken Incubate on the bed at 37°C and 200 rpm for 24 hours.
  • the OD 600 of the fermentation broth after 24 hours of cultivation was measured using a spectrophotometer.
  • the fermentation products (tetrahydropyrimidine and hydroxypyrimidine) in the fermentation broth of Bacillus clausii cultured for 24 hours were detected and identified by HPLC, and the Agilent 1260 high-performance liquid phase detection system was used for detection.
  • the detection conditions were:
  • Liquid chromatography column Zorbax SB C18 column (column length 150mm, column inner diameter 4.6mm; packing particle size 5 ⁇ m);
  • tetrahydropyrimidine standard aqueous solution and hydroxypyrimidine standard aqueous solution both were purchased from Sigma-Aldrich, the article number of tetrahydropyrimidine was E2271, and the article number of hydroxypyrimidine was 70709.
  • the peak time of the tetrahydropyrimidine standard is 7.8 min, and the peak time of the hydroxypyrimidine standard is about 7.0 min (HPLC peaks are not shown).
  • Table 1 OD 600 and tetrahydropyrimidine detection results of the fermentation broth of Bacillus claus ET01 under different NaCl concentration conditions
  • Bacillus clausii of the present invention grows normally in the absence of NaCl addition (0g/L NaCl), but no tetrahydropyrimidine is produced; too high in NaCl addition (100g/LNaCl)
  • the strain OD 600 is low, indicating that the strain grows poorly, and only a weak accumulation of tetrahydropyrimidine, the overall yield is low; while in the case of 30-70g/L, the strain not only grows normally, but also the tetrahydropyrimidine
  • the constant is higher, especially in the case of 50g/L NaCl, the strain OD 600 is the highest, indicating that the strain grows well, and the production of tetrahydropyrimidine is higher than that of other salt concentrations, reaching 25g/L.
  • Bacillus clausii TDB1-4 (CCTCC NO: M2015430) and Bacillus clausii ET01 of the present invention were used for fermentation culture in LB medium with NaCl concentration of 0 g/L and 50 g/L, respectively.
  • the strain was cultured according to the method of Example 2 and the OD 600 and tetrahydropyrimidine after 24 hours of fermentation culture were tested. The results are shown in Table 2.
  • Bacillus clausii ET01 and Bacillus clausii TDB1-4 are similar; but in the LB medium of 50 g/L NaCl, the Bacillus clausii ET01 grows well and produces tetrahydropyrimidine; while Bacillus clausii TDB1-4 grows poorly and does not produce tetrahydropyrimidine.
  • Bacillus clausii ET01 of the present invention has a certain salt tolerance, and can produce tetrahydropyrimidine.
  • Bacillus clausii ET01 of the present invention to synthesize tetrahydropyrimidine can reach 25.0g/L, compared with other tetrahydropyrimidine-producing strains reported in the prior art (for example, Halomonas in CN103451137A, 18g/L ) Has obvious improvement and has the prospect of being used for industrial production of tetrahydropyrimidine.
  • Hydropyrimidine does not require high cost separation and purification processes such as ion exchange chromatography and molecular sieve. It can save costs, has simple operation, and has good industrial application prospects.

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Abstract

一种产四氢嘧啶的克劳氏芽孢杆菌ET01及使用该菌株发酵生产四氢嘧啶的方法，该菌株保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏编号为CGMCC No.16310。该菌株能够在低盐浓度刺激下产生较高产量的四氢嘧啶，可用于工业发酵生产四氢嘧啶。

Description

一株克劳氏芽孢杆菌及使用其生产四氢嘧啶的方法 技术领域
本发明属于微生物领域，具体而言，本发明涉及一株生产四氢嘧啶的克劳氏芽孢杆菌(Bacillus clausii)，以及使用该菌株生产四氢嘧啶的应用及方法。
背景技术
四氢嘧啶(Ectoine)是许多耐盐和嗜盐微生物为维持渗透压平衡而在细胞内产生的一种相容性溶质，能够为处于外界高温、冷冻、射线、干燥等极端条件刺激下的细胞、蛋白质、细胞膜和核酸等提供保护作用。此外，四氢嘧啶对阿尔兹海默氏症、帕金森病等神经性疾病均有一定的疗效，并且最新研究发现四氢嘧啶能够提高皮肤的再生能力和延缓皮肤的老化。因此，四氢嘧啶在精细化工和生物医药等行业将具有广泛的应用前景。
目前，四氢嘧啶的生产方法主要通过嗜盐微生物(特别是盐单胞菌)的高密度发酵来获得。通常而言，这个过程通过采用被称为“细菌挤奶法/泌乳法”的方式进行生产，即高渗透压下培养细菌、然后低渗冲击释放溶质、再将菌体重新高渗培养、低渗冲击释放溶质，依次循环多至8-9次，获得产物。该方法对反应器材料的稳定性有较高要求，而且这种不连续的生产流程及高浓度的盐增加了下游纯化工艺的难度，另外高浓度的盐易对设备造成腐蚀，同时还会影响菌体的生长，影响四氢嘧啶的产量，导致生产成本的增加，影响了四氢嘧啶的大规模应用。
目前现有的生产菌株及使用其生产四氢嘧啶的方法严重制约了四氢嘧啶的工业化生产和大规模应用，因此开发一株新型的四氢嘧啶高产菌株以简化生产工艺、提高合成效率、降低生产成本，对四氢嘧啶的应用有重要的实践意义。
克劳氏芽孢杆菌是一种益生菌株，不仅可满足安全性要求较高的生产，而且发酵工艺较为成熟，发酵培养需求简单，还可满足发酵操作简便、成本低的工业应用条件，但是目前的克劳氏芽孢杆菌多用于蛋白酶 工业生产，尚未发现将其用于工业生产四氢嘧啶的相关报道。
发明内容
目前现有的四氢嘧啶生产工艺复杂、合成效率低、生产成本高，严重制约了四氢嘧啶的工业化生产和大规模应用。因此，本发明的目的是提供一株生物安全性高、四氢嘧啶产量高、发酵操作简便且培养成本低廉的生产四氢嘧啶的菌株及其发酵生产四氢嘧啶的方法，以克服现有技术的不足。
在第一方面，本发明提供了一株产四氢嘧啶的克劳氏芽孢杆菌ET01(Bacillus clausii ET01)，保藏编号为CGMCC NO.16310。本发明所述的菌株可在无机盐浓度为0-100g/L的液体培养基中生长，并在低盐浓度(小于100g/L且大于0g/L，例如50g/L)下高产四氢嘧啶(多达25g/L)。
在第二方面，本发明提供了第一方面所述的克劳氏芽孢杆菌ET01在生产四氢嘧啶方面的应用。
在第三方面，本发明提供了一种四氢嘧啶的生产方法，所述方法包括：
将第一方面的克劳氏芽孢杆菌ET01在无机盐浓度小于100g/L的克劳氏芽孢杆菌培养基中进行有氧发酵，从而获得四氢嘧啶。
本发明的有益效果如下：与现有技术中其它产四氢嘧啶菌株(例如CN103451137A中的盐单胞菌)相比，本发明的克劳氏芽孢杆菌ET01具有较高的四氢嘧啶合成能力(25g/L)，可在低盐浓度(小于100g/L)培养条件下发酵生产良好，降低了其它嗜盐菌株在发酵生产中由于盐度过高造成发酵设备腐蚀以及污染环境的风险；使用本发明的克劳氏芽孢杆菌ET01进行有氧发酵后，菌株发酵副产物及其它杂质含量较低，且无羟基嘧啶产生，经简单分离纯化后获得的四氢嘧啶纯度可达98％以上，降低了分离提纯成本，具有良好的工业应用前景。
附图说明
图1是在100倍显微镜下观察到的克劳氏芽孢杆菌ET01。
具体实施方式
本发明中，本发明人采用梯度稀释法，从威海近海海盐场泥中分离 筛选出一株高产四氢嘧啶的细菌。将该菌株在LB固体培养基(10g/L NaCl)上在38℃下培养24小时后，形成乳白色菌落，边缘不整齐，表面有突起；在显微镜100倍下观察，菌体呈长杆状(如图1所示)；经革兰氏染色呈阳性，为革兰氏阳性菌。结合该菌的16s rDNA序列(SEQ ID NO:1)并参照《伯杰细菌鉴定手册》，科学出版社，1984年中记载的标准，将该菌株鉴定并命名为克劳氏芽孢杆菌ET01(Bacillus clausii ET01)。将该菌株保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)(北京市朝阳区北辰西路1号院)，保藏日期为2018年8月17日，分类学名称为克劳氏芽孢杆菌(Bacillus clausii)，保藏编号为CGMCC NO.16310。
在本发明中，术语“克劳氏芽孢杆菌培养基”是指本领域技术人员熟知的可用于培养克劳氏芽孢杆菌的培养基，例如含有碳源、氮源和无机盐的营养培养基(如LB培养基或营养肉汤培养基)。所述培养基的配方可为本领域已知的配方，例如通常使用的LB培养基的配方为：0.8-1wt％蛋白胨，0.5-0.8wt％酵母粉，1-1.5wt％氯化钠，pH为6.8-7.0；营养肉汤培养基的配方为0.8-1wt％蛋白胨，0.3-0.5wt％牛肉膏，0.5-0.8wt％氯化钠，pH为7.2-7.6。但是本领域技术人员可根据需要对培养基中具体的碳源、氮源和无机盐及各自的含量进行选择和调整，尤其是对无机盐的种类和浓度进行选择和调整，以适于对本发明的克劳氏芽孢杆菌进行培养和应用，例如将LB培养基中的氯化钠浓度调整为5wt％用于生产四氢嘧啶。
在本发明中，术语“碳源”是指为微生物提供生长繁殖所需碳元素的营养物质。所述碳源的实例包括但不限于葡萄糖、糊精、蔗糖、味精、糖蜜、甘油、乳酸、麦芽糊精、麦芽糖。在一些实施方式中，所述“碳源”优选为葡萄糖。
在本发明中，术语“氮源”是指为微生物提供生长繁殖所需氮元素的营养物质。所述氮源的实例包括但不限于酵母抽提物、酵母膏、胰蛋白胨、味精、氯化铵、硫酸铵、大豆蛋白胨、豆粉、玉米粉、花生饼、棉籽粕。在一些实施方式中，所述“氮源”优选为酵母抽提物、酵母膏、胰蛋白胨、氯化铵或硫酸铵，或它们的任意组合。
在本发明中，术语“无机盐”是指无机化合物中的矿物质盐类，如钠盐、钾盐、钙盐、磷酸盐、硫酸盐等。所述无机盐的实例包括但不限于：氯化钠、磷酸氢二钾、磷酸二氢钾、七水硫酸镁、七水硫酸铁、二水氯化钙、氯化钾、硫酸钠、磷酸氢二钠。
在本发明中，术语“种子培养基”是指对菌株进行活化时所用的培养基，所述种子培养基可以是本领域常规使用的普通培养基，例如LB培养基或营养肉汤培养基。本领域技术人员可根据需要对培养基中具体成分和各自的含量进行选择，以适于本发明的克劳氏芽孢杆菌的活化，尤其是对无机盐的种类和浓度进行选择和调整。
因此，本发明提供了一株产四氢嘧啶的克劳氏芽孢杆菌ET01(Bacillus clausii ET01)，保藏编号为CGMCC NO.16310。本发明所述的菌株在无机盐浓度为0-100g/L的液体培养基中生长，并在低盐浓度(小于100g/L且大于0g/L)下高产四氢嘧啶。在优选的实施方式中，所述克劳氏芽孢杆菌ET01可在无机盐浓度为0-100g/L，优选30-70g/L，更优选50g/L的液体培养基中生长。
在一个实施方式中，本发明还提供了本发明的克劳氏芽孢杆菌ET01在生产四氢嘧啶方面的应用。
在一个实施方式中，本发明提供了一种四氢嘧啶的生产方法，所述方法包括：将本发明的克劳氏芽孢杆菌ET01在无机盐浓度小于100g/L的克劳氏芽孢杆菌培养基中进行有氧发酵，从而获得四氢嘧啶。
在优选的实施方式中，所述克劳氏芽孢杆菌培养基为本领域用于培养克劳氏芽孢杆菌的液体培养基，例如含有碳源、氮源和无机盐的营养培养基，如LB培养基或营养肉汤培养基。本领域技术人员能够根据需要对所述培养基的具体成分和含量进行选择和调整，这些选择和调整方案也在本发明的范围内。
在优选的实施方式中，所述无机盐选自氯化钠、磷酸氢二钾、磷酸二氢钾、七水硫酸镁、七水硫酸铁、二水氯化钙、氯化钾、硫酸钠、磷酸氢二钠，或它们的任意组合。在优选的实施方式中，所述无机盐为氯化钠。在优选的实施方式中，所述无机盐的盐浓度为小于100g/L且大于 0g/L，优选30-70g/L，更优选50g/L。
在优选的实施方式中，所述碳源选自葡萄糖、糊精、蔗糖、味精、糖蜜、甘油、乳酸、麦芽糊精、麦芽糖，或它们的任意组合。
在优选的实施方式中，所述氮源选自酵母抽提物、酵母膏、胰蛋白胨、味精、氯化铵、硫酸铵、大豆蛋白胨、豆粉、玉米粉、花生饼、棉籽粕，或它们的任意组合。在优选的实施方式中，所述氮源选自酵母抽提物、酵母膏、胰蛋白胨、氯化铵或硫酸铵，或它们的任意组合。
在优选的实施方式中，所述无机盐浓度小于100g/L的克劳氏芽孢杆菌培养基为NaCl浓度小于100g/L的LB培养基。
在一些实施方式中，可采用本领域通常使用的有氧发酵设备或方法，进行有氧发酵。本领域技术人员可以根据实际发酵情况选择合适的发酵设备和发酵条件。例如在摇瓶有氧发酵的情况下，发酵条件为在28-38℃、优选37℃，150-220rpm、优选200rpm下培养12-24h。应当指出的是，本领域技术人员在本发明的基础上对有氧发酵条件进行的任何优化和调整也属于本发明范围内。
在优选的实施方式中，所述四氢嘧啶的生产方法包括将本发明所述的克劳氏芽孢杆菌ET01在氯化钠浓度小于100g/L、优选30-70g/L、更优选50g/L的LB培养基或营养肉汤培养基中，在28-38℃、优选37℃，150-220rpm、优选200rpm下培养12-24h。
在优选的实施方式中，通过对有氧发酵的发酵液(也称为“有氧发酵液”)进行分离和/或纯化从而获得四氢嘧啶。可使用本领域通常用于分离和/或纯化的设备或方法对发酵液中的四氢嘧啶进行分离和/或纯化，例如离心、过滤、萃取等。在优选的实施方式中，采用萃取法获得四氢嘧啶，例如采用80％乙醇萃取所述有氧发酵液中的四氢嘧啶。
在进一步优选的实施方式中，所述采用80％乙醇萃取所述有氧发酵液中的四氢嘧啶包括如下步骤：将有氧发酵液用等体积的80％乙醇水溶液静置12-24h，离心，取上清获得四氢嘧啶。在加入80％乙醇水溶液之前，也可对发酵液进行离心，分离菌体和/或菌液，进行萃取。
在优选的实施方式中，所述方法进一步包括对所述克劳氏芽孢杆菌 进行活化。在一个优选的实施方式中，所述活化包括：将所述克劳氏芽孢杆菌ET01接种于种子培养基进行摇床培养，在培养温度为28-38℃，转速150-220rpm条件下培养12-24h。
在优选的实施方式中，所述种子培养基为LB培养基(0.8-1wt％蛋白胨，0.5-0.8wt％酵母粉，1-1.5wt％氯化钠；pH为6.8-7.0)或营养肉汤培养基(0.8-1wt％蛋白胨，0.3-0.5wt％牛肉膏，0.5-0.8wt％氯化钠；pH为7.2-7.6)。
在优选的实施方式中，由本发明的方法制备得到的四氢嘧啶的纯度为98％以上。
实施例
为了使本发明的目的、技术方案及优点更加清楚，以下结合附图及具体实施方式，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施方式仅仅用以解释本发明，并不用于限定本发明。下述实施例中所述实验方法和设备，如无特殊说明，均为常规方法和设备。下述实施例中所用的实验材料，如无特别说明，全部试剂购自OXOID公司。
实施例中所用的试剂：
1.LB培养基：胰蛋白胨10g/L；酵母浸粉5g/L；氯化钠0-100g/L；pH 7.0，121℃灭菌15min。
2.LB固体培养基：胰蛋白胨10g/L；酵母浸粉5g/L；琼脂15g/L；氯化钠20-100g/L；pH 7.0，121℃灭菌15min。
实施例1克劳氏芽孢杆菌ET01的获得
取威海近海盐场泥样1g用无菌水稀释至100mL，取200μL稀释液涂布于20g/L的LB固体培养基上，置于37℃恒温培养箱中培养24h，挑取单菌落经过多轮划线培养，最终分离得到纯化菌株。
对该菌株进行细菌形态鉴定。将该菌株在10g/L NaCl的LB固体培养基上于38℃培养24小时后，形成乳白色菌落，边缘不整齐，表面有突起；在显微镜100倍下观察，菌体呈长杆状(如图1所示)，经革兰氏染色呈阳性，为革兰氏阳性菌。根据菌体形态及16S rDNA测序比对，将菌株鉴定并命名为克劳氏芽孢杆菌ET01(Bacillus clausii ET01)，对其进 行保藏，保藏编号为CGMCC NO.16310。
实施例2克劳氏芽孢杆菌的培养条件及产物检测
将甘油管中保藏的克劳氏芽孢杆菌ET01以2v/v％的量接种于60mL 10g/L NaCl的LB培养基，在摇床上37℃，200rpm过夜培养活化。使用分光光度计测量发酵液在600nm波长下吸光值(OD 600)为2.3。
将经活化的克劳氏芽孢杆菌ET01以1v/v％的量分别接种于60mL含0g/L、30g/L、50g/L、70g/L及100g/L NaCl的LB培养基中，在摇床上37℃，200rpm培养24h。
为了检测本发明的克劳氏芽孢杆菌ET01在各个盐浓度下的生长情况，使用分光光度计测量培养24h后发酵液的OD 600。通过HPLC对培养24h的克劳氏芽孢杆菌的发酵液中的发酵产物(四氢嘧啶和羟基嘧啶)进行检测鉴定，使用安捷伦1260高效液相检测系统进行检测，检测条件为：
a)液相色谱柱：Zorbax SB C18柱(柱长150mm，柱内径4.6mm；填料粒径5μm)；
b)流动相：2％乙腈：98％水；
c)流速：0.2mL/min；
d)柱温：30℃；
e)进样量：20μL；
f)检测波长：210nm。
g)0.1mg/mL的四氢嘧啶标品水溶液和羟基嘧啶标品水溶液：均购自Sigma-Aldrich，四氢嘧啶的货号为E2271，羟基嘧啶的货号为70709。
先对四氢嘧啶和羟基嘧啶的标品进行检测，四氢嘧啶标品出峰时间为7.8min，羟基嘧啶标品出峰时间为7.0min左右(HPLC峰图未示出)。
对在不同NaCl浓度下培养24h的发酵液中的四氢嘧啶和羟基嘧啶进行鉴定，具体步骤如下：分别取1mL发酵液加入等体积的80％乙醇溶液萃取，8000rpm离心10min取上清进行上述HPLC检测。结果所有样品均未在7.0min处检测到明显的羟基嘧啶产物峰(HPLC峰图未示出)，说明本发明的克劳氏芽孢杆菌几乎不生产羟基嘧啶；而除了0g/L NaCl的样品以外，其它样品均在7.8min处检测到明显的四氢嘧啶产物峰，对HPLC峰图结果进行数值分析，数据结果在表1中示出。
表1：克劳氏芽孢杆菌ET01在不同NaCl浓度条件下的发酵液的OD 600和四氢嘧啶检测结果
NaCl(g/L) OD 600 四氢嘧啶(g/L)
0 2.3 0
30 5.7 13.2
50 6.1 25.0
70 2.8 14.8
100 0.6 4.9
由上述结果可以看出：本发明的克劳氏芽孢杆菌在无NaCl添加(0g/L NaCl)的情况下，菌株生长正常，但无四氢嘧啶产生；在NaCl添加过高(100g/LNaCl)的情况下，菌株OD 600较低，说明菌株生长较差，且仅微弱的四氢嘧啶积累，产量整体较低；而在30-70g/L的情况下，菌株不仅生长正常，而且四氢嘧啶常量较高，尤其是在50g/L NaCl的情况下，菌株OD 600最高，说明菌株生长良好，且四氢嘧啶产量较其它盐浓度培养时最高，达到25g/L。
实施例3克劳氏芽孢杆菌TDB1-4的培养及产物检测
使用克劳氏芽孢杆菌TDB1-4(CCTCC NO：M2015430)和本发明的克劳氏芽孢杆菌ET01分别于NaCl浓度为0g/L和50g/L的LB培养基中进行发酵培养。按照实施例2的方法对菌株进行培养并且对发酵培养24小时后的OD 600及四氢嘧啶进行检测，结果如表2所示。
表2克劳氏芽孢杆菌ET01和TDB1-4的OD 600及四氢嘧啶检测结果
Figure PCTCN2019128701-appb-000001
由表2可以看出，在无NaCl添加情况下，本发明的克劳氏芽孢杆菌ET01与克劳氏芽孢杆菌TDB1-4生长情况相近；但是在50g/LNaCl的LB培养基中，本发明的克劳氏芽孢杆菌ET01生长良好，且产四氢嘧啶；而克劳氏芽孢杆菌TDB1-4生长较差并且并不产生四氢嘧啶。这说明本发明的克劳氏芽孢杆菌ET01具有一定的耐盐能力，而且能够产生四氢嘧啶。本发明的克劳氏芽孢杆菌ET01合成四氢嘧啶的能力可达到25.0g/L，相比于现有技术中报道的其它产四氢嘧啶菌株(例如CN103451137A中的盐单胞菌， 18g/L)具有明显的改善，具有用于工业生产四氢嘧啶的前景。
实施例4对克劳氏芽孢杆菌ET01发酵液中其它代谢产物的检测
根据GB/T18246-2000和GB5009.157-2016，对在50g/L NaCl的LB培养基中培养24h的克劳氏芽孢杆菌发酵液中的氨基酸、有机酸等产物进行检测，结果如表3所示。
表3发酵前后培养基中的氨基酸和有机酸的含量
Figure PCTCN2019128701-appb-000002
由表3可以看出，本发明的克劳氏芽孢杆菌ET01的发酵过程中无大量其它代谢产物积累，这有利于后续对四氢嘧啶的分离和纯化。
选用50g/L NaCl的LB培养基对本发明的克劳氏芽孢杆菌ET01进行有氧发酵培养并获得发酵液，然后仅经80％乙醇萃取，烘干结晶后即可得到纯度达98％以上的四氢嘧啶，无需进行离子交换层析、分子筛等费用较高的高分离纯化过程，可节约成本，操作简单，具有良好的工业应用前景。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或 改进，均属于本发明要求保护的范围。

Claims (10)

  1. 一株产四氢嘧啶的克劳氏芽孢杆菌ET01(Bacillus clausii ET01)，保藏编号为CGMCC NO.16310。
  2. 如权利要求1所述的克劳氏芽孢杆菌ET01在生产四氢嘧啶方面的应用。
  3. 一种四氢嘧啶的生产方法，所述方法包括：将如权利要求1所述的克劳氏芽孢杆菌ET01在无机盐浓度小于100g/L的克劳氏芽孢杆菌培养基中进行有氧发酵，从而获得四氢嘧啶。
  4. 如权利要求3所述的方法，其中，所述克劳氏芽孢杆菌培养基为用于培养克劳氏芽孢杆菌的含有碳源、氮源和无机盐的培养基；其中，
    所述碳源优选选自葡萄糖、糊精、蔗糖、味精、糖蜜、甘油、乳酸、麦芽糊精、麦芽糖，或它们的任意组合；
    所述氮源优选选自酵母抽提物、酵母膏、胰蛋白胨、味精、氯化铵、硫酸铵、大豆蛋白胨、豆粉、玉米粉、花生饼、棉籽粕，或它们的任意组合；
    所述无机盐优选选自氯化钠、磷酸氢二钾、磷酸二氢钾、七水硫酸镁、七水硫酸铁、二水氯化钙、氯化钾、硫酸钠、磷酸氢二钠，或它们的任意组合。
  5. 如权利要求4所述的方法，其中，所述碳源为葡萄糖；所述氮源选自酵母抽提物、酵母膏、胰蛋白胨、氯化铵或硫酸铵，或它们的任意组合；所述无机盐为氯化钠。
  6. 如权利要求3或4所述的方法，其中，所述克劳氏芽孢杆菌培养基为LB培养基或营养肉汤培养基。
  7. 如权利要求3-6中任一项所述的方法，其中，所述无机盐浓度为小于100g/L，优选30-70g/L，更优选50g/L。
  8. 如权利要求3-7中任一项所述的方法，其中，所述有氧发酵的条件为在28-38℃，150-220rpm下培养12-24h。
  9. 如权利要求3所述的方法，其中，将所述克劳氏芽孢杆菌ET01在氯化钠浓度小于100g/L、优选30-70g/L、更优选50g/L的LB培养基或营养肉汤培养基中，在28-38℃、优选37℃，150-220rpm、优选200rpm下培养12-24h。
  10. 如权利要求3-9中任一项所述的方法，其中，所述方法进一步包括对所述克劳氏芽孢杆菌ET01进行活化。
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