A fermentation preparation method of adenosine cyclic phosphate

Abstract

The present invention discloses a method for preparing adenosine cyclic phosphate. In this method, a fermentation medium containing trace elements is used for semi-continuous fermentation of Arthrobacter to prepare adenosine cyclic phosphate. The single-batch fermentation time of the method of the present invention is shortened from 72 h to 36 h, the adenosine cyclic phosphate is increased from 11.6 g / L to 16.5 g / L, continuous fermentation can be carried out for more than 5 batches, and the average production intensity reaches 0.358 g / L / h, which is 57.71% higher than that of batch fermentation. This method can significantly shorten the fermentation working hours, improve the production intensity, and reduce the production cost.

Description

Technical Field

[0001] The present invention belongs to the field of microbial fermentation, and particularly relates to a method for fermentative preparation of adenosine cyclic phosphate (cAMP). Background Art

[0002] Adenosine cyclic phosphate is a physiologically active substance widely existing in the human body. As an intracellular second messenger, it plays an important regulatory role in carbohydrate metabolism, fat metabolism, nucleic acid synthesis, protein synthesis, etc. The largest use of adenosine cyclic phosphate is for the treatment of chronic congestive heart failure, pulmonary heart disease, myocardial infarction, myocarditis and cardiogenic shock; it has a certain effect on improving symptoms such as palpitations, shortness of breath and chest tightness in rheumatic heart disease; it can improve the curative effect of combined chemotherapy for acute leukemia and can also be used for the induction remission of acute leukemia; domestic research has found that adenosine cyclic phosphate also has a certain curative effect on senile chronic bronchitis, various hepatitis and psoriasis. Adenosine cyclic phosphate can also be used as a pharmaceutical intermediate to prepare dibutyryl cyclic adenosine monophosphate and meglumine cyclic adenosine monophosphate to improve liposolubility, so as to exert more effective physiological and pharmacological effects. In addition, adenosine cyclic phosphate can also be used as a livestock and poultry feed additive, which can simulate the role of growth hormone in vitro, promote the growth of livestock and poultry, and increase the output of high-quality poultry products. At present, the domestic demand for adenosine cyclic phosphate is increasing at a rate of 20% per year, and the global market is also increasing at a rate of 15%, with an annual consumption of up to hundreds of tons, having a good market prospect.

[0003] All industrialized productions at home and abroad all adopt chemical synthesis method, using AMP as raw material and adopting high-efficiency separation columns for intermediate separation. The large solvent loss, low yield, high cost, small output, and serious environmental pollution restrict its large-scale production. In contrast, the production of cAMP by microbial fermentation method has mild conditions, few by-products and little environmental pollution. It is a green industry characterized by biotechnology and has very important practical significance under the background of low-carbon economy. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a preparation method of adenosine cyclic phosphate in view of the deficiencies of the prior art.

[0005] The idea of the present invention is as follows: An Arthrobacter sp. A302 strain (CGMCC No. 3584) for producing adenosine cyclic phosphate has been screened in the early stage of our laboratory (already recorded in CN 102268385 A), and the fermentation medium has been optimized (already recorded in CN102899373A), but the production level and intensity of adenosine cyclic phosphate are not high. Therefore, the present invention hopes to further optimize the medium formula by changing the fermentation mode, so as to greatly improve the fermentation level and efficiency, shorten the fermentation time and reduce the production cost.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A preparation method of adenosine cyclic phosphate, using Arthrobacter sp. A302 CGMCC No. 3584 as the fermentation strain, and preparing adenosine cyclic phosphate by adding trace elements to the fermentation medium and adopting a semi-continuous fermentation method.

[0008] Among them, the trace elements include Fe 2+ , Mn 2+ , Co 2+ , Cu 2+ , Zn 2+ , Mo 6+ , B 3+ , F - Any one or a combination of several of them; in the fermentation medium, the total concentration of the trace elements is 0.001 g / L to 1 g / L. Preferably 0.1 - 0.5 g / L

[0009] Among them, the fermentation medium includes: glucose 5 - 50 g / L, dipotassium hydrogen phosphate 2 - 20 g / L, potassium dihydrogen phosphate 2 - 20 g / L, magnesium sulfate 0.2 - 2 g / L, peptone 1 - 10 g / L, biotin 0.001 - 0.01 g / L, hypoxanthine 1 - 10 g / L, trace elements 0.001 - 1 g / L, the solvent is water, and the pH is 5 - 9.

[0010] Among them, the fermentation preparation method specifically includes the following steps:

[0011] (1) Inoculate Arthrobacter into a fermenter containing the fermentation medium for fermentation. After the growth of adenosine cyclic phosphate is stable, release part of the fermentation broth;

[0012] (2) Add fresh fermentation medium to the fermenter to the initial volume of the fermentation medium, continue fermentation culture. After the growth of adenosine cyclic phosphate is stable, release part of the fermentation broth again;

[0013] (3) Repeat step (2) 2 - 20 times.

[0014] Specifically, in step (1), the inoculation amount of Arthrobacter is 10% (v:v).

[0015] Specifically, in step (1), the volume of the fermenter is 20 L to 2 T, and the fermentation medium accounts for 50% - 80% of the volume of the fermenter, preferably 60%.

[0016] Specifically, for the fermentation, the fermentation temperature is 25 - 35 °C, and the single-batch fermentation time is 10 - 100 h. The preferred fermentation temperature is 30 °C, and the single-batch fermentation time is between 36 h.

[0017] Further, the single-batch fermentation time is determined by the following factors: The growth of cyclic adenosine monophosphate (cAMP) is stable when the increase in cAMP concentration within 6 hours does not exceed 0.5 g / L.

[0018] Specifically, the partial fermentation broth is 10% - 90% of the volume of the fermentation medium, preferably 80%.

[0019] Specifically, the number of repetitions is determined by the following factor: The fermentation concentration of cAMP drops to less than 80% of the cAMP concentration at the end of the first batch of fermentation.

[0020] Beneficial effects: The present invention adopts a semi-continuous fermentation method. By adding trace elements for semi-continuous fermentation, the single-batch fermentation time is shortened from 72 h to 36 h, the cAMP is increased from 11.6 g / L to 16.5 g / L, and continuous fermentation can be carried out for more than 5 batches. This method can significantly shorten the fermentation working hours, improve the production intensity, and reduce the production cost. Specific embodiments

[0021] In the following examples, the experimental methods are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial sources unless otherwise specified.

[0022] According to the following examples, the present invention can be better understood. However, those skilled in the art can easily understand that the content described in the examples is only used to illustrate the present invention and should not and will not limit the present invention described in detail in the claims.

[0023] The analysis methods used in the following examples are as follows:

[0024] The product cAMP in the fermentation broth was determined by an Agilent 1100 high-performance liquid chromatograph (HPLC). The chromatographic column was Lichrospher C18 (4.6 x 250 mm, 5 μm) from Hanbang Technology Co., Ltd., Huaiyin, Jiangsu. The column temperature was 30 °C, the detector was an ultraviolet detector (254 nm), and the mobile phase was V (methanol): V (triethylamine phosphate solution with pH 6.6) = 30:70; the flow rate was 0.8 mL / min; the injection volume was 20 μL. The standard product of cAMP was accurately weighed and prepared into a standard product solution with a mass concentration of about 50 mg / L using double-distilled triple-deionized water; the pretreated sample was diluted to an appropriate concentration with deionized water and filtered through a 0.45 μm microporous membrane, and then used as the sample solution to be detected. The cAMP yield of the fermentation sample was calculated based on the peak area.

[0025] Example 1:

[0026] Preparation of fermentation medium: glucose 40 g / L, dipotassium hydrogen phosphate 10 g / L, potassium dihydrogen phosphate 10 g / L, magnesium sulfate 1 g / L, peptone 5 g / L, biotin 0.005 g / L, hypoxanthine 6 g / L, FeSO 4 0.03 g / L, Na 2 MoO 4 0.001 g / L, H 3 BO 3 0.002 g / L, MnSO 4 0.01 g / L, CoCl 2 0.001 g / L, CuCl 2 0.005 g / L, ZnSO 4 0.01 g / L, NaF 0.003 g / L, pH 7, sterilize at 121 °C for 15 min, cool down to about 30 °C for standby.

[0027] Load 60 L of the medium into a 100 L stirred tank, inoculate 10% (v:v) Arthrobacter strain after sterilization, and carry out fermentation culture at 30 °C and pH 7. Sample at regular intervals to measure the concentration of cyclic adenosine monophosphate. After 72 h of culture, the concentration of cyclic adenosine monophosphate stabilizes at about 16.5 g / L. Discharge 48 L of the fermentation broth, and keep 12 L in the tank as the seed liquid for the next batch of fermentation. Prepare 48 L of fermentation medium, sterilize it and transfer it into the fermentation tank for further culture. After 36 h, the concentration of cyclic adenosine monophosphate reaches 16.3 g / L. Discharge 48 L of the fermentation broth again, and supplement 48 L of sterilized fresh medium in the same way. Repeat the fed-batch operation 5 times. After the 2nd and 3rd fed-batch operations, the concentration of cyclic adenosine monophosphate is about 16.5 g / L. After the 4th fed-batch operation, the concentration of cyclic adenosine monophosphate drops to 15.4 g / L. After the 5th fed-batch operation, the concentration of cyclic adenosine monophosphate decreases significantly, only 12.3 g / L, and the semi-continuous fermentation is stopped. The average production intensity of cyclic adenosine monophosphate in the whole semi-continuous fermentation is 0.358 g / L / h.

[0028] Comparative Example 1: Compared with Example 1, only FeSO 4

[0029] Preparation of fermentation medium: glucose 40 g / L, dipotassium hydrogen phosphate 10 g / L, potassium dihydrogen phosphate 10 g / L, magnesium sulfate 1 g / L, peptone 5 g / L, biotin 0.005 g / L, hypoxanthine 6 g / L, FeSO 4 0.03 g / L, pH 7, sterilize at 121 °C for 15 min, cool down to about 30 °C for standby.

[0030] 60 L of culture medium was loaded into a 100 L stirring tank, and after sterilization, 10% (v:v) Arthrobacter strain was inoculated. Fermentation culture was carried out at 30 °C and pH 7. Samples were taken at regular intervals to measure the concentration of adenosine cyclic phosphate. After 72 h of culture, the concentration of adenosine cyclic phosphate was stable at about 11.8 g / L. 48 L of fermentation broth was discharged, and 12 L was left in the tank as the seed liquid for the next batch of fermentation. 48 L of fermentation medium was prepared, sterilized and then pumped into the fermentation tank for further culture. After 36 h, the concentration of adenosine cyclic phosphate reached 11.9 g / L. Again, 48 L of fermentation broth was discharged, and 48 L of sterilized fresh medium was added in the same way. Such cyclic feeding was carried out 5 times. After the second and third feedings, the concentration of adenosine cyclic phosphate was about 11.8 g / L. After the fourth feeding, the concentration of adenosine cyclic phosphate decreased to 11.4 g / L. After the fifth feeding, the concentration of adenosine cyclic phosphate decreased significantly, only 9.3 g / L, and semi-continuous fermentation was stopped. The average production intensity of adenosine cyclic phosphate during the whole semi-continuous fermentation was 0.260 g / L / h.

[0031] Comparative Example 2: Compared with Example 1, only Na was added as trace elements 2 MoO 4

[0032] Preparation of fermentation medium: 40 g / L of glucose, 10 g / L of dipotassium hydrogen phosphate, 10 g / L of potassium dihydrogen phosphate, 1 g / L of magnesium sulfate, 5 g / L of peptone, 0.005 g / L of biotin, 6 g / L of hypoxanthine, Na 2 MoO 4 0.001 g / L, pH 7, sterilized at 121 °C for 15 min, and cooled to about 30 °C for standby.

[0033] 60 L of culture medium was loaded into a 100 L stirring tank, and after sterilization, 10% (v:v) Arthrobacter strain was inoculated. Fermentation culture was carried out at 30 °C and pH 7. Samples were taken at regular intervals to measure the concentration of adenosine cyclic phosphate. After 72 h of culture, the concentration of adenosine cyclic phosphate was stable at about 12.4 g / L. 48 L of fermentation broth was discharged, and 12 L was left in the tank as the seed liquid for the next batch of fermentation. 48 L of fermentation medium was prepared, sterilized and then pumped into the fermentation tank for further culture. After 36 h, the concentration of adenosine cyclic phosphate reached 12.4 g / L. Again, 48 L of fermentation broth was discharged, and 48 L of sterilized fresh medium was added in the same way. Such cyclic feeding was carried out 5 times. After the second and third feedings, the concentration of adenosine cyclic phosphate was about 12.4 g / L. After the fourth feeding, the concentration of adenosine cyclic phosphate decreased to 11.8 g / L. After the fifth feeding, the concentration of adenosine cyclic phosphate decreased significantly, only 9.5 g / L, and semi-continuous fermentation was stopped. The average production intensity of adenosine cyclic phosphate during the whole semi-continuous fermentation was 0.271 g / L / h.

[0034] Comparative Example 3: Compared with Example 1, only H was added as trace elements 3 BO 3

[0035] Preparation of fermentation medium: glucose 40 g / L, dipotassium hydrogen phosphate 10 g / L, potassium dihydrogen phosphate 10 g / L, magnesium sulfate 1 g / L, peptone 5 g / L, biotin 0.005 g / L, hypoxanthine 6 g / L, H 3 BO 3 0.002 g / L, pH 7, sterilized at 121 °C for 15 min, cooled to about 30 °C for standby.

[0036] Load 60 L of the medium into a 100 L stirred tank. After sterilization, inoculate 10% (v:v) of Arthrobacter strain, and carry out fermentation culture at 30 °C and pH 7. Samples are taken at regular intervals to measure the concentration of cyclic adenosine monophosphate. After 72 h of culture, the concentration of cyclic adenosine monophosphate stabilizes at about 12.2 g / L. Then, 48 L of the fermentation broth is discharged, and 12 L is retained in the tank as the seed liquid for the next batch of fermentation. Prepare 48 L of fermentation medium, sterilize it, and pump it into the fermentation tank for re-culture. After 36 h, the concentration of cyclic adenosine monophosphate reaches 12.3 g / L. Again, 48 L of the fermentation broth is discharged, and 48 L of sterilized fresh medium is added in the same way. Such cyclic feeding is carried out 5 times. After the second and third feedings, the concentration of cyclic adenosine monophosphate is about 12.2 g / L. After the fourth feeding, the concentration of cyclic adenosine monophosphate drops to 11.5 g / L. After the fifth feeding, the concentration of cyclic adenosine monophosphate drops significantly, only 9.2 g / L, and the semi-continuous fermentation is stopped. The average production intensity of cyclic adenosine monophosphate in the whole semi-continuous fermentation is 0.265 g / L / h.

[0037] Comparative Example 4: Compared with Example 1, only MnSO is added as trace element 4

[0038] Preparation of fermentation medium: glucose 40 g / L, dipotassium hydrogen phosphate 10 g / L, potassium dihydrogen phosphate 10 g / L, magnesium sulfate 1 g / L, peptone 5 g / L, biotin 0.005 g / L, hypoxanthine 6 g / L, MnSO 4 0.01 g / L, pH 7, sterilized at 121 °C for 15 min, cooled to about 30 °C for standby.

[0039] 60 L of a culture medium was loaded into a 100 L stirring tank, and after sterilization, 10% (v:v) Arthrobacter strain was inoculated. Fermentation culture was carried out at 30 °C and pH 7. Samples were taken at regular intervals to determine the concentration of cyclic adenosine monophosphate. After 72 h of culture, the concentration of cyclic adenosine monophosphate stabilized at about 11.9 g / L. 48 L of the fermentation broth was discharged, and 12 L was retained in the tank as the seed liquid for the next batch of fermentation. 48 L of the fermentation medium was prepared, sterilized, and then pumped into the fermentation tank for further culture. After 36 h, the concentration of cyclic adenosine monophosphate reached 11.9 g / L. Again, 48 L of the fermentation broth was discharged, and 48 L of sterilized fresh medium was supplemented in the same way. Such cyclic feeding was carried out 5 times. After the second and third feedings, the concentration of cyclic adenosine monophosphate was about 11.9 g / L. After the fourth feeding, the concentration of cyclic adenosine monophosphate decreased to 11.3 g / L. After the fifth feeding, the concentration of cyclic adenosine monophosphate decreased significantly, only 9.2 g / L, and the semi-continuous fermentation was stopped. The average production intensity of cyclic adenosine monophosphate in the whole semi-continuous fermentation was 0.252 g / L / h.

[0040] Comparative Example 5: Compared with Example 1, only CoCl was added as a trace element 2

[0041] Preparation of the fermentation medium: 40 g / L of glucose, 10 g / L of dipotassium hydrogen phosphate, 10 g / L of potassium dihydrogen phosphate, 1 g / L of magnesium sulfate, 5 g / L of peptone, 0.005 g / L of biotin, 6 g / L of hypoxanthine, CoCl 2 0.001 g / L, pH 7, sterilized at 121 °C for 15 min, and cooled to about 30 °C for standby.

[0042] 60 L of a culture medium was loaded into a 100 L stirring tank, and after sterilization, 10% (v:v) Arthrobacter strain was inoculated. Fermentation culture was carried out at 30 °C and pH 7. Samples were taken at regular intervals to determine the concentration of cyclic adenosine monophosphate. After 72 h of culture, the concentration of cyclic adenosine monophosphate stabilized at about 13.1 g / L. 48 L of the fermentation broth was discharged, and 12 L was retained in the tank as the seed liquid for the next batch of fermentation. 48 L of the fermentation medium was prepared, sterilized, and then pumped into the fermentation tank for further culture. After 36 h, the concentration of cyclic adenosine monophosphate reached 13.0 g / L. Again, 48 L of the fermentation broth was discharged, and 48 L of sterilized fresh medium was supplemented in the same way. Such cyclic feeding was carried out 5 times. After the second and third feedings, the concentration of cyclic adenosine monophosphate was about 13.1 g / L. After the fourth feeding, the concentration of cyclic adenosine monophosphate decreased to 12.1 g / L. After the fifth feeding, the concentration of cyclic adenosine monophosphate decreased significantly, only 10.3 g / L, and the semi-continuous fermentation was stopped. The average production intensity of cyclic adenosine monophosphate in the whole semi-continuous fermentation was 0.286 g / L / h.

[0043] Comparative Example 6: Compared with Example 1, only CuCl was added as a trace element 2

[0044] Preparation of fermentation medium: glucose 40 g / L, dipotassium hydrogen phosphate 10 g / L, potassium dihydrogen phosphate 10 g / L, magnesium sulfate 1 g / L, peptone 5 g / L, biotin 0.005 g / L, hypoxanthine 6 g / L, CuCl 2 0.005 g / L, pH 7, sterilized at 121 °C for 15 min, cooled to about 30 °C for standby.

[0045] 60 L of the medium was filled into a 100 L stirred tank. After sterilization, 10% (v:v) Arthrobacter strain was inoculated, and fermentation culture was carried out at 30 °C and pH 7. Samples were taken at regular intervals to measure the concentration of cyclic adenosine monophosphate. After 72 h of culture, the concentration of cyclic adenosine monophosphate was stable at about 12.5 g / L. 48 L of the fermentation broth was discharged, and 12 L was retained in the tank as the seed liquid for the next batch of fermentation. 48 L of the fermentation medium was prepared, sterilized and then pumped into the fermentation tank for further culture. After 36 h, the concentration of cyclic adenosine monophosphate reached 12.51.9 g / L, and 48 L of the fermentation broth was discharged again. 48 L of sterilized fresh medium was supplemented in the same way. Such cyclic feeding was carried out 5 times. After the second and third feedings, the concentration of cyclic adenosine monophosphate was about 12.5 g / L. After the fourth feeding, the concentration of cyclic adenosine monophosphate decreased to 10.4 g / L. After the fifth feeding, the concentration of cyclic adenosine monophosphate decreased significantly, only 9.2 g / L, and the semi-continuous fermentation was stopped. The average production intensity of cyclic adenosine monophosphate in the whole semi-continuous fermentation was 0.266 g / L / h.

[0046] Comparative Example 7: Compared with Example 1, only ZnSO was added as trace elements 4

[0047] Preparation of fermentation medium: glucose 40 g / L, dipotassium hydrogen phosphate 10 g / L, potassium dihydrogen phosphate 10 g / L, magnesium sulfate 1 g / L, peptone 5 g / L, biotin 0.005 g / L, hypoxanthine 6 g / L, ZnSO 4 0.01 g / L, pH 7, sterilized at 121 °C for 15 min, cooled to about 30 °C for standby.

[0048] 60 L of a culture medium was loaded into a 100 L stirred tank, and after sterilization, 10% (v:v) Arthrobacter strain was inoculated, followed by fermentation culture at 30 °C and pH 7. Samples were taken at regular intervals to measure the concentration of adenosine cyclic phosphate. After 72 h of culture, the concentration of adenosine cyclic phosphate stabilized at around 12.0 g / L. Then, 48 L of the fermentation broth was discharged, and 12 L remained in the tank as the seed liquor for the next batch of fermentation. 48 L of the fermentation medium was prepared, sterilized, and then pumped into the fermentation tank for further culture. After 36 h, the concentration of adenosine cyclic phosphate reached 11.9 g / L. Again, 48 L of the fermentation broth was discharged, and 48 L of sterilized fresh medium was added in the same way. Such cyclic feeding was carried out 5 times. After the second and third feedings, the concentration of adenosine cyclic phosphate was around 12.0 g / L. After the fourth feeding, the concentration of adenosine cyclic phosphate decreased to 10.2 g / L. After the fifth feeding, the concentration of adenosine cyclic phosphate decreased significantly, only reaching 9.2 g / L, and the semi-continuous fermentation was stopped. The average production intensity of adenosine cyclic phosphate throughout the semi-continuous fermentation was 0.257 g / L / h.

[0049] Comparative Example 8: Compared with Example 1, only NaF was added as the trace element.

[0050] Preparation of the fermentation medium: 40 g / L of glucose, 10 g / L of dipotassium hydrogen phosphate, 10 g / L of potassium dihydrogen phosphate, 1 g / L of magnesium sulfate, 5 g / L of peptone, 0.005 g / L of biotin, 6 g / L of hypoxanthine, 0.003 g / L of NaF, pH 7, sterilized at 121 °C for 15 min, and cooled to about 30 °C for standby.

[0051] 60 L of a culture medium was loaded into a 100 L stirred tank, and after sterilization, 10% (v:v) Arthrobacter strain was inoculated, followed by fermentation culture at 30 °C and pH 7. Samples were taken at regular intervals to measure the concentration of adenosine cyclic phosphate. After 72 h of culture, the concentration of adenosine cyclic phosphate stabilized at around 13.2 g / L. Then, 48 L of the fermentation broth was discharged, and 12 L remained in the tank as the seed liquor for the next batch of fermentation. 48 L of the fermentation medium was prepared, sterilized, and then pumped into the fermentation tank for further culture. After 36 h, the concentration of adenosine cyclic phosphate reached 13.3 g / L. Again, 48 L of the fermentation broth was discharged, and 48 L of sterilized fresh medium was added in the same way. Such cyclic feeding was carried out 5 times. After the second and third feedings, the concentration of adenosine cyclic phosphate was around 13.2 g / L. After the fourth feeding, the concentration of adenosine cyclic phosphate decreased to 12.4 g / L. After the fifth feeding, the concentration of adenosine cyclic phosphate decreased significantly, only reaching 10.4 g / L, and the semi-continuous fermentation was stopped. The average production intensity of adenosine cyclic phosphate throughout the semi-continuous fermentation was 0.290 g / L / h.

[0052] Comparative Example 9: Compared with Example 1, only FeSO, Na, and MoO were added as the trace elements. 4 , Na 2 MoO 4

[0053] Preparation of fermentation medium: glucose 40 g / L, dipotassium hydrogen phosphate 10 g / L, potassium dihydrogen phosphate 10 g / L, magnesium sulfate 1 g / L, peptone 5 g / L, biotin 0.005 g / L, hypoxanthine 6 g / L, FeSO 4 0.03 g / L, Na 2 MoO 4 0.001 g / L, pH 7, sterilize at 121 °C for 15 min, cool down to about 30 °C for standby.

[0054] Load 60 L of medium into a 100 L stirred tank. After sterilization, inoculate 10% (v:v) of Arthrobacter strain, and carry out fermentation culture at 30 °C and pH 7. Sample at regular intervals to determine the concentration of cyclic adenosine monophosphate. After 72 h of culture, the concentration of cyclic adenosine monophosphate stabilizes at about 12.8 g / L. Discharge 48 L of fermentation broth, and retain 12 L in the tank as the seed broth for the next batch of fermentation. Prepare 48 L of fermentation medium, sterilize it, and pump it into the fermentation tank for re-culture. After 36 h, the concentration of cyclic adenosine monophosphate reaches 12.8 g / L, and 48 L of fermentation broth is discharged again. Supplement 48 L of sterilized fresh medium in the same way. Repeat the fed-batch operation 5 times. After the second and third fed-batches, the concentration of cyclic adenosine monophosphate is about 12.8 g / L. After the fourth fed-batch, the concentration of cyclic adenosine monophosphate drops to 11.4 g / L. After the fifth fed-batch, the concentration of cyclic adenosine monophosphate decreases significantly, only 10.1 g / L, and the semi-continuous fermentation is stopped. The average production intensity of cyclic adenosine monophosphate in the whole semi-continuous fermentation is 0.278 g / L / h.

[0055] Comparative Example 10: Compared with Example 1, only MnSO 4 and CoCl 2

[0056] Preparation of fermentation medium: glucose 40 g / L, dipotassium hydrogen phosphate 10 g / L, potassium dihydrogen phosphate 10 g / L, magnesium sulfate 1 g / L, peptone 5 g / L, biotin 0.005 g / L, hypoxanthine 6 g / L, MnSO 4 0.01 g / L, CoCl 2 0.001 g / L, pH 7, sterilize at 121 °C for 15 min, cool down to about 30 °C for standby.

[0057] 60 L of a culture medium was loaded into a 100 L stirring tank, and after sterilization, 10% (v:v) Arthrobacter strain was inoculated. Fermentation culture was carried out at 30 °C and pH 7. Samples were taken at regular intervals to determine the concentration of adenosine cyclic phosphate. After 72 h of culture, the concentration of adenosine cyclic phosphate stabilized at about 13.6 g / L. 48 L of the fermentation broth was discharged, and 12 L was retained in the tank as the seed liquor for the next batch of fermentation. 48 L of the fermentation medium was prepared, sterilized, and then pumped into the fermentation tank for further culture. After 36 h, the concentration of adenosine cyclic phosphate reached 13.6 g / L. Again, 48 L of the fermentation broth was discharged, and 48 L of sterilized fresh medium was added in the same way. Such cyclic feeding was carried out 5 times. After the second and third feedings, the concentration of adenosine cyclic phosphate was about 13.6 g / L. After the fourth feeding, the concentration of adenosine cyclic phosphate decreased to 12.4 g / L. After the fifth feeding, the concentration of adenosine cyclic phosphate decreased significantly, only 10.8 g / L, and the semi-continuous fermentation was stopped. The average production intensity of adenosine cyclic phosphate in the whole semi-continuous fermentation was 0.297 g / L / h.

[0058] Comparative Example 11: Compared with Example 1, only CuCl was added as a trace element 2 , ZnSO 4 , NaF

[0059] Preparation of the fermentation medium: glucose 40 g / L, dipotassium hydrogen phosphate 10 g / L, potassium dihydrogen phosphate 10 g / L, magnesium sulfate 1 g / L, peptone 5 g / L, biotin 0.005 g / L, hypoxanthine 6 g / L, CuCl 2 0.005 g / L, ZnSO 4 0.01 g / L, NaF 0.003 g / L, pH 7, sterilized at 121 °C for 15 min, and cooled to about 30 °C for standby.

[0060] 60 L of a culture medium was loaded into a 100 L stirring tank, and after sterilization, 10% (v:v) Arthrobacter strain was inoculated. Fermentation culture was carried out at 30 °C and pH 7. Samples were taken at regular intervals to determine the concentration of adenosine cyclic phosphate. After 72 h of culture, the concentration of adenosine cyclic phosphate stabilized at about 14.0 g / L. 48 L of the fermentation broth was discharged, and 12 L was retained in the tank as the seed liquor for the next batch of fermentation. 48 L of the fermentation medium was prepared, sterilized, and then pumped into the fermentation tank for further culture. After 36 h, the concentration of adenosine cyclic phosphate reached 14.1 g / L. Again, 48 L of the fermentation broth was discharged, and 48 L of sterilized fresh medium was added in the same way. Such cyclic feeding was carried out 5 times. After the second and third feedings, the concentration of adenosine cyclic phosphate was about 14.0 g / L. After the fourth feeding, the concentration of adenosine cyclic phosphate decreased to 12.6 g / L. After the fifth feeding, the concentration of adenosine cyclic phosphate decreased significantly, only 11.2 g / L, and the semi-continuous fermentation was stopped. The average production intensity of adenosine cyclic phosphate in the whole semi-continuous fermentation was 0.306 g / L / h.

[0061] Comparative Example 12: Compared with Example 1, only Na was added as a trace element2 MoO 4 ,H 3 BO 3 ,MnSO 4 ,CoCl 2

[0062] Preparation of fermentation medium: glucose 40 g / L, dipotassium hydrogen phosphate 10 g / L, potassium dihydrogen phosphate 10 g / L, magnesium sulfate 1 g / L, peptone 5 g / L, biotin 0.005 g / L, hypoxanthine 6 g / L, Na 2 MoO 4 0.001 g / L, H 3 BO 3 0.002 g / L, MnSO 4 0.01 g / L, CoCl 2 0.001 g / L, pH 7, sterilized at 121 °C for 15 min, cooled to about 30 °C for standby.

[0063] Load 60 L of medium into a 100 L stirred tank. After sterilization, inoculate 10% (v:v) of Arthrobacter strain, and carry out fermentation culture at 30 °C and pH 7. Samples were taken at regular intervals to determine the concentration of cyclic adenosine monophosphate. After 72 h of culture, the concentration of cyclic adenosine monophosphate stabilized at about 14.5 g / L. 48 L of fermentation broth was discharged, and 12 L was retained in the tank as the seed broth for the next batch of fermentation. Prepare 48 L of fermentation medium, sterilize it and transfer it to the fermentation tank for further culture. After 36 h, the concentration of cyclic adenosine monophosphate reached 14.4 g / L, and 48 L of fermentation broth was discharged again. 48 L of sterilized fresh medium was supplemented in the same way. Such cyclic feeding was carried out 5 times. After the 2nd and 3rd feedings, the concentration of cyclic adenosine monophosphate was about 14.5 g / L. After the 4th feeding, the concentration of cyclic adenosine monophosphate decreased to 13.5 g / L. After the 5th feeding, the concentration of cyclic adenosine monophosphate decreased significantly, only 11.1 g / L, and semi-continuous fermentation was stopped. The average production intensity of cyclic adenosine monophosphate during the whole semi-continuous fermentation was 0.315 g / L / h.

[0064] Comparative Example 13: Compared with Example 1, only MnSO 4 ,CoCl 2 ,CuCl 2 ,ZnSO 4 ,NaF

[0065] Preparation of fermentation medium: glucose 40 g / L, dipotassium hydrogen phosphate 10 g / L, potassium dihydrogen phosphate 10 g / L, magnesium sulfate 1 g / L, peptone 5 g / L, biotin 0.005 g / L, hypoxanthine 6 g / L, MnSO 4 0.01 g / L, CoCl 2 0.001 g / L, CuCl 20.005 g / L, ZnSO 4 0.01 g / L, NaF 0.003 g / L, pH 7, sterilized at 121 °C for 15 min, cooled to about 30 °C for standby.

[0066] 60 L of the culture medium was loaded into a 100 L stirred tank. After sterilization, 10% (v:v) of the Arthrobacter strain was inoculated, and fermentation culture was carried out at 30 °C and pH 7. Samples were taken at regular intervals to measure the concentration of cyclic adenosine monophosphate. After 72 h of culture, the concentration of cyclic adenosine monophosphate stabilized at about 15.1 g / L. 48 L of the fermentation broth was discharged, and 12 L was retained in the tank as the seed liquid for the next batch of fermentation. 48 L of the fermentation medium was prepared, sterilized, and then pumped into the fermentation tank for further culture. After 36 h, the concentration of cyclic adenosine monophosphate reached 15.1 g / L, and 48 L of the fermentation broth was discharged again. 48 L of the sterilized fresh medium was supplemented in the same way. Such cyclic feeding was carried out 5 times. After the second and third feedings, the concentration of cyclic adenosine monophosphate was about 15.1 g / L. After the fourth feeding, the concentration of cyclic adenosine monophosphate decreased to 14.4 g / L. After the fifth feeding, the concentration of cyclic adenosine monophosphate decreased significantly, only 11.4 g / L, and the semi-continuous fermentation was stopped. The average production intensity of cyclic adenosine monophosphate in the whole semi-continuous fermentation was 0.329 g / L / h.

[0067] Example 14: Compared with Example 1, only Na 2 MoO 4 ,H 3 BO 3 ,MnSO 4 ,CoCl 2 ,CuCl 2 ,ZnSO 4

[0068] Preparation of the fermentation medium: glucose 40 g / L, dipotassium hydrogen phosphate 10 g / L, potassium dihydrogen phosphate 10 g / L, magnesium sulfate 1 g / L, peptone 5 g / L, biotin 0.005 g / L, hypoxanthine 6 g / L, Na 2 MoO 4 0.001 g / L, H 3 BO 3 0.002 g / L, MnSO 4 0.01 g / L, CoCl 2 0.001 g / L, CuCl 2 0.005 g / L, ZnSO 4 0.01 g / L, pH 7, sterilized at 121 °C for 15 min, cooled to about 30 °C for standby.

[0069] 60 L of culture medium was loaded into a 100 L stirring tank. After sterilization, 10% (v:v) Arthrobacter strain was inoculated, and fermentation culture was carried out at 30 °C and pH 7. Samples were taken at regular intervals to determine the concentration of cyclic adenosine monophosphate. After 72 h of culture, the concentration of cyclic adenosine monophosphate stabilized at about 15.7 g / L. 48 L of fermentation broth was discharged, and 12 L was retained in the tank as the seed liquid for the next batch of fermentation. 48 L of fermentation medium was prepared, sterilized, and then pumped into the fermentation tank for further culture. After 36 h, the concentration of cyclic adenosine monophosphate reached 15.8 g / L. Again, 48 L of fermentation broth was discharged, and 48 L of sterilized fresh medium was supplemented in the same way. Such cyclic feeding was carried out 5 times. After the second and third feedings, the concentration of cyclic adenosine monophosphate was about 15.7 g / L. After the fourth feeding, the concentration of cyclic adenosine monophosphate decreased to 14.8 g / L. After the fifth feeding, the concentration of cyclic adenosine monophosphate decreased significantly, only 12.5 g / L, and semi-continuous fermentation was stopped. The average production intensity of cyclic adenosine monophosphate in the whole semi-continuous fermentation was 0.332 g / L / h.

[0070] Comparative Example 15: Compared with Example 1, only FeSO 4 , Na 2 MoO 4 , MnSO 4 , CoCl 2 , CuCl 2 , ZnSO 4 , NaF

[0071] Preparation of fermentation medium: glucose 40 g / L, dipotassium hydrogen phosphate 10 g / L, potassium dihydrogen phosphate 10 g / L, magnesium sulfate 1 g / L, peptone 5 g / L, biotin 0.005 g / L, hypoxanthine 6 g / L, FeSO 4 0.03 g / L, Na 2 MoO 4 0.001 g / L, MnSO 4 0.01 g / L, CoCl 2 0.001 g / L, CuCl 2 0.005 g / L, ZnSO 4 0.01 g / L, NaF 0.003 g / L, pH 7, sterilized at 121 °C for 15 min, and cooled to about 30 °C for standby.

[0072] 60 L of culture medium was loaded into a 100 L stirring tank. After sterilization, 10% (v:v) Arthrobacter strain was inoculated, and fermentation culture was carried out at 30 °C and pH 7. Samples were taken at regular intervals to measure the concentration of adenosine cyclic phosphate. After 72 h of culture, the concentration of adenosine cyclic phosphate was stable at about 16.2 g / L. 48 L of fermentation broth was discharged, and 12 L was retained in the tank as the seed liquid for the next batch of fermentation. 48 L of fermentation medium was prepared, sterilized, and then pumped into the fermentation tank for further culture. After 36 h, the concentration of adenosine cyclic phosphate reached 16.3 g / L. Again, 48 L of fermentation broth was discharged, and 48 L of sterilized fresh medium was supplemented in the same way. Such cyclic feeding was carried out 5 times. After the second and third feedings, the concentration of adenosine cyclic phosphate was about 16.2 g / L. After the fourth feeding, the concentration of adenosine cyclic phosphate decreased to 15.2 g / L. After the fifth feeding, the concentration of adenosine cyclic phosphate decreased significantly, only 12.6 g / L, and the semi-continuous fermentation was stopped. The average production intensity of adenosine cyclic phosphate in the whole semi-continuous fermentation was 0.355 g / L / h.

[0073] Comparative Example 16: Compared with Example 1, the concentration of trace elements was twice that of Example 1.

[0074] Preparation of fermentation medium: glucose 40 g / L, dipotassium hydrogen phosphate 10 g / L, potassium dihydrogen phosphate 10 g / L, magnesium sulfate 1 g / L, peptone 5 g / L, biotin 0.005 g / L, hypoxanthine 6 g / L, FeSO 4 0.06 g / L, Na 2 MoO 4 0.002 g / L, H 3 BO 3 0.004 g / L, MnSO 4 0.02 g / L, CoCl 2 0.002 g / L, CuCl 2 0.01 g / L, ZnSO 4 0.02 g / L, NaF 0.006 g / L, pH 7, sterilized at 121 °C for 15 min, and cooled to about 30 °C for standby.

[0075] 60 L of a culture medium was loaded into a 100 L stirring tank. After sterilization, 10% (v:v) Arthrobacter strain was inoculated, and fermentation culture was carried out at 30 °C and pH 7. Samples were taken at regular intervals to measure the concentration of cyclic adenosine monophosphate. After 72 h of culture, the concentration of cyclic adenosine monophosphate stabilized at about 15.5 g / L. 48 L of the fermentation broth was discharged, and 12 L was retained in the tank as the seed liquor for the next batch of fermentation. 48 L of the fermentation medium was prepared, sterilized, and then pumped into the fermentation tank for further culture. After 36 h, the concentration of cyclic adenosine monophosphate reached 15.5 g / L. Again, 48 L of the fermentation broth was discharged, and 48 L of sterilized fresh medium was added in the same way. Such cyclic feeding was carried out 5 times. After the 2nd and 3rd feedings, the concentration of cyclic adenosine monophosphate was about 15.5 g / L. After the 4th feeding, the concentration of cyclic adenosine monophosphate decreased to 14.2 g / L. After the 5th feeding, the concentration of cyclic adenosine monophosphate decreased significantly, only 11.8 g / L, and the semi-continuous fermentation was stopped. The average production intensity of cyclic adenosine monophosphate in the whole semi-continuous fermentation was 0.336 g / L / h.

[0076] Comparative Example 17: Compared with Example 1, the concentration of trace elements was four times that of Example 1

[0077] Preparation of the fermentation medium: glucose 40 g / L, dipotassium hydrogen phosphate 10 g / L, potassium dihydrogen phosphate 10 g / L, magnesium sulfate 1 g / L, peptone 5 g / L, biotin 0.005 g / L, hypoxanthine 6 g / L, FeSO 4 0.12 g / L, Na 2 MoO 4 0.004 g / L, H 3 BO 3 0.008 g / L, MnSO 4 0.04 g / L, CoCl 2 0.004 g / L, CuCl 2 0.02 g / L, ZnSO 4 0.04 g / L, NaF 0.012 g / L, pH 7, sterilized at 121 °C for 15 min, and cooled to about 30 °C for standby.

[0078] 60 L of a culture medium was loaded into a 100 L stirred tank. After sterilization, 10% (v:v) of Arthrobacter sp. strain was inoculated, and fermentation culture was carried out at 30 °C and pH 7. Samples were taken at regular intervals to determine the concentration of adenosine 3',5'-cyclic monophosphate (cAMP). After 72 h of culture, the cAMP concentration stabilized at about 15.0 g / L. Then, 48 L of the fermentation broth was discharged, and 12 L remained in the tank as the seed liquid for the next batch of fermentation. 48 L of the fermentation medium was prepared, sterilized, and pumped into the fermentation tank for further culture. After 36 h, the cAMP concentration reached 15.1 g / L. Again, 48 L of the fermentation broth was discharged, and 48 L of sterilized fresh medium was supplemented in the same way. Such cyclic feeding was carried out 5 times. After the 2nd and 3rd feeding, the cAMP concentration was about 15.0 g / L. After the 4th feeding, the cAMP concentration decreased to 13.5 g / L. After the 5th feeding, the cAMP concentration decreased significantly, only reaching 11.6 g / L, and the semi-continuous fermentation was stopped. The average production intensity of cAMP during the whole semi-continuous fermentation was 0.326 g / L / h.

[0079] Comparative Example 18: Compared with Example 1, trace elements were not added to the fermentation medium.

[0080] Preparation of the fermentation medium: 40 g / L of glucose, 10 g / L of dipotassium hydrogen phosphate, 10 g / L of potassium dihydrogen phosphate, 1 g / L of magnesium sulfate, 5 g / L of peptone, 0.005 g / L of biotin, 6 g / L of hypoxanthine, pH 7, sterilized at 121 °C for 15 min, and cooled to about 30 °C for standby.

[0081] 60 L of a culture medium was loaded into a 100 L stirred tank. After sterilization, 10% (v:v) of Arthrobacter sp. strain was inoculated, and fermentation culture was carried out at 30 °C and pH 7. Samples were taken at regular intervals to determine the concentration of adenosine 3',5'-cyclic monophosphate (cAMP). After 72 h of culture, the cAMP concentration stabilized at about 11.6 g / L. Then, 48 L of the fermentation broth was discharged, and 12 L remained in the tank as the seed liquid for the next batch of fermentation. 48 L of the fermentation medium was prepared, sterilized, and pumped into the fermentation tank for further culture. After 36 h, the cAMP concentration reached 11.3 g / L. Again, 48 L of the fermentation broth was discharged, and 48 L of sterilized fresh medium was supplemented in the same way. Such cyclic feeding was carried out 5 times. After the 2nd and 3rd feeding, the cAMP concentration was about 11.6 g / L. After the 4th feeding, the cAMP concentration decreased to 10.4 g / L. After the 5th feeding, the cAMP concentration decreased significantly, only reaching 8.6 g / L, and the semi-continuous fermentation was stopped. The average production intensity of cAMP during the whole semi-continuous fermentation was 0.274 g / L / h.

[0082] Comparative Example 19: Compared with Example 1, the fermentation method was batch fermentation.

[0083] Preparation of the fermentation medium: 40 g / L of glucose, 10 g / L of dipotassium hydrogen phosphate, 10 g / L of potassium dihydrogen phosphate, 1 g / L of magnesium sulfate, 5 g / L of peptone, 0.005 g / L of biotin, 6 g / L of hypoxanthine, FeSO4 0.03 g / L, Na 2 MoO 4 0.001 g / L, H 3 BO 3 0.002 g / L, MnSO 4 0.01 g / L, CoCl 2 0.001 g / L, CuCl 2 0.005 g / L, ZnSO 4 0.01 g / L, NaF 0.003 g / L, pH 7, sterilized at 121 °C for 15 min, cooled to about 30 °C for standby.

[0084] 60 L of the culture medium was loaded into a 100 L stirring tank. After sterilization, 10% (v:v) of Arthrobacter strains were inoculated, and fermentation culture was carried out at 30 °C and pH 7. Samples were taken at regular intervals to measure the concentration of cyclic adenosine monophosphate. After 72 h of culture, the concentration of cyclic adenosine monophosphate was stabilized at about 16.4 g / L. The fermentation was ended and the tank was emptied. The production intensity of cyclic adenosine monophosphate in batch fermentation was 0.227 g / L / h.

[0085] The present invention provides an idea and method for a preparation method of cyclic adenosine monophosphate. There are many specific methods and ways to implement this technical solution. The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be realized by the prior art.

Claims

1. A method for fermentative preparation of adenosine cyclic phosphate, characterized in that, using Arthrobacter sp. A302 CGMCC No. 3584 as the fermentation strain, adenosine cyclic phosphate is prepared by adding trace elements to the fermentation medium and adopting a semi-continuous fermentation method; Among them, the trace element is Fe 2+ , Mn 2+ , Co 2+ , Cu 2+ , Zn 2+ , Mo 6+ , B 3+ , F - ; in the fermentation medium, the total concentration of the trace elements is 0.062 g / L.

2. The fermentative preparation method according to claim 1, characterized in that, the fermentation medium comprises: glucose 5 - 50 g / L, dipotassium hydrogen phosphate 2 - 20 g / L, potassium dihydrogen phosphate 2 - 20 g / L, magnesium sulfate 0.2 - 2 g / L, peptone 1 - 10 g / L, biotin 0.001 - 0.01 g / L, hypoxanthine 1 - 10 g / L, trace elements 0.062 g / L, the solvent is water, and the pH is 5 - 9.

3. The fermentative preparation method according to claim 1, characterized in that, it comprises the following steps: (1) Inoculate Arthrobacter into a fermenter containing the fermentation medium for fermentation. After the growth of adenosine cyclic phosphate is stable, part of the fermentation broth is discharged; (2) Add fresh fermentation medium to the fermenter to the initial volume of the fermentation medium, continue fermentation culture. After the growth of adenosine cyclic phosphate is stable, part of the fermentation broth is discharged again; (3) Repeat step (2) 2 - 20 times.

4. The fermentative preparation method according to claim 3, characterized in that, in step (1), the inoculation amount of Arthrobacter is 10% (v:v).

5. The fermentative preparation method according to claim 3, characterized in that, in step (1), the volume of the fermenter is 20 L - 2 T, and the fermentation medium accounts for 50% - 80% of the volume of the fermenter.

6. The fermentative preparation method according to claim 3, characterized in that, for the fermentation, the fermentation temperature is 25 - 35 °C, and the single-batch fermentation time is 10 - 100 h.

7. The fermentative preparation method according to claim 3 or 6, characterized in that, the single-batch fermentation time is determined by the following factor: the growth of adenosine cyclic phosphate is stable when the increase amplitude of the adenosine cyclic phosphate concentration within 6 hours does not exceed 0.5 g / L.

8. The fermentative preparation method according to claim 3, characterized in that, the part of the fermentation broth is 10% - 90% of the volume of the fermentation medium.

9. The fermentative preparation method according to claim 3, characterized in that, the number of repetitions is determined by the following factor: the fermentation concentration of adenosine cyclic phosphate drops to less than 80% of the adenosine cyclic phosphate concentration at the end of the first batch of fermentation.