A method for producing adenosine by fermentation

Abstract

The invention discloses a method for producing adenosine by fermentation, which involves a deep liquid fermentation medium formula, a fermentation control method and a feeding method for producing adenosine by the strain, and the color purity level of the fermentation liquid finally obtained is close to 90%, which is conducive to extraction and purification to obtain a finished adenosine product of pharmacopoeia standard. The method uses high-concentration glucose as a carbon source and yeast extract, urea, ammonium sulfate and the like as a nitrogen source for deep liquid fermentation, and the level of a 50L fermentation tank can reach 11.2g / L, and the level of a 1T fermentation tank can reach 10.5g / L.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation, and in particular relates to a method for producing adenosine by fermentation. Background Art

[0002] Adenosine, chemical name 6-amino-9-β-D-ribofuranosyl-9-hydrogen purine, is an important nucleotide derivative and is the product of adenine nucleotide dephosphorylation. As an endogenous nucleoside distributed throughout human cells, it can directly enter the myocardium and generate adenylic acid through phosphorylation, participate in myocardial energy metabolism, and also participate in dilating coronary vessels and increasing blood flow. It can be used to treat supraventricular tachycardia, supraventricular arrhythmias related to the atrioventricular chamber, and treat angina pectoris, myocardial infarction, coronary insufficiency, arteriosclerosis, essential hypertension, cerebrovascular disorders, sequelae of stroke, progressive muscular atrophy, etc. Adenosine has physiological effects on the cardiovascular system and many other systems and tissues of the body. Adenosine plays an important role in biochemistry, including transferring energy in the form of adenosine triphosphate (ATP) or adenosine diphosphate (ADP), or transmitting signals in the form of cyclic adenosine monophosphate (cAMP). In addition, adenosine is an inhibitory neurotransmitter and plays an important role in neurotransmission.

[0003] At present, the existing technology for producing adenosine is RNA hydrolysis, chemical method and fermentation method. The chemical synthesis method for synthesizing adenosine has a series of problems such as high cost, cumbersome reaction and low yield. RNA hydrolysis method will obtain 4 kinds of nucleoside substances at the same time, which brings difficulties to the subsequent separation. In comparison, the microbial fermentation method has a lower cost for producing adenosine, the raw materials are easy to obtain, and the efficiency of producing adenosine is higher, so it has an absolute advantage in the production of adenosine.

[0004] However, there are also many problems in the fermentation process of adenosine. One is that adenosine is mostly produced by genetically engineered strains or strains after physical and chemical mutagenesis. For example, the adenosine fermentation strain of Bacillus subtilis should have the following characteristics: lack of inosine dehydrogenase, that is, xanthine deficiency; lack of adenosine deaminase, cutting off the pathway from adenylic acid to inosinic acid. Lack of adenosine phosphorylase reduces the decomposition of adenosine; relieves the feedback inhibition of the end product pathway enzymes of adenosine monophosphate. High activity of 5′-nucleotidase rapidly decomposes adenylic acid into adenosine. However, the inventors found that nutritional deficiency strains generally have problems such as genetic traits and unstable yields, serious strain degeneration, high reverse mutation rate, poor product quality stability, and are not conducive to industrial production. In addition, the glucose tolerance of the bacteria disclosed in the prior art is not high, and glucose tolerance is an important indicator of the industrial application prospects of strains. Strains with good glucose tolerance can tolerate both high concentrations of substrates and high concentrations of osmotic pressure, which is of great significance for increasing the concentration of products. Secondly, the development of adenosine for injection requires very high quality of raw materials. The original strains usually have the problem of low purity of adenosine fermentation, which complicates the downstream process and has high purification costs. Thirdly, adenosine fermentation requires very rich nutrition (organic nitrogen source), which makes the cost of adenosine fermentation very high. Fourthly, the high-level adenosine-producing bacteria reported to be available only exist at the laboratory level and have not reached the large-scale production stage. Industrial scale-up is a very big difficulty for genetically engineered bacteria. Therefore, it is very necessary to develop a fermentation method that can stably produce adenosine and can be stably scaled up for industrialization. Summary of the invention

[0005] The object of the present invention is to provide a fermentation method which can stably produce adenosine and can be stably scaled up for industrialization.

[0006] A method for producing adenosine by fermentation, the method comprising the following steps: using adenosine-producing Bacillus subtilis as a fermentation strain, controlling the glucose concentration of the fermentation liquid to be 5-60 g / L, such as 5-10 g / L, 10-20 g / L, 20-30 g / L, 30-40 g / L, 40-50 g / L, 50-60 g / L, and the pH to be 6.1-7.3, such as 6.1, 6.2, 6.2, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3 during the fermentation process.

[0007] As a specific embodiment, the glucose concentration is 10-60 g / L, preferably 20-60 g / L, more preferably 30-60 g / L, such as 30-40 g / L, 40-50 g / L, 50-60 g / L.

[0008] As a specific implementation manner, the glucose is supplemented in a pulsed manner.

[0009] Further, preferably, when the glucose concentration is lower than 10-15 g / L, glucose is supplemented to make the glucose concentration 80-120 g / L.

[0010] As a specific implementation manner, the pH is preferably 6.3-6.4, and the pH is adjusted by adding ammonia water.

[0011] As a specific embodiment, the fermentation strain adopts Bacillus subtilis HDCC112-21027, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), with the deposit number CGMCC NO.23448 and the deposit date of September 18, 2021.

[0012] As a specific embodiment, the Bacillus subtilis is inoculated into the fermentation medium with a seed liquid at an inoculation rate of 5-20% (V / V) for fermentation culture, the temperature is controlled at 25-40°C, the dissolved oxygen is controlled to be ≥10-40%, and the total fermentation time is 48-64h.

[0013] As a specific embodiment, the liquid fermentation medium formula contains the following components: 5-240 g / L of glucose monohydrate, 0-50 g / L of yeast extract, 0-50 g / L of yeast powder, 0-30 g / L of ammonium sulfate, 0-7 g / L of potassium dihydrogen phosphate, 0-5 g / L of potassium chloride, 0-0.05 g / L of manganese sulfate, 0-14 g / L of urea, 0-5 g / L of magnesium sulfate, and 0-1 g / L of sodium chloride. If any of the components involve 0, then 0 is not taken.

[0014] Furthermore, the liquid fermentation medium preferably comprises the following components: 180 g / L glucose monohydrate, 20 g / L yeast extract, 30 g / L yeast powder, 10 g / L ammonium sulfate, 3.5 g / L potassium dihydrogen phosphate, 2 g / L potassium chloride, 0.02 g / L manganese sulfate, 7 g / L urea, 2 g / L magnesium sulfate, 0.5 g / L sodium chloride, and ammonia water to adjust the pH to 7.0.

[0015] Further, as a specific embodiment, the seed liquid is obtained by culturing Bacillus subtilis in a seed culture medium; taking qualified liquid seeds, inoculating them into a liquid fermentation medium at a ratio of 0.1-2.0% (V / W), controlling the temperature to 36±1°C, controlling the dissolved oxygen to be greater than 10-40%, culturing for 5-20h, and controlling the seed liquid OD≥8.0; the culture medium during the seed culture process is LB culture medium, and the LB culture medium contains 5g / L yeast extract, 10g / L peptone, 10g / L sodium chloride, and 10g / L glucose, and the pH is adjusted to 7.0.

[0016] This patent provides a method for producing adenosine by high-concentration glucose-tolerant Bacillus subtilis, which involves the formula of deep liquid fermentation medium, fermentation control method and feeding method for producing adenosine by this strain of bacteria. The color purity level of the fermentation liquid finally obtained is close to 90%, which is conducive to the extraction and purification of adenosine products that meet the pharmacopoeia standards. This method uses high-concentration glucose as a carbon source and yeast extract, urea, ammonium sulfate, etc. as nitrogen sources for deep liquid fermentation. The level of a 50L fermentation tank can reach 11.2g / L, and the level of a 1T fermentation tank can reach 10.5g / L.

[0017] The strain of Bacillus subtilis resistant to high-concentration glucose for producing high-purity adenosine in the present invention is unique among the currently disclosed adenosine production strains. Compared with the low-concentration sugar of other disclosed strains, the color purity of the fermentation liquid is low, which is not conducive to subsequent purification. From the comparison of fine control process, this strain has the advantages of wide tolerance to sugar concentration range, not easy to be contaminated, simple control process, high color purity of the final fermentation liquid, etc., and can realize industrial production. DETAILED DESCRIPTION

[0018] In order to enable those skilled in the art to better understand the technical solutions in this application, the present invention will be described more clearly and completely below in conjunction with the specific embodiments of this application. Obviously, the described embodiments are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of this invention.

[0019] Bacillus subtilis HDCC112-21027 described in the embodiment is deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC NO.23448 and a deposit date of September 18, 2021. The fermentation broth obtained in the embodiment of the present invention was analyzed by liquid chromatography with reference to Part II of the 2020 edition of the Chinese Pharmacopoeia.

[0020] Example 1

[0021] 1. Resuscitation and activation of bacterial strains: Take the working bacterial strain glycerol tube (CGMCC NO.23448), thaw it at room temperature, draw 0.1 ml of bacterial suspension and inoculate it onto the LB solid plate, spread it evenly, and culture it in a 35°C incubator for 24 hours to obtain a single colony of activated and revived bacteria.

[0022] 2. Liquid seed preparation: Take a single colony that has been activated and revived, pick up a single colony with an inoculation shovel, inoculate it into a 500ml Erlenmeyer flask containing 100ml LB liquid medium, wrap it up and place it on a shaker at 35℃ and 250rpm for 20h, and control the OD value of the seed liquid to be ≥0.5. Before disinfection of the LB liquid medium, adjust the pH to 7.0, and the disinfection conditions are 121-123℃ and 30min.

[0023] 3. Seed cultivation in seed tank: Take the qualified liquid seeds and inoculate them into a 50L automatic control fermentation tank with 20L liquid fermentation medium at a ratio of 0.5% (V / W). Control the temperature at 36°C and the dissolved oxygen at more than 20%. Adjust the air flow and stirring speed during the process. Cultivate in the tank for 9 hours, control the seed liquid OD ≥ 8.0, the seed tank formula is LB medium, adjust the pH to 7.0 before disinfection, and the disinfection conditions are 121-123°C, 30min.

[0024] 4. Fermentation tank culture: Take the qualified seed tank seeds and add them to a 50L automatic control fermentation tank filled with 25L liquid fermentation medium at a ratio of 10% (V / V). The temperature is controlled at 36℃, the dissolved oxygen is controlled to be ≥20%, and the air flow and stirring speed are adjusted during the process. The glucose concentration of the fermentation liquid is controlled to be 30-40g / L during the process, and the pH is automatically controlled at 6.3-6.4 by ammonia flow addition. After 48h of cultivation, the tank is released. The liquid fermentation medium formula is composed of: monohydrate glucose 180g / L, yeast paste 20g / L, yeast powder 30g / L, ammonium sulfate 10g / L, potassium dihydrogen phosphate 3.5g / L, potassium chloride 2g / L, manganese sulfate 0.02g / L, urea 7g / L, magnesium sulfate 2g / L, sodium chloride 0.5g / L, and the pH is adjusted to 7.0 with ammonia after disinfection. The disinfection conditions are 121-123℃, 30min.

[0025] 5. Sample processing and testing: After the fermentation is completed, take 2 ml of the fermentation liquid, centrifuge at 10000 rpm for 10 min to remove the bacteria, and then take the supernatant and filter it through a 0.45 μm microporous filter membrane. Refer to the "Chinese Pharmacopoeia" 2020 Edition Part II for liquid chromatography analysis and determination. The final potency level reached 11.2 g / L and the purity was 90.1%.

[0026] Example 2

[0027] 1. The preparation of seed solution is the same as in Example 1;

[0028] 2. Fermentation tank culture: Take the qualified seeds from the seed tank and add them into a 50L automatic control fermentation tank with 25L liquid fermentation medium at a ratio of 10% (V / V). The temperature is controlled at 36°C, the dissolved oxygen is controlled to be ≥20%, and the air flow and stirring speed are adjusted during the process. The glucose concentration of the fermentation liquid is controlled to be 5-10g / L, 10-20g / L, 20-30g / L, 30-40g / L, 40-50g / L, 50-60g / L, and the pH is automatically controlled at 6.3-6.4 by ammonia flow addition. After 48 hours of culture, the tank is released.

[0029] 3. The liquid fermentation medium formula is composed of: glucose monohydrate 180g / L, yeast extract 20g / L, yeast powder 30g / L, ammonium sulfate 10g / L, potassium dihydrogen phosphate 3.5g / L, potassium chloride 2g / L, manganese sulfate 0.02g / L, urea 7g / L, magnesium sulfate 2g / L, sodium chloride 0.5g / L. After disinfection, the pH of ammonia water is adjusted to 7.0, and the disinfection conditions are 121-123℃, 30min. The final tank level is shown in Table 1 below, and the purity is between 85% and 91%.

[0030] Table 1 Fermentation levels corresponding to different glucose concentrations

[0031] Glucose concentration (g / L) Fermentation level (g / L) 5-10 5.4 10-20 5.3 20-30 7.2 30-40 11.2 40-50 10.4 50-60 9.8

[0032] Example 3

[0033] 1. The preparation of seed solution is the same as in Example 1;

[0034] 2. Fermentation tank culture: Take the seeds from the seed tank that have passed the culture, and add them into a 50L automatic control fermentation tank filled with 25L liquid fermentation medium at a ratio of 10% (V / V). The temperature is controlled at 36°C, the dissolved oxygen is controlled to be ≥20%, and the air flow and stirring speed are adjusted during the process. The glucose concentration of the fermentation liquid is controlled to be 30-40g / L during the process, and the pH is automatically controlled at 5.5, 5.8, 6.1, 6.4, 6.7, 7.0, and 7.3 by ammonia flow addition. The tank is released after 48 hours of culture; the liquid fermentation medium formula is composed of: monohydrate glucose 180g / L, yeast paste 20g / L, yeast powder 30g / L, ammonium sulfate 10g / L, potassium dihydrogen phosphate 3.5g / L, potassium chloride 2g / L, manganese sulfate 0.02g / L, urea 7g / L, magnesium sulfate 2g / L, sodium chloride 0.5g / L. After disinfection, the pH of the ammonia water was adjusted to 7.0, and the disinfection conditions were 121-123°C, 30 min. The final tank discharge levels were shown in Table 2 below, and the purity was between 85% and 91%.

[0035] Table 2 Fermentation levels corresponding to different pH values

[0036] pH Fermentation level (g / L) 5.5 0.3 5.8 1.2 6.1 6.8 6.4 10.9 6.7 9.9 7.0 8.4 7.3 5.3

[0037] Example 4

[0038] 1. The preparation of seed solution is the same as in Example 1;

[0039] 2. Fermentation tank culture: Take the qualified seed tank seeds and add them into a 50L automatic control fermentation tank filled with 25L liquid fermentation medium at a ratio of 10% (V / V). The temperature is controlled at 36℃, the dissolved oxygen is controlled to be ≥20%, and the air flow and stirring speed are adjusted during the process. The glucose concentration of the fermentation liquid is controlled to be 30-40g / L during the process, and the pH is automatically controlled at 6.4 by 30% sodium hydroxide solution. After 48h of cultivation, the tank is released; the liquid fermentation medium formula is composed of: monohydrate glucose 180g / L, yeast paste 20g / L, yeast powder 30g / L, ammonium sulfate 10g / L, potassium dihydrogen phosphate 3.5g / L, potassium chloride 2g / L, manganese sulfate 0.02g / L, urea 7g / L, magnesium sulfate 2g / L, sodium chloride 0.5g / L. After disinfection, the pH of ammonia water is adjusted to 7.0, and the disinfection conditions are 121-123℃, 30min. The final tank release level is 7.2g / L, and the purity is 85.7%.

[0040] Example 5

[0041] 1. The preparation of seed solution is the same as in Example 1;

[0042] 2. Fermentation tank culture: Take the seeds from the seed tank that have passed the culture and add them into a 50L automatic control fermentation tank filled with 25L liquid fermentation medium at a ratio of 2%, 5%, 10%, 15%, and 20% (V / V). The temperature is controlled at 36℃, the dissolved oxygen is controlled to be ≥20%, and the air flow and stirring speed are adjusted during the process. The glucose concentration of the fermentation liquid is controlled to be 30-40g / L during the process, and the pH is automatically controlled at 6.4 by ammonia solution. After 48 hours of culture, the tank is released; the liquid fermentation medium formula is composed of: monohydrate glucose 180g / L, yeast paste 20g / L, yeast powder 30g / L, ammonium sulfate 10g / L, potassium dihydrogen phosphate 3.5g / L, potassium chloride 2g / L, manganese sulfate 0.02g / L, urea 7g / L, magnesium sulfate 2g / L, sodium chloride 0.5g / L. After disinfection, the pH of ammonia water is adjusted to 7.0, and the disinfection conditions are 121-123℃, 30min. The final tank release levels are shown in Table 3 below, with purities ranging from 85% to 91%.

[0043] Table 3 Fermentation levels corresponding to different transplanting amounts

[0044] Transplanting amount% Fermentation level (g / L) 2 1.1 5 7.8 10 10.9 15 10.5 20 9.9

[0045] Example 6

[0046] 1. The preparation of seed solution is the same as in Example 1;

[0047] 2. Fermentation tank culture: Take the seeds from the seed tank that have passed the culture and add them to a 50L automatic control fermentation tank filled with 25L liquid fermentation medium at a ratio of 10% (V / V). The temperature is controlled at 36°C, the dissolved oxygen is controlled to be ≥20%, and the air flow and stirring speed are adjusted during the process. The glucose concentration of the fermentation liquid is controlled to be 30-40g / L during the process, and the pH is automatically controlled at 6.4 by ammonia solution flow addition. After 48h of culture, the tank is released; the liquid fermentation medium formula is composed of: monohydrate glucose concentration as a gradient, yeast paste 20g / L, yeast powder 30g / L, ammonium sulfate 10g / L, potassium dihydrogen phosphate 3.5g / L, potassium chloride 2g / L, manganese sulfate 0.02g / L, urea 7g / L, magnesium sulfate 2g / L, sodium chloride 0.5g / L. After disinfection, the ammonia water is adjusted to pH 7.0, and the disinfection conditions are 121-123°C, 30min. The final tank release level is shown in Table 4 below, and the purity is between 85% and 91%.

[0048] Table 4 Fermentation levels corresponding to different basic glucose concentrations

[0049] Basal glucose concentration g / L Fermentation level (g / L) 60 7.8 100 8.1 140 9.9 180 11.0 220 8.8 260 4.4

[0050] Example 7

[0051] 1. The seed solution was prepared as in Example 1.

[0052] 2. Fermentation tank culture: Take the qualified seed tank seeds and add them into a 50L automatic control fermentation tank with 25L liquid fermentation medium at a ratio of 10% (V / V). The temperature is controlled at 36℃, the dissolved oxygen is controlled to be ≥20%, and the air flow and stirring speed are adjusted during the process. The process control method for the glucose concentration of the fermentation liquid is: the glucose concentration is lower than 10g / L, and glucose is added once to make the glucose concentration of the fermentation liquid reach 80-120g / L, and this action is repeated after the glucose is consumed. The ammonia solution is automatically controlled to pH 6.4, and the tank is released after 48h of cultivation; the liquid fermentation medium formula is composed of: monohydrate glucose 180g / L, yeast extract 20g / L, yeast powder 30g / L, ammonium sulfate 10g / L, potassium dihydrogen phosphate 3.5g / L, potassium chloride 2g / L, manganese sulfate 0.02g / L, urea 7g / L, magnesium sulfate 2g / L, sodium chloride 0.5g / L. After disinfection, the pH of ammonia water was adjusted to 7.0, the disinfection conditions were 121-123°C, 30 min, the final tank discharge level was 10.5 g / L, and the purity was 92%.

Claims

1. A method for producing adenosine by fermentation, characterized in that: The method comprises the following steps: using adenosine-producing Bacillus subtilis as a fermentation strain, controlling the glucose concentration of the fermentation liquid to be 30-60 g / L and the pH to be 6.3-6.4 during the fermentation process, inoculating the seed liquid of the Bacillus subtilis into a liquid fermentation medium at an inoculation rate of 10-15% (v / v) for fermentation culture, wherein the monohydrate glucose content in the liquid fermentation medium is 140-180 g / L, and the fermentation strain is Bacillus subtilis ( Bacillus subtilis ) HDCC112-21027, deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC), with the deposit number CGMCC NO.23448 and the deposit date of September 18, 2021.

2. The method according to claim 1, characterized in that: The glucose supplementation method is pulse supplementation.

3. The method according to claim 1, characterized in that: The pH was adjusted by adding aqueous ammonia.

4. The method according to claim 1 or 2, characterized in that: The temperature of the fermentation process is controlled at 25-40°C, the dissolved oxygen is controlled at ≥10-40%, and the total fermentation time is 48-64 hours.

5. The method according to claim 4, characterized in that The liquid fermentation medium comprises the following components: 140-180 g / L of glucose monohydrate, 0-50 g / L of yeast extract, 0-50 g / L of yeast powder, 0-30 g / L of ammonium sulfate, 0-7 g / L of potassium dihydrogen phosphate, 0-5 g / L of potassium chloride, 0-0.05 g / L of manganese sulfate, 0-14 g / L of urea, 0-5 g / L of magnesium sulfate, and 0-1 g / L of sodium chloride.

6. The method according to claim 5, characterized in that The liquid fermentation medium comprises the following components: 180 g / L glucose monohydrate, 20 g / L yeast extract, 30 g / L yeast powder, 10 g / L ammonium sulfate, 3.5 g / L potassium dihydrogen phosphate, 2 g / L potassium chloride, 0.02 g / L manganese sulfate, 7 g / L urea, 2 g / L magnesium sulfate, 0.5 g / L sodium chloride, and the pH value of ammonia water is adjusted to 7.

0.

7. The method according to claim 6, characterized in that The seed liquid is prepared by culturing Bacillus subtilis in a seed culture medium, and then taking qualified liquid seeds, inoculating them into a fermentation tank filled with liquid fermentation culture medium at a ratio of 0.1-2.0% (V / W) for fermentation, controlling the temperature to 36±2°C, controlling the dissolved oxygen to be greater than 10-40%, culturing for 5-20 hours, and controlling the seed liquid OD to be ≥8.

0.

8. The method according to claim 7, characterized in that: The culture medium for the seed culture process is LB culture medium, and the pH is adjusted to 7.0; the LB culture medium contains 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, and 10 g / L glucose.