CN110863022A - Fermentation production process of purine - Google Patents

Abstract

The invention belongs to the technical field of microbial fermentation, in particular to a purine fermentation production process. The optimization of the fermentation production process includes the following steps: (1) activating the marine Bacillus sp. JIN118 to obtain a single colony; (2) expanding the seed liquid to obtain the seed liquid; (3) inoculating the seed liquid into the fermentation culture Fermentation is carried out in the base to obtain a fermentation liquid; (4) the fermentation liquid is centrifuged and extracted, and primary separation is carried out to obtain pure purine; (5) the fermentation production process of purine is preliminarily optimized by single factor experiment; (6) orthogonal experiment is determined The optimal fermentation production process conditions. The fermentation process involved in the present invention greatly increases the yield of purine, and after optimization, the yield of purine is increased from 4.76 mg/L in the basic fermentation medium to 17.88 mg/L, and has the advantages of high fermentation yield, short period and easy control of conditions. And other advantages, the chemical synthesis method for purine synthesis consumes a lot of organic solvents, the source of raw materials is difficult, and the problems of environmental pollution are solved.

Description

Fermentation production process of purine

technical field

The invention belongs to the technical field of microbial fermentation, in particular to a purine fermentation production process.

Background technique

Purine is an organic synthesis intermediate and a very important chemical raw material. It is widely used in the production of dyes, pesticides, medicines and spices, and is in great social demand. In addition, many purine analogs are clinically used as antitumor drugs, such as natural caffeine, which has excitatory and diuretic effects on the human body. Therefore, purine compounds play a very important role in life and health.

At present, the methods for producing purine mainly include chemical synthesis. Since the chemical synthesis method requires a large amount of organic solvents, there are problems such as source of raw materials and environmental pollution. The production of purine by fermentation has not been reported yet, but generally speaking, the production of chemical raw materials by fermentation has the advantages of short cycle and easy control, and has broad application prospects.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a kind of optimization process of fermenting purine which improves purine yield and improves production efficiency.

The above-mentioned purpose of the present invention is a kind of fermentation production technology of purine realized by following technical scheme, specifically comprises the following steps:

(1) Bacillus sp.JIN118 is activated to obtain a single colony;

(2) picking activated single colony, carrying out seed liquid expansion culture, obtains seed liquid;

(3) seed liquid is inoculated into fermentation medium and fermented to obtain fermented liquid;

(4) centrifuging the fermentation broth, discarding the bacterial cells to obtain a supernatant; adding an equal volume of ethyl acetate to the supernatant for extraction, and the extract is subjected to primary separation by medium pressure preparative chromatography to obtain a crude product; the crude product is prepared using preparative Purine pure product was obtained by high performance liquid chromatography.

The specific structure of the above-mentioned purine compound is shown in formula I:

The beneficial effects of the present invention are as follows:

After the fermentation production process of the purine of the invention is optimized, the fermentation cycle is short, the fermentation cost is low, the fermentation conditions are simple and easy to control, and the like.

The beneficial effect of the invention is to solve the problems such as the consumption of a large amount of organic solvent, the difficulty in the source of raw materials, the environmental pollution and the like in the process of obtaining the purine by the chemical synthesis method. The production of purine by fermentation has the advantages of high yield, short cycle and easy control.

The invention provides a purine fermentation production process, the invention aims to further improve the purine yield by optimizing the purine fermentation production process, and the purpose is to solve the process of synthesizing purine compounds by a chemical synthesis method. and other technical issues.

The invention provides a fermentation production process of purine. The optimal fermentation production process conditions of purine are preliminarily determined by single factor experiment, and the optimal fermentation production process conditions are further determined by combining orthogonal experiments as follows: (NH ₄ ) ₂ SO ₄ As the nitrogen source, the addition amount was 11 g/L, the temperature was 37 °C, the rotation speed was 150 r/min, the incubation time was 72 h, and the pH was 7.0. After optimization, the yield of purine is increased from 4.76 mg/L in the basic fermentation medium to 17.88 mg/L. The invention has the advantages of high fermentation yield, short period, easy control of conditions and the like, and solves the problem that the chemical synthesis method consumes a lot of money for purine synthesis. Problems such as organic solvent, difficult source of raw materials and environmental pollution will become an important method for industrial production of purine. Moreover, in the present invention, Bacillus sp. JIN118 can stably produce purine, which is convenient for the later industrial production and development of purine.

Description of drawings

Fig. 1 HPLC detection result of purine-containing components;

Fig. 2 is the purine purity detection result of the present invention;

Fig. 3 is the standard curve of purine standard substance of the present invention;

Fig. 4 is the screening result of nitrogen source of the present invention;

Fig. 5 is the optimization result of the concentration of (NH ₄ ) ₂ SO ₄ in the present invention;

Fig. 6 is the fermentation temperature optimization result of the present invention;

Fig. 7 is fermentation time optimization result of the present invention;

Fig. 8 is the optimization result of the rotation speed of the shaker culture of the present invention;

Fig. 9 is the initial pH optimization result of the present invention;

Figure 10 is a phylogenetic tree of the strain JIN118 of the present invention.

Detailed ways

The present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial sources.

The marine Bacillus sp. JIN118 of the present invention has been submitted for preservation, and the specific preservation information is as follows:

Name of the depositor: General Microbiology Center of China Microorganism Culture Collection Management Committee (CGMCC);

Depositary address: No. 3, No. 1, Beichen Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

16S rDNA sequence analysis

Use a sterile inoculation loop to pick a single colony into a PCR tube filled with 10 μL of sterile ultrapure water, mix well, boil at 100 °C for 5 min, cool at 4 °C for 5 min, and draw 4 μL of the supernatant as the DNA template for PCR amplification. Add the required reagents to the 50 μL system in Table 1, and perform PCR amplification under the conditions in Table 2. The PCR amplification products were subjected to nucleic acid electrophoresis and sequencing. The 16S rDNA sequence (1460 bp) of strain JIN118 was analyzed by BLAST in the NCBI nucleic acid database and a phylogenetic tree was drawn. The results are shown in Figure 10. After analysis, it can be determined that the strain is a bacterium of the genus Bacillus, named Bacillus sp.JIN118.

Table 1 PCR reaction system (50μL)

Table 2 PCR reaction conditions

Embodiment 1 strain activation and fermentation

(1) Strain activation

The marine Bacillus sp. JIN118 stored in a refrigerator at -80°C was spread on the basal fermentation solid medium, and cultured at 30°C for 16-24h. The basal fermentation medium (g/L) contains: 5 g of peptone, 10 g of yeast powder, 10 g of glucose, 1 g of potassium dihydrogen phosphate (KH ₂ PO ₄ ), 20 g of agar, and 1 L of Chen seawater.

(2) Seed cultivation

Use a sterilized toothpick to pick a single colony, inoculate it into a test tube containing 2 mL of basal fermentation medium, and culture with shaking at 30°C, 180 r/min for 16-24 hours. Then inoculate 1% of the inoculum into the basal fermentation medium with a liquid content of 40% (the formula of the basal fermentation medium is the same as that in step (1)), 30°C, 180r/min shaker culture for 24h to obtain seeds liquid.

(3) Fermentation culture

The seed solution was inoculated according to 5% of the inoculum, and cultured with shaking at 30°C and 180r/min for 72h.

(4) Pretreatment of fermentation broth

The fermentation broth was centrifuged at 8000 r/min for 10 min to obtain the fermentation supernatant. The supernatant was fully extracted three times by adding an equal volume of ethyl acetate, and the organic phases were combined and concentrated by rotary evaporation to obtain a crude extract of ethyl acetate paste.

Further, the step (4) is specifically as follows: the paste crude extract is subjected to rough separation by medium pressure preparative chromatography, the chromatographic column filler is silica gel powder, and the mobile phase is petroleum ether/ethyl acetate and dichloromethane/methanol, The purine-containing fractions were detected and combined by high performance liquid chromatography.

Further, the step (4) is specifically as follows: using a high performance liquid chromatograph to detect the purine content of the purine-containing component obtained after the medium-pressure preparation, and the detection result is shown in Figure 1 . Its purity reached 94%, and the purity test results are shown in Figure 2.

(5) Purine standard curve

For the preparation of the standard curve, accurately weigh 3 mg of the purine standard, dissolve it in methanol and dilute to 120 μg/mL, and dilute it with methanol to different concentrations of 60 μg/mL, 30 μg/mL, 15 μg/mL, 7.5 μg/mL, The peak areas of 5 gradient concentration standard solutions were measured by high performance liquid chromatograph, 20 μL of each sample was injected, and 3 parallel measurements were taken, and the average peak area was taken. Taking the concentration of the standard solution as the abscissa and the chromatographic peak area as the ordinate, the linear equation of the measured purine standard is y=145746.80+88082.67x, and the correlation coefficient R ² =0.99954. The results are shown in Figure 3.

Content detection: In this experiment, the external standard method is used to detect the standard substance and the substance to be tested by high performance liquid chromatography, and the content of the substance to be tested can be determined according to the peak area ratio of the peaks corresponding to the same retention time. The measurement conditions are chromatographic column Innoval ODS-2C18 (4.6×250mm, 5μm), detection wavelength is 222nm, mobile phase is 10%-90% methanol water, flow rate is 0.8mL/min, and injection volume is 20μL.

The solution concentration and peak area of table 3 purine standard

Example 2 Single factor experiment

The nitrogen source and the best fermentation conditions were preliminarily determined by single factor experiments, and the standard and the substance to be tested were detected by high performance liquid chromatography using the external standard method. According to the linear equation of purine standard, y=145746.80+88082.67x, the yield of purine is calculated, the peak area is the y value, and the x value is calculated, that is, the content of purine.

(1) Screening of nitrogen sources: Take the basal fermentation medium as a control, take the strain Bacillus sp. JIN118, the bacterial age is 24h, and the inoculation amount is 5% of the seed liquid to inoculate the basal fermentation medium containing 0.5% of different nitrogen sources The nitrogen sources were peptone, beef extract, yeast extract, urea, ammonium chloride (NH ₄ Cl), and ammonium sulfate (NH ₄ ) ₂ SO ₄ , respectively, at 30 °C, 180 r/min shaker for 48 h. The yield of purine was measured, and the results are shown in Figure 4. The results show that the yield of purine in the basal fermentation medium is 4.76 mg/L, and the optimal nitrogen source is preliminarily determined to be (NH ₄ ) ₂ SO ₄ , and the yield of purine is 13.45mg/L.

(2) Optimization of the concentration of (NH ₄ ) ₂ SO ₄ : On the basis of determining the optimal nitrogen source of the fermentation medium, the concentration of different optimal nitrogen sources was changed, and ammonium sulfate (NH ₄ ) ₂ SO ₄ was added to 2g/ L, 5g/L, 8g/L, 11g/L, 15g/L. 30 ℃, 180r/min shaker culture for 48h. The yield of purine was measured, and the results were shown in Figure 5. It was preliminarily determined that the optimum addition amount of (NH ₄ ) ₂ SO ₄ was 11 g/L, and the yield of purine was 13.46 mg/L.

(3) Fermentation temperature: On the basis of determining the nitrogen source of the fermentation medium, the bacterial strain Bacillus sp. JIN118 was taken, the bacterial age was 24h, and the inoculation amount was 5% of the seed liquid into the fermentation medium. 200mL/500mL, respectively, under the conditions of 22℃, 27℃, 32℃, 37℃, 42℃, shaking culture at 180r/min for 48h. The yield of purine was measured, and the results were shown in Figure 6. It was preliminarily determined that the optimal fermentation culture temperature was 32°C, and the yield of purine was 14.33 mg/L.

(4) Fermentation time: take the strain Bacillus sp. JIN118, the bacterial age is 24h, and the inoculation amount is 5% of the seed liquid to inoculate the fermentation medium. Culture for 24h, 48h, 72h, 96h, 120h, 144h. The yield of purine was measured, and the results were shown in Figure 7. It was preliminarily determined that the optimal fermentation culture time was 72 h, and the yield of purine was 15.34 mg/L.

(5) Shaker rotation speed: take the strain Bacillus sp.JIN118, the bacterial age is 24h, the inoculation amount is 5% of the seed liquid and inoculated into the fermentation medium, and the liquid amount is 40%, respectively at 100r/min, 150r/min Under the conditions of /min, 200r/min and 250r/min, the cells were incubated at 32°C with shaking on a shaker for 72h. The yield of purine was measured, and the results were shown in Figure 8. It was preliminarily determined that the optimal shaking speed was 200 r/min, and the yield of purine was 15.82 mg/L.

(6) Initial pH: in 6 fermentation media, the initial pH was adjusted to 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 with 2 mol/L NaOH and 2 mol/L HCl solution, respectively, and used after sterilization. Take the strain Bacillus sp.JIN118, the bacterial age is 24h, and the inoculation amount is 5% of the seed liquid into the above fermentation medium. The yield of purine was measured, and the results were shown in Figure 9. It was preliminarily determined that the optimal initial pH value of the fermentation culture was 8.0, and the yield of purine was 13.87 mg/L.

Example 3 Orthogonal experiment

Orthogonal experiment: based on the single factor optimization results, the present invention further determines the optimal culture conditions for fermentation to produce purine by designing a 5-factor 4-level L ₁₆ (4 ⁵ ) orthogonal experiment, and the investigated factors are nitrogen source concentration, temperature and time. , rotational speed, pH, and experimental design factors and levels are shown in Table 4.

A total of 16 groups of experiments were done, and the culture conditions of each group were as follows: 32°C, 200 r/min, and cultured for 72 hours. The yield of purine is calculated according to the linear equation of purine standard y=145746.80+88082.67x, the peak area is the y value, and the x value is obtained, that is, the content of purine. The orthogonal experimental design and results of L ₁₆ (4 ⁵ ) are shown in Table 5. According to the orthogonal experimental results, the optimal experimental combination is A3B4C4D1E3, that is, the optimal fermentation and culture conditions are: the nitrogen source is (NH ₄ ) ₂ SO ₄ , the addition amount is 11 g/L, the temperature is 37° C., the rotational speed is 150 r/min, the time is 72 h, and the pH is 7.0.

Bacillus sp. JIN118 was re-fermented under the optimal fermentation conditions, and its purine yield was determined to be 17.88 mg/L.

Table 4 L ₁₆ (4 ⁵ ) orthogonal experimental factors and levels

Table 5 L ₁₆ (4 ⁵ ) orthogonal experimental design and results

The invention provides a purine fermentation production process, the invention aims to optimize the purine fermentation production process, achieve the purpose of greatly increasing the purine yield, and provide an effective technology for industrial production. Combining single factor experiment and orthogonal experiment to determine the optimal purine fermentation production process conditions, the optimal results are: nitrogen source is (NH ₄ ) ₂ SO ₄ , (NH ₄ ) ₂ SO ₄ addition amount is 11g/L, temperature The temperature is 37°C, the rotating speed is 150r/min, the time is 72h, and the pH is 7.0. After the fermentation production process of purine of the present invention is optimized, the yield of purine is increased from 4.76 mg/L in the basic fermentation medium to 17.88 mg/L. The process optimization greatly improves the fermentation yield and fermentation efficiency. Large-scale industrial production has important research significance.

The above descriptions are only preferred specific embodiments of the present invention, but do not limit the present invention to the scope of the described embodiments. Equivalent replacement or modification of the technical solutions and inventive concepts of the present invention shall be included within the protection scope of the present invention.

Claims (8)

1. a fermentation production technique of purine, is characterized in that, comprises concrete steps as follows: S1 strain activation: spread the marine Bacillus sp. JIN118 stored in the -80℃ refrigerator on the basal fermentation solid medium, and cultivate at 30℃ for 16-24h; S2 Seed culture: Use a sterilized toothpick to pick a single colony, inoculate it into a test tube containing 2 mL of basal fermentation medium, and inoculate with shaking at 30°C, 180 r/min for 16-24 hours, and then inoculate with 1% of the inoculum To the basal fermentation medium containing 40% of the liquid, 30 ° C, 180 r/min shaker shaking culture for 24 h, to obtain seed liquid; S3 Fermentation culture: inoculate the seed liquid in the basic fermentation medium containing a certain amount of nitrogen source according to the inoculum amount of 5%, and shake the culture at 22-42°C, 100-250r/min for 24-144h; the fermentation pH value is 5 -10; S4 fermentation broth pretreatment: fermentation broth was centrifuged at 8000r/min for 10min to obtain fermentation supernatant, supernatant was fully extracted three times by adding equal volume of ethyl acetate, the organic phases were combined and concentrated by rotary evaporation to obtain ethyl acetate paste-like crude The extract, the crude extract in paste form was subjected to rough separation by medium pressure preparative chromatography, the column packing was silica gel powder, the mobile phase was petroleum ether/ethyl acetate and dichloromethane/methanol, and the content of purine was detected by high performance liquid chromatography. ; S5. Purine standard curve preparation: take the concentration of the standard solution as the abscissa and the chromatographic peak area as the ordinate to make a graph, and use the external standard method to detect the standard and the substance to be tested by high performance liquid chromatography. According to the same retention time The peak area ratio of the corresponding peak is used to determine the content of the analyte; S6. The single factor test preliminarily optimizes the fermentation production process of purine; S7. On the basis of the single factor test results, combined with the orthogonal experiment to further determine the optimal fermentation production process conditions. 2. the fermentation production technique of purine according to claim 1, is characterized in that, optimum nitrogen source is ammonium sulfate, and addition is 11g/L. 3. The fermentation production process of purine according to claim 1, characterized in that, in the step S3, the fermentation culture temperature is 37°C, the culture time is 72h, and the shaker culture rotation speed is 150r/min. 4. the fermentation production technology of purine according to claim 1, is characterized in that, optimum fermentation culture initial pH value is 7.0. 5. the fermentation production technique of a kind of purine according to claim 1, is characterized in that, described basal fermentation medium (g/L) formula comprises: peptone 5g, yeast powder 10g, glucose 10g, potassium dihydrogen phosphate 1g, Chen seawater 1L. 6 . The purine fermentation production process according to claim 1 , wherein the linear equation of the purine standard product is y=145746.80+88082.67×, and the correlation coefficient R ² =0.99954. 7 . 7. the fermentation production technique of a kind of purine according to claim 1 is characterized in that, the analytical chromatographic column of described purine is: Innoval ODS-2C18 (4.6 x 250mm, 5 μm), detects wavelength 222nm, and mobile phase is 10%-90% methanol water, the flow rate is 0.8mL/min, the injection volume is 20μL, and the peak time is 18.3582min. 8. the purine compound that the described fermentation process of claim 1-7 prepares, concrete structure is shown in formula I:

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