CN111411134B - A preparation method for producing purine by fermentation of marine bacillus sp.JIN118 - Google Patents

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for producing purine by fermenting Bacillus sp.JIN118 and a preparation method thereof. The preparation method comprises the following steps: (1) Activating and fermenting Bacillus sp.JIN118 to obtain fermentation liquor; (2) Centrifuging the fermentation liquor, and discarding thalli to obtain a supernatant; (3) Adding ethyl acetate with the same volume into the supernatant for extraction, and performing primary separation on the extract by a medium-pressure preparation chromatography to obtain a crude product; (4) And separating the crude product by using preparative high performance liquid chromatography to obtain a pure purine product. The Bacillus sp.JIN118 can stably produce purine, and the preparation method is quick and effective, can provide raw materials for preparing purine derivatives with strong antibacterial effect and antitumor activity, and has wide industrial application value.

Description

A method for preparing purine produced by fermentation of marine Bacillus sp. JIN118

Technical Field

The invention belongs to the technical field of microbial fermentation, and specifically relates to a preparation method for producing purine by fermenting marine bacillus sp. JIN118.

Background Art

Purine (C ₅ H ₄ N ₄ ) exists in the body mainly in the form of purine nucleotides, and plays a very important role in energy supply, metabolic regulation and coenzyme synthesis.

Due to the different positions of the H atoms attached to the N atom, purine has four possible tautomers, among which isomers 3 and 4 have a very small probability of existing. In the crystalline state, they mainly exist in the form of 2, and in the solution, they mainly exist in the form of 1 and 2 in a nearly equimolecular ratio.

Purine is an organic synthesis intermediate and a very important chemical raw material. It is widely used in dyes, pesticides, medicines, spices and other products, and has a great social demand. Most purine derivatives have good biological activity. At the same time, purine and its derivatives are high-energy density compounds and can be used to prepare high-energy density materials. They have a wide range of application values in military and fireworks.

In the prior art, there are many studies on the preparation and application of purine nucleotides and purine compounds, but there are few studies on the biosynthesis of purine itself. At present, purine is mainly obtained by chemical synthesis, and there is no report on its isolation from plants or microbial fermentation products.

The invention provides a strain capable of stably producing purine and a method for preparing purine with simple process, safety, reliability, low cost and little pollution.

Summary of the invention

In view of the shortcomings of the prior art, the present invention provides a method for preparing purine by fermenting marine Bacillus sp. JIN118.

The inventive concept of the present invention is that in the prior art, there are few research reports on the separation and preparation of purines from natural products, and the purines currently available on the market are mainly obtained by chemical synthesis. In particular, there are no reports on the preparation of purines from microbial metabolites. The inventors isolated and screened a marine bacillus sp. JIN118 from a sea mud sample in the Arctic Ocean. The bacillus was fermented in liquid, and purines with high purity were detected and separated from its metabolites.

To achieve the above object, the present invention adopts the following technical scheme: A preparation method for producing purine by fermentation of marine Bacillus sp. JIN118 is specifically as follows:

(1) activating and fermenting marine Bacillus sp. JIN118 to obtain a fermentation liquid;

(2) centrifuging the fermentation broth, discarding the bacterial cells, and obtaining the supernatant;

(3) the supernatant was extracted with an equal volume of ethyl acetate, and the extract was subjected to primary separation by medium pressure preparative chromatography to obtain a crude product;

(4) The crude product is separated by preparative high performance liquid chromatography to obtain pure purine.

Furthermore, in the step (1), the activation and fermentation process of marine Bacillus sp. JIN118 is as follows: taking out the freeze-dried storage tube from -80°C, inoculating it onto the slant of the test tube, culturing it at 30°C for 16-24h, picking a small amount of bacteria from the slant of the test tube and inoculating it into a triangular flask containing seed culture solution, culturing it at 30°C and 180r/min on a shaker for 24h, and then inoculating it into a triangular flask containing fermentation medium at a 5% inoculation rate, and culturing it at 30°C and 180r/min for 72h.

Furthermore, the step (1) specifically includes the following steps:

(1.1) Bacterial Activation

The marine Bacillus sp. JIN118 stored in a -80°C refrigerator was spread on a solid culture medium and cultured at 30°C for 24 hours. The solid culture medium formula contained: 1L deionized water, 10g peptone, 5g yeast powder, 10g NaCl, and 15g agar.

(1.2) Seed liquid culture

Use a sterilized toothpick to pick a single colony and inoculate it into a test tube containing 2 mL of culture medium. Incubate it in a shaker at 30°C and 180 r/min for 24 hours. Then inoculate it into a fermentation medium with a liquid volume of 40% at a 1% inoculum volume and incubate it in a shaker at 30°C and 180 r/min for 24 hours. The fermentation medium formula contains: 1 L of seawater and 35 g of potato dextrose broth medium.

(1.3) Fermentation culture

The seed liquid was inoculated at 5% inoculum and cultured at 30°C and 180 r/min with shaking for 72 h.

Furthermore, the step (2) is specifically as follows: taking the fermentation liquid and centrifuging it at 8000 rpm for 10 minutes, discarding the precipitate and retaining the supernatant.

Furthermore, the step (3) is specifically as follows: the supernatant is fully extracted three times with an equal volume of ethyl acetate, the organic phases are combined, and the crude ethyl acetate extract is concentrated by rotary evaporation to obtain the crude paste extract. The crude paste extract is roughly separated by medium pressure preparative chromatography, the chromatographic column filler is silica gel powder, the mobile phase is petroleum ether/ethyl acetate and dichloromethane/methanol, and the purine-containing components are combined by high performance liquid chromatography.

Furthermore, the step (4) is specifically as follows: using a preparative high performance liquid chromatograph to separate and prepare the purine-containing components obtained after the medium pressure preparation, the chromatographic column is Hypersil BDS C8 (10mmx250mm, 10μm), the mobile phase is 5% methanol water, the flow rate is 2mL/min, the detection wavelength is 222nm, the sample injection concentration is 70mg/mL, and the injection volume is 60μL.

The present invention also claims protection for the purine compound obtained by the above preparation method. The specific structure of the compound is shown in Formula I:

The beneficial effects of the present invention are as follows:

(1) The purine preparation process of the present invention is simple and easy to operate, with the characteristics of short fermentation cycle, low fermentation cost, easy handling of fermentation liquid and convenient preparation.

(2) The beneficial effect of the present invention is to solve the shortcomings of the chemical synthesis of purine compounds, such as complicated operation methods, many reaction by-products, and difficulty in separation and purification.

(3) The present invention provides a new method for preparing purine from the metabolites of marine Bacillus sp. JIN118, which has the characteristics of short fermentation cycle, low fermentation cost, easy processing of fermentation broth, and convenient preparation. It effectively solves the technical problem that there are many by-products in the synthesis reaction of purine compounds and it is not easy to separate and purify. In the present invention, marine Bacillus sp. JIN118 stably produces purine, and the preparation method achieves the purpose of quickly and effectively obtaining purine with a purity of 95%, providing raw materials for the future preparation of purine analogs with strong antibacterial and anti-tumor activities, and has a wide range of industrial application value.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a phylogenetic tree of strain JIN118 of the present invention;

FIG2 is a HPLC test result of a pure compound of the present invention;

FIG3 is a HPLC purity test result of the pure compound of the present invention;

FIG4 is a mass spectrum of a purine compound of the present invention;

FIG5 is a hydrogen spectrum of the purine compound of the present invention;

FIG6 is a carbon spectrum of the purine compound of the present invention.

DETAILED DESCRIPTION

The present invention is described in detail below through specific examples, but the protection scope of the present invention is not limited. Unless otherwise specified, the experimental methods used in the present invention are conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial channels. The marine bacillus sp. JIN118 of the present invention has been submitted for deposit, and the specific deposit information is as follows:

Deposit number: CCTCC NO: M 2019988; Deposit date: December 2, 2019; Deposit unit: China Center for Type Culture Collection; Deposit address: Wuhan University, Wuhan, China.

Example 1 16S rDNA sequence analysis

Use a sterile inoculation loop to pick a single colony and place it in a PCR tube containing 10 μL sterile ultrapure water, mix well, boil at 100°C for 5 min, cool at 4°C for 5 min, and take 4 μL of supernatant as a DNA template for PCR amplification. Add the required reagents according to the 50 μL system in Table 1, and perform PCR amplification according to the conditions in Table 2. The PCR amplification product was subjected to nucleic acid electrophoresis and sequencing, and the sequence (1460 bp) of the 16S rDNA 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 1. The analysis confirmed that the strain was a bacterium of the genus Bacillus and was named Bacillus sp. JIN118.

Table 1 PCR reaction system (50 μL)

Table 2 PCR reaction conditions

Example 2 Isolation and purification of purine compounds

(1) Activation and fermentation of Bacillus sp. JIN118

Take out the freeze-dried storage tube from -80℃, inoculate it into the slant of the test tube, culture at 30℃ for 24h, then pick a small amount of bacteria from the slant of the test tube and inoculate it into a conical flask containing seed culture solution, culture it on a shaker at 30℃ and 180r/min for 24h, inoculate it into a conical flask containing fermentation medium at a rate of 5%, and culture it at 30℃ and 180r/min for 72h.

(2) Fermentation broth extraction

The fermentation broth was centrifuged at 8000 rpm for 10 min, the cells were discarded, and the supernatant was added with an equal volume of ethyl acetate to fully extract until no purine was detected in the water layer. The extract was rotary evaporated to obtain a paste crude extract. The paste crude extract was roughly separated by medium pressure preparative chromatography, the chromatographic column filler was silica gel powder, the mobile phase was petroleum ether/ethyl acetate (V/V) 2:1 and dichloromethane/methanol (V/V) 69:1, 9:1, 1:1, respectively, and the fractions were collected based on TLC and concentrated under reduced pressure to obtain four components.

(3) Separation and preparation of purine compounds

Each component was detected by high performance liquid chromatography, and the dichloromethane/methanol (V/V) 69:1 component containing purine was selected to prepare a pure product, and its preparation conditions were determined. Detection conditions: The analytical column was Innova! ODS-2C18 (4.6x250mm, 5μm), the detection wavelength was 222nm, the mobile phase was 5% methanol water, the flow rate was 0.8mL/min, the sample concentration was 70mg/mL, and the injection volume was 20μL. The results showed that the separation of purine was best when the mobile phase was 5% methanol water isocratic elution, and the detection results are shown in Figure 2.

The purine-containing components obtained after medium pressure preparation were separated and prepared by preparative high performance liquid chromatography, the chromatographic column was Hypersil BDS C8 (10mmx250mm, 10μm), the mobile phase was 5% methanol water, the flow rate was 2mL/min, the detection wavelength was 222nm, the sample injection concentration was 70mg/mL, and the injection volume was 60μL. The enriched fractions were used to obtain pure purine compounds with a purity of 95%, and the purity test is shown in Figure 3.

Example 3 Structural Identification of Purine Compounds

The pure purine compound is a white powder. According to the high-resolution mass spectrum (ESI-MS), the molecular weight of the purine compound is M=121.0508[M+H] ⁺ , as shown in Figure 4. ¹ H-NMR (CH ₃ OD, 400MHz) δ: 9.09 (s, 1H), 8.93 (s, 1H), 8.56 (s, 1H), ¹ H-NMR results are shown in Figure 5. ¹³ C-NMR (CD ₃ OD, 400MHz) δ: 155.00, 152.16, 146.40, 145.15, 130.16, ¹³ C-NMR results are shown in Figure 6. Therefore, it is inferred that the compound belongs to the alkaloid compound purine, the molecular formula is C ₅ H ₄ N ₄ , and the structural formula is shown in Formula I.

The present invention prepares purine compounds from marine Bacillus sp. JIN118 for the first time, and provides a new way for marine Bacillus sp. JIN118 to produce purine compounds and a new method for preparing purine, which provides new ideas for the future research direction of marine microbial metabolites. It also lays the foundation for the future development of purine and purine analogs with novel structures and unique mechanisms of action.

The above description is only a preferred specific implementation mode of the present invention, but does not limit the present invention to the described embodiments. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solutions and inventive concepts of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Sequence Listing

<110> Dalian Minzu University

<120> A method for producing purine by fermentation of marine Bacillus sp. JIN118

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1460

<212> DNA

<213> Marine Bacillus sp. JIN118

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tagaccgggc ggcgtgccta atacatgcaa gtcgagcgga cagatgggag cttgctccct 60

gatgttagcg gcggacgggt gagtaacacg tgggtaacct gcctgtaaga ctgggataac 120

tccgggaaac cggggctaat accggatggt tgtttgaacc gcatggttca gacataaaag 180

gtggcttcgg ctaccactta cagatggacc cgcggcgcat tagctagttg gtgaggtaac 240

ggctcaccaa ggcaacgatg cgtagccgac ctgagagggt gatcggccac actgggactg 300

agacacggcc cagactccta cgggaggcag cagtagggaa tcttccgcaa tggacgaaag 360

tctgacggag caacgccgcg tgagtgatga aggttttcgg atcgtaaagc tctgttgtta 420

gggaagaaca agtgccgttc aaatagggcg gcaccttgac ggtacctaac cagaaagcca 480

cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta 540

ttgggcgtaa agggctcgca ggcggtttct taagtctgat gtgaaagccc ccggctcaac 600

cggggagggt cattggaaac tggggaactt gagtgcagaa gaggagagtg gaattccacg 660

tgtagcggtg aaatgcgtag agatgtggag gaacaccagt ggcgaaggcg actctctggt 720

ctgtaactga cgctgaggag cgaaagcgtg gggagcgaac aggattagat accctggtag 780

tccacgccgt aaacgatgag tgctaagtgt tagggggttt ccgcccctta gtgctgcagc 840

taacgcatta agcactccgc ctggggagta cggtcgcaag actgaaactc aaaggaattg 900

acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc gaagaacctt 960

accaggtctt gacatcctct gacaatccta gagataggac gtccccttcg ggggcagagt 1020

gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1080

cgagcgcaac ccttgatctt agttgccagc attcagttgg gcactctaag gtgactgccg 1140

gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgacctgggc 1200

tacacacgtg ctacaatggg cagaacaaag ggcagcgaaa ccgcgaggtt aagccaatcc 1260

cacaaatctg ttctcagttc ggatcgcagt ctgcaactcg actgcgtgaa gctggaatcg 1320

ctagtaatcg cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc 1380

cgtcacacca cgagagtttg taacacccga agtcggtgag gtaacctttt aggagccagc 1440

cgccgaaggt gacaagatgt 1460

Claims (6)

1. A method for producing purine by fermentation of marine Bacillus sp. JIN118, characterized in that it comprises the following steps: (1) activating and fermenting Bacillus sp. JIN118 to obtain a fermentation liquid; (2) centrifuging the fermentation broth, discarding the bacterial cells, and obtaining the supernatant; (3) the supernatant was extracted with an equal volume of ethyl acetate, and the extract was subjected to primary separation by medium pressure preparative chromatography to obtain a crude product; (4) separating the crude product by preparative high performance liquid chromatography to obtain pure purine; The deposit number of the marine Bacillus sp. JIN118 is CCTCC NO: M 2019988. 2. The method for producing purine by fermentation of marine Bacillus sp. JIN118 according to claim 1, characterized in that the activation and fermentation process of marine Bacillus sp. JIN118 is as follows: taking out the freeze-dried storage tube from -80°C, inoculating it to the slant of the test tube, culturing at 30°C for 16-24 hours, then picking a small amount of bacteria from the slant of the test tube and inoculating it into a triangular flask containing seed culture solution, culturing at 30°C and 180r/min on a shaker for 24 hours, and then inoculating it into a triangular flask containing fermentation medium according to an inoculum amount of 5%, and culturing at 30°C and 180r/min for 72 hours. 3. The method for producing purine by fermentation of marine Bacillus sp. JIN118 according to claim 1, characterized in that the centrifugation condition of the fermentation liquid is: centrifugation at 8000 rpm for 10 min. 4. The method for producing purine by fermentation of marine Bacillus sp. JIN118 according to claim 1, characterized in that the supernatant is extracted in batches by adding equal volumes of ethyl acetate until no purine is detected in the water layer, and the extract is rotary evaporated to obtain a paste-like crude extract. 5. The method for producing purine by fermentation of marine Bacillus sp. JIN118 according to claim 4, characterized in that the paste crude extract is roughly separated by medium pressure preparative chromatography, the chromatographic column filler is silica gel powder, the mobile phase is petroleum ether/ethyl acetate and dichloromethane/methanol, and the purine-containing components are combined by high performance liquid chromatography. 6. The method for producing purine by fermentation of marine Bacillus sp. JIN118 according to claim 1, characterized in that the purine-containing components obtained after medium-pressure preparation are separated and prepared using a preparative high performance liquid chromatograph, the chromatographic column is Hypersil BDS C8, the mobile phase is 5% methanol water, the flow rate is 2 mL/min, the detection wavelength is 222 nm, the sample injection concentration is 70 mg/mL, and the injection volume is 60 μL.

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