CN103509840A - Method for increasing yield of anidulafungin precursor compound Echinocandin B - Google Patents

Abstract

The invention provides a method for increasing the yield of an anidulafungin precursor compound Echinocandin B (ECB). The method comprises the following steps: preparing a fermentation culture medium for culturing Aspergillus nidulans for synthesizing ECB, inoculating aspergillus nidulans, culturing for 3 days at 28-37 DEG C, and continuously culturing at 25 DEG C till the fermentation is accomplished. Compared with constant temperature culture, the method adopts the strategy that the ECB is cultured at 37 DEG C at the early three days and is cultured at 25 DEG C at the later 9 days, so that the yield of the ECB is increased to be 1,237mg/L from 728mg/L; in addition, the yield of the ECB is increased to be 850mg/L from 540mg/L by adding 2g/L proline on the sixth day; and the yield of the ECB can be respectively increased by 21.9% and 55.2% by adding 2g/L threonine and 2g/L ornithine into the culture medium.

Description

A method for increasing the yield of anidulungin precursor compound Echinocandin B

(1) Technical field

The invention relates to a cultivation method for increasing the yield of anidulungin precursor compound Echinocandin B. the

(2) Background technology

In recent years, organ transplantation has led to the extensive use of immunosuppressants, the application of chemotherapy and more aggressive medical methods and other reasons have caused an increase in immunocompromised patients, and the incidence of fungal infections has increased significantly, especially the incidence of deep fungal infections The incidence and fatality rate are increasing year by year. According to the statistics of the World Health Organization (WHO) in 2004, the annual number of systemic fungal infections in the world is 335,000. The most common of these is systemic candidiasis, accounting for about 70% of all such infections, and the mortality rate it causes is as high as about 40%. With the continuous advancement of medicine, after the 1990s, various antifungal drugs came out continuously, bringing revolutionary progress in the treatment of various fungal diseases. As the demand for antifungal drugs is increasing year by year, the market size is getting stronger. the

Echinocandins (Echinocandin) antibiotics are a group of natural products discovered in the 1970s, which have similar structural features of cyclic polypeptide cores and different fatty acid side chains, and can non-competitively inhibit fungal cell wall β-1, 3-glucan synthase activity, so as to achieve the purpose of antifungal. Among them, Anidulafungin is the third-generation systemic antifungal echinocandin derivative drug following Caspofungin and Micafungin. On February 21, 2006, it was issued by Anifungin produced by Pfizer has passed the US FDA certification, mainly for the treatment of candidemia and other forms of candida infection (abdominal abscess, peritonitis) and esophageal candida, etc. Anifungin is a precursor compound Echinocandin B (ECB) through the action of the acylase produced by Actinomycetes Utahii to remove the side chain linoleoyl group, and then in DMF with the active intermediate 4"-pentyloxy- [1,1',4',1"]triphenyl-4-formic acid-2,4,5-trichloro-phenyl ester reaction in the system. The chemical structural formulas of Anifungin and ECB are shown in formulas 1 and 2. the

According to literature and patent reports, ECB is currently prepared by microbial fermentation at home and abroad, and the main one is the fermentation of Aspergillus nidulans. Maria Papagianni et al. reviewed the effects of factors such as inoculum size, carbon and nitrogen source types and concentrations, pH, temperature, and stirring rate on the growth of Aspergillus nidulans during the fermentation process, indicating that neutral pH is conducive to bacterial growth and spore formation, and Its specific growth rate will increase with the increase of temperature, but the consequence of increased dehydration of the bacteria after temperature increase will eventually lead to a decrease in the synthesis of target compounds. ECB is the main precursor compound for the synthesis of Anifungin, and the level of its fermentation unit will directly affect the market prospect of Anifungin. Scholars such as Fritz Benz determined the molecular structure of ECB through infrared, nuclear magnetic resonance and other methods, and found that its hydrolyzed components include linoleic acid, 4-hydroxy-L-proline, 4-carbonyl-L-proline, L-threonine, (2S,3S,4S)-4-methyl-3-hydroxyproline, etc., and the addition of carbonate, phosphate, sulfate and nitrate to the fermentation medium has a positive effect on ECB The increase in production has a promoting effect. the

At present, there are few literature reports on the fermentation of ECB, and most of the related literatures focus on the fermentation of the precursor compounds of caspofungin and micafungin. When American scholars studied the fermentation of the precursor compound Pneumocandin B ₀ of caspofungin, they found that appropriately increasing the concentration of magnesium ions could stimulate the growth of the bacteria, but the increase of the concentration of magnesium ions would reduce the synthesis of the precursor compound. On the contrary, ammonium sulfate and Potassium dihydrogen phosphate can promote the production of the precursor substance. People such as Petersen investigated the impact of the addition of proline on Pneumocandin B ₀ , and found that when proline was not added in the medium, its titer was only 274mg/L, and its titer was only 274mg/L after adding 10g/L proline. increased to 494mg/L; domestic Liu Liang et al. also found that adding 3g/L proline to the medium can effectively increase the proportion of Pneumocandin B ₀ in the fermentation product.

In addition to amino acids, Petersen et al. also investigated the effect of divalent metal ions on the titer of Pneumocandin B ₀ and found that adding 36 μM Zn ²⁺ reduced the titer of B ₀ from 629 mg/L to 312 mg/L; while adding 36 μM Co ²⁺ and Ni ²⁺ reduced the potency of B ₀ by 33% and 15%, respectively; Cu ²⁺ had little effect on the fermentation product, and the analysis showed that the reduction in potency was due to the effect of metal ions on the synthesis of Pneumocandin B ₀ It is caused by the inhibition of the activity of the related enzyme α-ketoglutarate-dependent dioxygenase. Munekazu Kanda et al. optimized the fermentation medium when fermenting and producing the precursor of micafungin, and found that the addition of magnesium sulfate was higher than 0.2%, and when the phosphate concentration was 0.5%, the enzyme activity of its key enzymes increased from 12U/ml To 16U/ml, increased by 30%.

At present, the main problem is that the level of fermentation preparation of ECB in China is still low. Therefore, it is of great significance for the industrial production of Anifungin to optimize the fermentation process and increase the yield of ECB by means of fermentation metabolism regulation. the

(3) Contents of the invention

The object of the present invention is to improve the ECB fermentation culture condition, provide a kind of change culture temperature in the fermentation process and add precursor amino acid in right amount and right amount in culture medium, as proline, ornithine, threonine, linoleic acid etc., techniques to ultimately increase the level of ECB fermentation. the

The technical scheme adopted in the present invention is:

A method for increasing the yield of anidulungin precursor compound Echinocandin B (ECB), the method comprising: preparing a fermentation medium for cultivating Aspergillus nidulans (Aspergillus nidulans) to synthesize ECB according to a conventional method, and inserting Aspergillus nidulans , after cultivating at 28-37°C for 3 days, continue culturing at 25°C until the end of fermentation. the

Preferably, precursor amino acids are added on days 0 to 9 of the culture, and the precursor amino acids are one of the following or a mixture of two or more of them: proline, ornithine, threonine, linoleic acid, The added amount is 2-8g/L fermentation medium. The amino acid added in the present invention is directly added by the method of solid addition, and a certain amount of amino acid is weighed and directly added into the liquid culture medium. The fermentation culture described in the present invention is added precursor amino acid between 0-9 days, wherein 0 means that the precursor amino acid is added at the beginning of fermentation, which is easily understood by those skilled in the art. the

Preferably, the composition of the fermentation medium is as follows: peanut oil 20g/L, glycerol 10g/L, peptone 10g/L, L-proline 2g/L, mannitol 90g/L, soybean powder 40g/L, _{K2HPO 4} 3H ₂ O8g/L, MgSO ₄ 7H ₂ O0.5g/L, MnSO ₄ H ₂ O0.1g/L, FeSO ₄ 7H ₂ O0.05g/L, CaCl ₂ 0.3g/L, solvent is Water, pH 7.0.

Specifically, the method can be as follows:

(1) Aspergillus nidulans was inoculated into the seed medium at a culture temperature of 25°C and a shaker speed of 220r/min for 2 days to obtain a seed liquid; the composition of the seed medium was as follows: glucose 10g/L, glycerol 10g/L, Cottonseed powder 25g/L, solvent is water, pH6.8～7.0, sterilized at 115℃ for 30min;

(2) Inoculate the seed liquid into the fermentation medium with an inoculation amount of 10% by volume, and cultivate it for 3 days at 28-37°C with a shaker speed of 220r/min, and then continue to grow at 25°C with a shaker speed of 220r/min Until the total fermentation time is 12 days, and add 2g/L proline on the 6th day of fermentation, or add 2g/L threonine or ornithine on the 0th day of fermentation; the composition of the fermentation medium is as follows: Peanut oil 20g/L, glycerin 10g/L, peptone 10g/L, L-proline 2g/L, mannitol 90g/L, soybean powder 40g/L, K ₂ HPO ₄ ·3H ₂ O8g/L, MgSO ₄ · 7H ₂ O 0.5g/L, MnSO ₄ H ₂ O 0.1g/L, FeSO ₄ 7H ₂ O 0.05g/L, CaCl ₂ 0.3g/L, solvent is water, pH7.0;

(3) The anidifungin precursor compound Echinocandin B is obtained by separating and purifying the fermentation product. the

The ECB content in the fermentation broth was detected by HPLC method. Sample pretreatment: Take 5ml of fermentation broth and place it in a 10ml centrifuge tube, centrifuge at 5000rpm for 5min, discard the precipitate, and collect the supernatant; mix the supernatant with absolute ethanol at a volume ratio of 1:4, centrifuge at 10000rpm for 10min, and collect the supernatant It was filtered with a 0.45 μm microfiltration membrane, and the filtrate was analyzed by Shimadzu HPLC. Preparation of HPLC mobile phase: Accurately weigh 0.30g KH ₂ PO ₃ , 0.35g Na ₂ HPO ₃ , dissolve them in ultrapure water and set the volume to 500ml, filter with a 0.45μm microfiltration membrane; the volume ratio of the filtrate to chromatographically pure acetonitrile is 3: 7 Mix and degas using ultrasound. HPLC analysis conditions: Shimadzu LC-20AT pump, Shimadzu SPD-20A ultraviolet-visible light detector, chromatographic column is C18 column (Dalian Elite 4.6mm×250mm), mobile phase ratio is acetonitrile:methanol:water=2:7 : 1, the flow rate is 1.0ml/min, the ultraviolet detection wavelength is 222nm, the injection volume is 20μL, and the column temperature is 40°C.

The effects and benefits of the present invention are: using the method of the present invention, by changing the culture temperature after 3-9 days of ECB fermentation culture; and adding precursor amino acids on the 0-9 day of ECB fermentation culture to improve the fermentation level of ECB. It can be seen from the examples of the present invention that compared with constant temperature culture, the strategy of culturing at 37°C for the first three days and culturing at 25°C for the next 9 days can increase the ECB production from 728mg/L to 1237mg/L; in addition, Adding 2g/L proline on the 6th day can increase the production of ECB from 540mg/L to 850mg/L; adding 2g/L threonine or 2g/L ornithine to the medium can increase the production of ECB by 21.9 % and 55.2%. the

(4) Description of drawings

Fig. 1 is the influence of temperature on ECB output;

Figure 2 shows the effect of proline addition time and concentration on ECB yield; A: addition on day 0; B: addition on day 3; C: addition on day 6; D: addition on day 9. the

Figure 3 shows the effect of ornithine addition time and concentration on the yield of ECB; A: addition on day 0; B: addition on day 3; C: addition on day 6; D: addition on day 9. the

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

Example 1:

The strain used in the examples of the present invention is Aspergillus nidulans (Aspergillus nidulans) ZJB09223 (CCTCC NO: M2012300), which was deposited in the China Center for Type Culture Collection on July 25, 2012, and has been applied for separately as a new strain. the

(1) Aspergillus nidulans ZJB09223 seed culture:

Take a ring of dark green colony from the PDA slant culture medium and insert it into the seed medium, to the seed medium, culture at a temperature of 25° C., and a shaker speed of 220 r/min, and cultivate for 2 days to obtain a seed liquid; the composition of the seed medium is as follows : Glucose 10g/L, glycerin 10g/L, cottonseed powder 25g/L, solvent is water, pH6.8～7.0, sterilized at 115℃ for 30min;

(2) Aspergillus nidulans ZJB09223 fermentation culture:

The composition of the fermentation medium is as follows: peanut oil 20g/L, glycerol 10g/L, peptone 10g/L, L-proline 2g/L, mannitol 90g/L, soybean powder 40g/L, K ₂ HPO ₄ ·3H ₂ O8g/L, MgSO ₄ 7H ₂ O0.5g/L, MnSO ₄ H ₂ O0.1g/L, FeSO ₄ 7H ₂ O0.05g/L, CaCl ₂ 0.3g/L, solvent is water, pH7 .0. The seed solution cultivated for 2 days was inserted into the fermentation medium with 10% inoculum amount, and the culture time was 12 days under the condition of 25-37°C and the shaker speed of 220r/min.

After 6 days of fermentation culture, samples were taken to measure the ECB content, and the ECB fermentation units after 12 days were shown in Figure 1. It can be seen from the results that the temperature has a great influence on the fermentation unit of ECB during the fermentation process, the optimum temperature is 25°C, and the ECB output is 728 mg/L at this time. the

Example 2:

According to the results of Example 1, the temperature was selected to be changed during the cultivation process, and the experiment was started at 25°C, 28°C, and 37°C for fermentation and cultivation, and then the temperature was adjusted after 3 days, 6 days, and 9 days, respectively. The adjustment method is: adjust 28°C and 37°C to 25°C and cultivate until the end of fermentation; adjust 25°C to 28°C and cultivate until the end of fermentation, and measure the ECB content. The results are shown in Table 1. High-temperature (28-37°C) cultivation followed by low-temperature (25°C) cultivation can greatly increase the yield of ECB; culturing at 37°C for the first 3 days and culturing at 25°C for the next 9 days can increase the fermentation level of ECB to 1237mg/L, 69.9% higher than 25°C constant temperature culture. the

Table 1: Effects of different temperature adjustment methods and adjustment time on ECB output

Example 3:

It is reported in relevant literature that proline plays an important role in the growth and metabolism of fungal microorganisms. The experiment investigated the effects of different addition times and different concentrations of proline on the fermentation results. the

The composition of the original fermentation medium of ECB in the experiment is: peanut oil 20g/L, glycerin 10g/L, peptone 10g/L, L-proline 5g/L, mannitol 90g/L, soybean powder 40g/L, K ₂ HPO ₄ 3H ₂ O8g/L, MgSO ₄ 7H ₂ O0.5g/L, MnSO ₄ H ₂ O0.1g/L, FeSO ₄ 7H ₂ O0.05g/L, CaCl ₂ 0.3g/L, the solvent is Water, pH 7.0. The seeds cultivated for 2 days were inserted into the fermentation medium with 10% inoculum amount, and cultivated for 12 days under the conditions of 25°C and shaking table speed of 220r/min.

Taking the above-mentioned fermentation medium as a control, proline was added to the fermentation broth at different times to make the final concentrations 2g/L, 5g/L, and 8g/L respectively, and the effects of different addition times and addition concentrations of proline were investigated. The effect of ECB yield, the results are shown in Fig. 2. the

It can be seen from Figure 2 that the ECB production of the control group added with 5g/L proline on the 0th day was about 540mg/L, while at different addition times, the addition of 2g/L proline showed better results. Effect. When 2 g/L was added on day 0, the final production of ECB reached about 800 mg/L, and its production decreased with the increase of proline concentration (Figure 2A), and when 2 g/L was added on day 3, it finally reached 700 mg/L About L (Figure 2B), adding 2g/L on the 6th day reached about 850mg/L (Figure 2C), adding 2g/L on the 9th day was about 840mg/L (Figure 2D). From the above results, it can be seen that adding 2g/L proline on the 6th day has the best effect, which can increase the yield of ECB by 57.4%. the

Example 4:

In addition to proline, the present invention also investigates the influence of different addition time and concentration of threonine on the yield of ECB fermentation. the

The composition of the original fermentation medium of ECB in the experiment is: peanut oil 20g/L, glycerin 10g/L, peptone 10g/L, L-proline 5g/L, mannitol 90g/L, soybean powder 40g/L, K ₂ HPO ₄ 3H ₂ O8g/L, MgSO ₄ 7H ₂ O0.5g/L, MnSO ₄ H ₂ O0.1g/L, FeSO ₄ 7H ₂ O0.05g/L, CaCl ₂ 0.3g/L, solvent is Water, pH 7.0. The seeds cultivated for 2 days were inserted into the fermentation medium with 10% inoculum, and cultivated for 12 days at 25°C and the shaker speed was 220r/min.

Taking the above fermentation medium as a control, threonine was added to the fermentation broth at different times to make the final concentrations 2g/L, 5g/L, and 8g/L respectively, and the different addition times and concentrations of threonine were investigated. The impact on ECB yield, the results are shown in the table below. the

It can be concluded from the experimental results that excessively high concentration of threonine (8g/L) can inhibit the synthesis of ECB, and reduce the yield of ECB at different addition times, so the final addition concentration of threonine is 2g /L; It can be seen from the addition time that adding threonine on the 0th day, that is, adding threonine at the beginning of fermentation, can increase the ECB production from 524mg/L to about 639mg/L, an increase of 21.9%. the

Table 2: The effect of the addition time and concentration of threonine on the yield of ECB

Embodiment 5:

The present invention also investigates the influence of different addition time and concentration of ornithine on the yield of ECB fermentation. the

The composition of the original fermentation medium of ECB in the experiment is: peanut oil 20g/L, glycerol 10g/L, peptone 10g/L, L-proline 2g/L, mannitol 90g/L, soybean powder 40g/L, K ₂ HPO ₄ 3H ₂ O8g/L, MgSO ₄ 7H ₂ O0.5g/L, MnSO ₄ H ₂ O0.1g/L, FeSO ₄ 7H ₂ O0.05g/L, CaCl ₂ 0.3g/L, solvent is Water, pH 7.0. The seeds cultivated for 2 days were inserted into the fermentation medium with 10% inoculum, and cultivated for 12 days at 25°C and the shaker speed was 220r/min.

Using the above fermentation medium as a control, add ornithine to the fermentation broth at different times to make the final concentrations 2g/L, 5g/L, and 8g/L respectively, and investigate the different addition times and concentrations of ornithine. The influence of yield and cell growth, the result is shown in Figure 3. the

The results showed that when 2g/L ornithine was added on the 0th to 3rd day, the ECB production would be greatly increased, and the final production reached about 1200mg/L, which was 55.2% higher than that of the control group; with the increase of ornithine concentration, ECB output showed a downward trend. With the increase of ornithine concentration, the biomass showed an upward trend (Figure 3A, B), and the fermentation broth became viscous. When adding ornithine on the 6th day of fermentation, the ECB production will increase with the increase of ornithine concentration, adding 8g/L ornithine can increase the ECB production to about 1100mg/L, while adding ornithine on the 9th day Acid has little effect on the yield of ECB. From the perspective of biomass, the addition of ornithine at the late stage of fermentation (6th to 9th day) had little effect on the growth of Aspergillus nidulans (Fig. 3C, D). Therefore, analyzing the experimental results, this experiment chooses to add 2g/L ornithine at the beginning of fermentation. the

Claims (4)

1. A method for improving the output of anidulungin precursor compound Echinocandin B (ECB), the method being: preparing a fermentation medium for cultivating Aspergillus nidulans (Aspergillus nidulans) to synthesize ECB, inserting Aspergillus nidulans, After culturing at 28 to 37° C. for 3 days, continue culturing at 25° C. until the end of fermentation. 2. The method according to claim 1, characterized in that precursor amino acids are added on the 0th to 9th day of the culture, and the precursor amino acids are one of the following or a mixture of two or more of them: proline, avian Amino acid, threonine, linoleic acid, the addition amount is 2-8g/L fermentation medium. 3. The method according to claim 1 or 2, wherein the fermentation medium is composed as follows: peanut oil 20g/L, glycerol 10g/L, peptone 10g/L, L-proline 2g/L, mannitol 90g/L, soybean flour 40g/L, K ₂ HPO ₄ 3H ₂ O 8g/L, MgSO ₄ 7H ₂ O 0.5g/L, MnSO ₄ H ₂ O 0.1g/L, FeSO ₄ 7H ₂ O0. 05g/L, CaCl ₂ 0.3g/L, solvent is water, pH7.0. 4. The method according to claim 1, characterized in that the method is as follows: (1) Aspergillus nidulans was inoculated into the seed medium at a culture temperature of 25°C and a shaker speed of 220r/min for 2 days to obtain a seed liquid; the composition of the seed medium was as follows: glucose 10g/L, glycerol 10g/L, Cottonseed powder 25g/L, solvent is water, pH6.8～7.0, sterilized at 115℃ for 30min; (2) Inoculate the seed liquid into the fermentation medium with an inoculation amount of 10% by volume, and cultivate it for 3 days at 28-37°C with a shaker speed of 220r/min, and then continue to grow at 25°C with a shaker speed of 220r/min Until the total fermentation time is 12 days, and add 2g/L proline on the 6th day of fermentation, or add 2g/L threonine or ornithine on the 0th day of fermentation; the composition of the fermentation medium is as follows: Peanut oil 20g/L, glycerin 10g/L, peptone 10g/L, L-proline 2g/L, mannitol 90g/L, soybean powder 40g/L, K ₂ HPO ₄ ·3H ₂ O8g/L, MgSO ₄ · 7H ₂ O 0.5g/L, MnSO ₄ H ₂ O 0.1g/L, FeSO ₄ 7H ₂ O 0.05g/L, CaCl ₂ 0.3g/L, solvent is water, pH7.0; (3) The anidifungin precursor compound EchinocandinB is obtained by separating and purifying the fermentation product.

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