阅读说明：本技术 嘌呤的发酵生产工艺 (Fermentation production process of purine ) 是由 权春善 刘宝全 金梅姝 郭斌梅 陈苛蒙 金黎明 俞勇 郑立 于 2020-05-08 设计创作，主要内容包括：本发明涉及一种嘌呤的发酵生产工艺,包括以下步骤：(1)将海洋芽孢杆菌Bacillus sp.JIN118进行活化,得到单一菌落；(2)种子液扩大培养,得到种子液；(3)将种子液接种到发酵培养基中发酵,得到发酵液；(4)对发酵液离心、萃取,进行初级分离,获得嘌呤纯品；(5)单因素试验初步优化嘌呤的发酵生产工艺；(6)正交实验确定最优的发酵生产工艺条件。本发明涉及到的发酵工艺大幅提高了嘌呤的产量,经优化后嘌呤的产量由基础发酵培养基中的4.76mg/L提高到17.88mg/L,具有发酵产率高、周期短、条件易控制等优点,解决了化学合成法合成嘌呤耗费大量有机溶剂,原料来源困难及环境污染等问题。(The invention relates to a purine fermentation production process which comprises the following steps of (1) activating marine Bacillus sp.JIN118 to obtain a single colony, (2) carrying out amplification culture on seed liquid to obtain seed liquid, (3) inoculating the seed liquid into a fermentation culture medium to ferment to obtain fermentation liquid, (4) centrifuging and extracting the fermentation liquid to carry out primary separation to obtain a pure purine product, (5) primarily optimizing the purine fermentation production process through a single-factor test, and (6) determining the optimal fermentation production process conditions through an orthogonal test.)

1. A fermentation production process of purine is characterized by comprising the following specific steps:

activation of S1 strain: coating the marine Bacillus sp.JIN118 stored in a refrigerator at-80 ℃ on a basic fermentation solid culture medium, and culturing for 16-24h at 30 ℃;

s2 seed culture, wherein a single colony is picked by using a sterilized toothpick and inoculated into a test tube filled with 2m L basic fermentation medium, shaking culture is carried out for 16-24h at 30 ℃ and 180r/min, then the single colony is inoculated into 40% basic fermentation medium with the inoculum size of 1%, and shaking culture is carried out for 24h at 30 ℃ and 180r/min to obtain seed solution;

s3 fermentation culture: inoculating the seed liquid into a basic fermentation culture medium containing a certain amount of nitrogen source according to the inoculation amount of 5%, and performing shaking culture on a shaking table at 22-42 ℃ and 100-; fermenting at pH 5-10;

pretreatment of S4 fermentation liquor: centrifuging the fermentation liquor at 8000r/min for 10min to obtain fermentation supernatant, adding equal volume of ethyl acetate into the supernatant, extracting for three times, mixing organic phases, rotary evaporating and concentrating to obtain ethyl acetate paste crude extract, performing crude separation on the paste crude extract by medium pressure preparative chromatography, using silica gel powder as chromatographic column filler, using petroleum ether/ethyl acetate and dichloromethane/methanol as mobile phase, and detecting purine content by high performance liquid chromatography;

s5, purine standard curve preparation: plotting by taking the concentration of a standard substance solution as a horizontal coordinate and taking a chromatographic peak area as a vertical coordinate, respectively carrying out high performance liquid chromatography detection on the standard substance and a substance to be detected by adopting an external standard method, and determining the content of the substance to be detected according to the peak area proportion of the peak value corresponding to the same retention time;

s6, primarily optimizing a fermentation production process of purine by a single-factor test;

and S7, on the basis of the single-factor test result, further determining the optimal fermentation production process condition by combining an orthogonal experiment.

2. The process for the fermentative production of a purine according to claim 1, wherein the optimum nitrogen source is ammonium sulfate and the amount added is 11 g/L.

3. The process for the fermentative production of a purine according to claim 1, wherein in step S3, the fermentation temperature is 37 ℃, the cultivation time is 72h, and the rotation speed of shaking table cultivation is 150 r/min.

4. The process for the fermentative production of a purine according to claim 1, wherein the optimal fermentation culture initial pH is 7.0.

5. The process of claim 1, wherein the basic fermentation medium (g/L) comprises peptone 5g, yeast powder 10g, glucose 10g, potassium dihydrogen phosphate 1g, and aged seawater 1L.

6. The process of claim 1, wherein the linear equation of the purine standard is 145746.80+88082.67x, and the correlation coefficient R is²＝0.99954。

7. The fermentation production process of purine according to claim 1, wherein the analytical chromatographic column for purine is Innoval ODS-2C18(4.6x250mm, 5 μm), the detection wavelength is 222nm, the mobile phase is 10% -90% methanol water, the flow rate is 0.8m L/min, the sample injection amount is 20 μ L, and the peak-off time is 18.3582 min.

8. The purine compounds produced by the fermentation process of claims 1-7, having the specific structure as shown in formula I:

Technical Field

The invention belongs to the technical field of microbial fermentation, and particularly relates to a fermentation production process of purine.

Background

Purine is an organic synthetic intermediate, is a very important chemical raw material, is widely used for the production of products such as dyes, pesticides, medicines, spices and the like, and has great social demand. In addition, many purine analogues are clinically used as antitumor drugs, such as natural caffeine which has exciting and diuretic effects on human bodies. Therefore, purine compounds have a very important role in life health.

The current methods for producing purines are mainly chemical synthesis. The chemical synthesis method needs a large amount of organic solvent, and has the problems of raw material sources, environmental pollution and the like. The purine production by fermentation has not been reported, but the fermentation method has the advantages of short period, easy control and the like, and has wide application prospect.

Disclosure of Invention

The invention aims to provide an optimized process for fermenting purine, which can improve the purine yield and the production efficiency.

The fermentation production process of purine is realized by the following technical scheme, and specifically comprises the following steps:

(1) activating marine Bacillus sp.JIN118 to obtain a single bacterial colony;

(2) selecting the activated single bacterial colony, and performing seed liquid amplification culture to obtain a seed liquid;

(3) inoculating the seed liquid into a fermentation culture medium for fermentation to obtain a fermentation liquid;

(4) centrifuging the fermentation liquor, and discarding thalli to obtain a supernatant; adding equal volume of ethyl acetate into the supernatant for extraction, and carrying out primary separation on the extract by medium-pressure preparative chromatography to obtain a crude product; and separating the crude product by preparative high performance liquid chromatography to obtain a pure purine product.

The purine compound has a specific structure shown in formula I:

the invention has the following beneficial effects:

after the fermentation production process of purine is optimized, the method has the characteristics of short fermentation period, low fermentation cost, simple and easily-controlled fermentation conditions and the like.

The invention has the advantages of solving the problems of large consumption of organic solvent, difficult raw material source, environmental pollution and the like in the process of obtaining purine by a chemical synthesis method. The fermentation method for producing purine has the advantages of high yield, short period, easy control and the like.

The invention provides a purine fermentation production process, aims to further improve the purine yield by optimizing the purine fermentation production process, and aims to solve the technical problems of more byproducts, difficulty in purification, uncontrollable reaction conditions and the like in the process of synthesizing a purine compound by a chemical synthesis method.

The invention provides a fermentation production process of purine, which preliminarily determines the optimal fermentation production process conditions of purine through a single-factor experiment, and further determines the optimal fermentation production process conditions by combining an orthogonal testThe fermentation production process conditions are as follows: (NH)₄)₂SO₄The method is used as a nitrogen source, the addition amount of the nitrogen source is 11 g/L, the temperature is 37 ℃, the rotating speed is 150r/min, the culture time is 72h, and the pH is 7.0. the yield of the optimized purine is improved to 17.88 mg/L from 4.76 mg/L in a basic fermentation culture medium.

Drawings

FIG. 1 high performance liquid chromatography assay results for purine-containing components;

FIG. 2 shows the result of purine purity detection according to the present invention;

FIG. 3 is a standard curve for a purine standard of the present invention;

FIG. 4 shows the results of screening nitrogen sources according to the present invention;

FIG. 5 shows the present invention (NH)₄)₂SO₄Optimizing the concentration;

FIG. 6 shows the results of the fermentation temperature optimization according to the present invention;

FIG. 7 shows the results of the fermentation time optimization according to the present invention;

FIG. 8 shows the optimized results of the rotating speed of shaking table cultivation according to the present invention;

FIG. 9 shows the initial pH optimization results of the present invention;

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

Detailed Description

The invention is described in more detail below with reference to specific examples, without limiting the scope of the invention. Unless otherwise specified, the experimental methods adopted by the invention are all conventional methods, and experimental equipment, materials, reagents and the like used in the experimental method can be obtained from commercial sources.

The marine Bacillus sp.jin118 of the invention is submitted for preservation, and the specific preservation information is as follows:

the preservation number is: CCTCC NO: m2019988; the preservation date is as follows: 12 months and 2 days 2019; the preservation unit: china center for type culture Collection; and (4) storage address: wuhan university in Wuhan, China.

16S rDNA sequence analysis

Using a sterile inoculating loop to pick a single colony in a PCR tube filled with 10 mu L sterile ultrapure water, uniformly mixing, boiling at 100 ℃ for 5min, cooling at 4 ℃ for 5min, sucking 4 mu L supernatant as a DNA template for PCR amplification, adding required reagents according to a 50 mu L system in table 1, carrying out PCR amplification under the conditions in table 2, carrying out nucleic acid electrophoresis and sequencing on a PCR amplification product, analyzing a 16S rDNA sequence (1460bp) of a strain JIN118 in an NCBI nucleic acid database through B L AST and drawing a phylogenetic tree, and obtaining a result shown in figure 10.

TABLE 1 PCR reaction System (50. mu. L)

TABLE 2 PCR reaction conditions