A kind of preparation method of agmatine

Abstract

The invention discloses a method for preparing agmatine, including adjusting the pH value of a fermentation broth containing arginine decarboxylase, decolorizing with activated carbon and filtering with a ceramic membrane in sequence to obtain a filtrate, subjecting the filtrate to affinity chromatography with a nickel column, adding an arginine slurry to the nickel column, and then adding PLP in batches to collect the liquid flowing out of the nickel column, i.e., agmatine conversion liquid; the agmatine conversion liquid is subjected to a membrane, first decolorization, electrodialysis, first concentration, and first separation to obtain agmatine crude product; the crude product is then redissolved, decolorized for a second time, concentrated for a second time, stirred for crystallization, and separated for a second time to obtain agmatine fine product, and finally dried to obtain solid agmatine. The preparation method of the present invention is simple, has low energy consumption, is pollution-free, has a high conversion rate of agmatine, and can realize the industrialized production of agmatine.

Description

Technical Field

[0001] The invention relates to the technical field of agmatine, and in particular to a method for preparing agmatine. Background Art

[0002] Agmatine is also known as herring spermine, with the molecular formula C5H 14 N4, an endogenous clonidine displacing substance (CDS), is a non-catecholamine ligand of the α2-adrenergic receptor, an agonist of the imidazoline receptor. It exists in various tissues and cells of organisms, plays an important role in the metabolism of organisms, and has multiple biological functions. In the cardiovascular system, agmatine can reduce heart rate, mean arterial pressure, left ventricular pressure, peripheral vascular resistance index, etc.; in the urinary system, agmatine can increase glomerular filtration rate and absolute proximal impulse absorption; in the gastrointestinal tract, agmatine can increase the secretion of gastric acid and pepsin, thin the mucosal thickness, and aggravate stress-induced mucosal lesions; in the condition of glucose metabolism, agmatine can increase the secretion of insulin. In addition, recent studies have shown that agmatine can also inhibit the proliferation of various cells (including vascular smooth muscle cells, astrocytes, endothelial cells, hepatocellular carcinoma cells and renal tubular epithelial cells, etc.). As people deepen their research on the physiological effects of agmatine, agmatine has shown broad prospects for clinical application, especially in the nervous system. In the fields of food, chemical industry, drug preparation and clinical practice, there is huge demand at home and abroad, and the market application prospects are broad.

[0003] The production methods of agmatine mainly include chemical method and bioenzyme method. The chemical method has many steps, low yield, high cost, and is not suitable for large-scale industrial production. The bioenzyme method mainly uses arginine decarboxylase (EC 4.1.1.19), which is a 5'-pyridoxal phosphate (coenzyme PLP) dependent decarboxylase. The enzyme can use L-arginine as a substrate and generate agmatine after decarboxylation. However, the conversion rate needs to be improved. The patent with application number 201510535382.2 discloses a method for producing agmatine using a recombinant bacterium with high arginine decarboxylase production. The method overexpresses the gene (speA) encoding arginine decarboxylase from Escherichia coli BL21 in E. coli BL21 (DE3), and obtains a recombinant strain pET20b (+) -ADC containing arginine decarboxylase. After the enzyme is induced and expressed in Escherichia coli, it shows high activity. The fermented bacteria can be directly used for transformation. By optimizing the transformation conditions, the transformation cycle takes 4-6 hours at 37°C, the agmatine production reaches 14.3g / L, and the conversion rate is 96.5%. In addition to inducing expression through Escherichia coli, it has been discovered this year that Bacillus subtilis can also induce the expression of arginine decarboxylase. Bacillus subtilis is a Gram-positive bacterium with a cell wall that does not contain endotoxins and can form spores. It is a producer of some important industrial enzyme preparations. Liu Jinni constructed the expression vectors of hemA and heml genes, and electro-transformed the two expression vectors into B.subtilis 1A747 competent cells to obtain recombinant bacteria. Maltose was used to induce expression. The results showed that a large amount of glutamyl t-RNA reductase protein and glutamyl t-RNA synthetase protein were obtained, and the synthesis of arginine decarboxylase in Bacillus subtilis was promoted. The content of arginine decarboxylase in the fermentation supernatant of the recombinant bacteria was as high as 46.75mg / L. However, how to improve the conversion rate of arginine decarboxylase induced by Bacillus subtilis to produce agmatine is a problem that needs to be solved. Summary of the invention

[0004] In view of the above prior art, the purpose of the present invention is to provide a method for preparing agmatine. The preparation method of the present invention is simple, low in energy consumption, pollution-free, high in conversion rate of agmatine and low in cost.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The present invention provides a method for preparing agmatine, comprising the following steps:

[0007] (1) adjusting the pH value of the fermentation liquid containing arginine decarboxylase to 4.2-4.6, decolorizing with activated carbon and filtering with a ceramic membrane in sequence to obtain a filtrate, subjecting the filtrate to affinity chromatography with a nickel column, adding the arginine slurry to the nickel column, and then adding PLP in portions, controlling the pH value of the liquid in the nickel column to 4.8-5.2, and collecting the liquid flowing out of the nickel column, which is the agmatine conversion liquid;

[0008] (2) The agmatine conversion liquid is subjected to membrane, first decolorization, electrodialysis, first concentration, and first separation to obtain agmatine crude product; the crude product is then redissolved, second decolorization, second concentration, stirred for crystal growth, and second separation to obtain agmatine fine product, and finally dried to obtain solid agmatine.

[0009] Preferably, in step (1), the pH value of the fermentation broth is adjusted to 4.4; the pore size of the ceramic membrane is 100 nm, and the pressure is 0.5 MPa; and the filtrate affinity chromatography uses 50 mM PBS as a binding buffer.

[0010] Preferably, in step (1), the preparation method of the arginine slurry is: adding L-arginine to pure water, adding 30wt% concentrated hydrochloric acid to adjust the pH to 5.5-6.0, cooling to 38°C to obtain arginine slurry; the concentration of the arginine slurry is 300g / L.

[0011] Preferably, in step (1), the flow rate of the arginine slurry is 50 cm / h; and the amount of PLP added is 0.03-0.04 wt % of the arginine slurry.

[0012] Preferably, in step (2), the membrane filtering is to filter the agmatine conversion liquid through a layer of diatomaceous earth and a layer of activated carbon in sequence; the amount of the activated carbon accounts for 0.5wt% of the agmatine conversion liquid, and the amount of the diatomaceous earth accounts for 0.5wt% of the agmatine conversion liquid.

[0013] Preferably, in step (2), the first decolorization is performed by decolorizing with activated carbon at 75° C. for 40 min; the amount of activated carbon used is 1.5 wt % of the agmatine conversion solution.

[0014] Preferably, in step (2), the pH of the electrodialysis is controlled at 5.7-6.1, and the temperature is controlled at below 40°C; when the conductivity of the fresh water chamber is ≤1000 μs / cm, the electrodialysis is turned off and the fresh water is released; and the first concentration is to concentrate the fresh water released by the electrodialysis to 400-450 g / L by rotary evaporation.

[0015] Preferably, in step (2), the re-dissolution is as follows: the crude product is re-dissolved at 150 g / L, and the re-dissolution temperature is 60°C; the second decolorization is as follows: 5-10 wt‰ of activated carbon is added to the heavy solution, and decolorized at 60°C for 30 min to obtain a decolorized solution, and the decolorized solution is filtered to make the transmittance of the filtrate reach more than 98%; the second concentration is to concentrate the filtrate by rotary evaporation to 400-450 g / L.

[0016] Preferably, in step (2), the stirring and crystal growing comprises: crystallization at room temperature with stirring.

[0017] Preferably, in step (2), during the first separation, the crude product is washed with 0.5 to 1 times the volume of pure water, and the washing liquid is refluxed to the first decolorization for repeated use; during the second separation, the fine product obtained by stirring and growing crystals is washed with 1 to 2 times the volume of pure water, and the washing liquid is refluxed to the first decolorization for repeated use. The above operations are all called mother liquor recycling.

[0018] Beneficial effects of the present invention:

[0019] (1) The present invention adopts an immobilized enzyme method, wherein a fermentation broth containing arginine decarboxylase is filtered, and then the arginine decarboxylase is hung on a nickel column, and then an arginine slurry containing L-arginine is added to the nickel column, and L-arginine is converted into agmatine by the arginine decarboxylase.

[0020] (2) The preparation method of the present invention is simple, easy to operate, low in energy consumption and pollution-free; it can realize the industrial production of agmatine, and the yield of agmatine is significantly improved.

[0021] (3) The method of the present invention has low cost and only needs to use a nickel column as the conversion site. The nickel column can convert 10 to 15 tons of L-arginine from the beginning of use to failure. DETAILED DESCRIPTION

[0022] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0023] As described in the background technology, the arginine decarboxylase used for preparing agmatine by a bioenzymatic method using L-arginine as a substrate is usually produced by genetically engineered recombinant bacteria. Such arginine decarboxylase generally exists in the form of cell inclusion bodies. When preparing agmatine using L-arginine as a substrate, the arginine decarboxylase in the bacterial body needs to be subjected to a series of extraction and purification, and the process is relatively complicated.

[0024] Based on this, the present invention provides a method for preparing agmatine, which adopts an immobilized enzyme method to directly hang the fermented liquid after breaking the bacteria on a nickel column. The production bacteria of arginine decarboxylase have a Gly-Arg-Gly-8His sequence added to the end of the optimized gene during the early design. This sequence is similar to a "small tail". The arginine decarboxylase produced by the production bacteria carries this "small tail" and is hung on the nickel column through it. L-arginine is then added to the nickel column, and the L-arginine is converted into agmatine by the arginine decarboxylase on the nickel column. The present invention does not need to extract and purify the arginine decarboxylase in the fermented liquid, and the method for preparing agmatine is simple and easy to operate, low in cost, and no pollution is generated throughout the process.

[0025] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0026] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0027] Embodiment 1:

[0028] 1. Preparation of arginine decarboxylase producing bacteria

[0029] The nucleotide sequence of the SpeA gene obtained by the inventors from the existing database is shown as SEQ ID NO.1.

[0030] (1) In order to make the SpeA gene more suitable for the prokaryotic expression system, the present invention codon-optimized the nucleotide sequence composition of the above gene, and added a Gly-Arg-Gly-8His sequence to the end of the optimized SpeA gene, as shown in SEQ ID NO.2; the optimized SpeA gene (i.e., the target gene) was obtained, as shown in SEQ ID NO.3.

[0031] (2) The Pg3 promoter was optimized to obtain an optimized Pg3 promoter (shown in SEQ ID NO.4), the plasmid pHT304 was double-digested with NdeⅠ and HincⅡ, and the optimized Pg3 promoter was integrated into the double-digested plasmid pHT304 to obtain plasmid pHT304-Pg3 (shown in SEQ ID NO.5);

[0032] (3) The plasmid pHT304-Pg3 was double-digested with SphⅠ and SacⅠ, and the target gene (shown in SEQ ID NO.3) was integrated into the double-digested plasmid pHT304-Pg3 to obtain a recombinant expression vector (shown in SEQ ID NO.6).

[0033] 2. Construction of arginine decarboxylase producing bacteria

[0034] The recombinant expression vector was introduced into Bacillus subtilis to obtain transformants.

[0035] The transformant was spread on an LB plate, and after a single colony grew, it was inoculated on an AMP plate (LB plate containing 50 μg / ml AMP). After a single colony grew on the resistance plate, the single colony grown on the AMP plate was picked out and taken as a positive transformant.

[0036] The positive transformant was inoculated into a fermentation medium (the composition of the fermentation medium was: peptone 12g / L, yeast extract 8g / L, sodium chloride 3g / L, ammonium sulfate 2.5g / L, potassium hydrogen phosphate trihydrate 4g / L, ammonium ferric citrate 0.3g / L, citric acid 2.1g / L, glycerol 10g / L, magnesium sulfate heptahydrate 0.5g / L, kanamycin sulfate 100ppm, defoamer 0.5g / L, and the pH of the fermentation medium was adjusted to 6.9-7.0 with ammonia water before inoculation), the fermentation temperature was 37°C, the pH was 6.9-7.0, and the dissolved oxygen (DO) was 20-40%; when OD was 20%, the temperature was lowered to 28°C, and 0.2mM IPTG was added to induce fermentation; the induction culture time was ensured to be not less than 14h, and the fermentation was terminated when OD increased slowly or not for a long time. The fermentation liquid obtained after the culture was subjected to a bacteria-breaking treatment, and centrifuged at 4000 r / min for 15 min, and the supernatant was collected to obtain a fermentation liquid containing arginine decarboxylase.

[0037] Example 2

[0038] (1) The fermentation broth obtained in Example 1 was adjusted to pH 4.42 with concentrated hydrochloric acid, and then treated with activated carbon decolorization and ceramic membrane filtration (the pore size of the ceramic membrane is 100nm, and the pressure is 0.5MPa) to obtain a filtrate; the filtrate was added to a nickel column to allow arginine decarboxylase to hang on the column, and the binding buffer was 50mM PBS. Arginine was dissolved in a small amount of pure water to form a 300g / L seminal plasma, and 30% concentrated hydrochloric acid was added to adjust the pH to 5.7, cooled, and set to volume for standby use. After the arginine slurry was cooled to 38°C, it was added to the nickel column, and PLP was added in batches, with the total proportion of PLP being 0.03wt% of the mass of the seminal plasma, and the process pH was controlled at about 5.0 with concentrated hydrochloric acid. The liquid flowing out of the nickel column was collected, which was the agmatine conversion liquid.

[0039] (2) Membrane filtering: The agmatine conversion solution is filtered through a filter layer composed of 0.5 wt% diatomaceous earth and 0.5 wt% activated carbon (diatomaceous earth is laid on top of the activated carbon layer);

[0040] First decolorization: using 1.5 wt% of activated carbon in the agmatine conversion solution, decolorizing at 75°C for 40 min;

[0041] Electrodialysis: Pump the decolorized liquid into the electrodialysis equipment, adjust the pH of the liquid to 5.9 with dilute hydrochloric acid or ammonia water, start the electrodialysis, control the pH at 5.9, and the temperature below 40°C. When the conductivity of the dilute chamber is ≤1000μs / cm, turn off the electrodialysis and release the fresh water.

[0042] First concentration: Turn on the vacuum pump, pump fresh water into the rotary evaporator, then turn on the water bath, control the temperature of the water bath to 75°C, turn on the cooling water for condensation, concentrate to 450g / L, and then release the liquid.

[0043] First separation: Centrifuge the concentrate to obtain a crude product, wash the crystals with pure water of 1 times the volume of the wet product, and return the washing liquid (mother liquor) to the first decolorization step for repeated use (mother liquor reuse).

[0044] Redissolution: The crude product was re-dissolved at 150 g / L, and the re-dissolution temperature was 60°C.

[0045] Second decolorization: add activated carbon accounting for 10wt‰ of the heavy solution, decolorize at 60℃ for 30min, and filter the decolorized solution to make the transmittance of the obtained filtrate reach more than 98%.

[0046] Second concentration: Turn on the vacuum pump, pump the filtrate after decolorization and filtration into a rotary evaporator, then turn on the water bath, control the temperature of the water bath to 75°C, turn on the cooling water for condensation, concentrate to 450g / L, and then release the liquid.

[0047] Stirring crystal growth: Stir the seminal plasma slowly and cool it down to room temperature for crystallization.

[0048] Second separation: centrifuge the concentrate to obtain the fine product, wash the crystals with pure water twice the volume of the wet product until no sulfate residue is detected at the outlet, and return the washing liquid (mother liquor) to the first decolorization process for repeated use (mother liquor reuse).

[0049] Drying: Place the fine product in a drying device and dry it at 60℃.

[0050] Packaging: After the fine product is dried, it will be crushed and packaged according to the particle size requirements of customers and goods to obtain the finished product.

[0051] Comparative Example 1

[0052] (1) Preparation of Agmatine Conversion Solution: Same as step (1) in Example 2.

[0053] (2) Membrane filtering: The agmatine conversion solution is filtered through a filter layer composed of 0.5 wt % diatomaceous earth and 0.5 wt % activated carbon;

[0054] Decolorization: decolorization with 1.5wt% activated carbon at 75℃ for 40min;

[0055] Electrodialysis: Pump the decolorized liquid into the electrodialysis equipment, adjust the pH of the liquid to 5.9 with dilute hydrochloric acid or ammonia water, start the electrodialysis, control the pH at 5.9, and the temperature below 40°C. When the conductivity of the dilute chamber is ≤1000μs / cm, turn off the electrodialysis and release the fresh water.

[0056] Concentration: Turn on the vacuum pump, pump fresh water into the rotary evaporator, then turn on the water bath, control the temperature of the water bath to 75°C, turn on the cooling water for condensation, concentrate to 450g / L, and then release the liquid.

[0057] Separation: Centrifuge the concentrate to obtain agmatine, wash the crystals with pure water of 1 times the volume of the wet product, and reuse the crude mother liquor washing liquid in the decolorization step before electrodialysis.

[0058] Drying: Place agmatine in a drying device and dry at 60°C.

[0059] Packaging: After the fine product is dried, it will be crushed and packaged according to the particle size requirements of customers and goods to obtain the finished product.

[0060] Comparative Example 2

[0061] (1) Preparation of Agmatine Conversion Solution: Same as step (1) in Example 2.

[0062] (2) Decolorization: Add 10 wt‰ activated carbon to the agmatine conversion solution, decolorize at 60°C for 30 min, and filter the decolorized solution until the light transmittance of the filtrate reaches more than 98%.

[0063] Concentration: Turn on the vacuum pump and pump the filtrate into a rotary evaporator. Then turn on the water bath and control the temperature of the water bath to 75°C. Turn on the cooling water for condensation and concentrate to 450g / L. Then release the liquid.

[0064] Stirring crystal growth: Stir the seminal plasma slowly and cool it down to room temperature for crystallization.

[0065] Separation: Centrifuge the concentrate to obtain the fine product, wash the crystals with pure water twice the volume of the wet product until no sulfate residue is detected at the outlet, and reuse the mother liquor washing liquid for the decolorization step before electrodialysis.

[0066] Drying: Place the fine product in a drying device and dry it at 60℃.

[0067] Packaging: After the fine product is dried, it will be crushed and packaged according to the particle size requirements of customers and goods to obtain the finished product.

[0068] Comparative Example 3

[0069] Transformation using the original enzyme transformation method

[0070] 1. Substrate preparation: Dissolve 150 g of arginine in pure water to form a 300 g / L seminal plasma solution, add 30 wt% concentrated hydrochloric acid to adjust the pH to 5.7, and cool for later use.

[0071] 2. Conversion process: cool to 38°C, keep warm in a water bath, add equal amounts of agmatine enzyme in batches, the amount of agmatine enzyme accounts for 4wt% of the seminal plasma, add PLP in batches, the amount of PLP accounts for 0.04wt% of the seminal plasma, and control the pH of the process at about 5.5 with concentrated hydrochloric acid to obtain agmatine conversion solution.

[0072] 3. The extraction method is the same as step (2) in Example 2.

[0073] Comparative Example 4

[0074] The steps are the same as those in Example 2, except that the washing liquid (mother liquor) obtained from the first separation and the second separation is not refluxed to the first decolorization step for reuse (i.e., the mother liquor is not reused).

[0075] Comparative Example 5

[0076] The steps are the same as those of Comparative Example 3, except that the washing liquid (mother liquor) obtained from the first separation and the second separation is not refluxed to the first decolorization step for reuse (i.e., the mother liquor is not reused).

[0077] The standard solution was prepared with agmatine sulfate as the standard substance. The appropriately diluted conversion supernatant and the standard solution were respectively derivatized with OPA, filtered through a 0.22 um microporous membrane, and the agmatine content was determined by high performance liquid chromatography.

[0078] HPLC conditions:

[0079] Chromatographic column: Acclaim 120C18 (250 mm × 4.6 mm × 5 um).

[0080] Mobile phase: Mobile phase A: 10 mM KH2PO4, buffer (pH 5.3); Mobile phase B: acetonitrile-methanol-10 mM KH2PO4, (volume ratio 5:3:1), filtered with a 0.22 um filter membrane;

[0081] Column temperature: 35 °C;

[0082] Detection wavelength: excitation wavelength 330nm, emission wavelength 465nm;

[0083] Injection volume: 12u1 (8u1 sample + 4u10PA derivatization agent);

[0084] Flow rate: 1ml / min.

[0085] Method for determination of agmatine content: area method.

[0086] Yield = the mass of agmatine actually obtained / the mass of agmatine completely converted from L-arginine;

[0087] Conversion rate = mass of agmatine / mass of L-arginine;

[0088] Purity = mass of target product / total mass.

[0089] The finished agmatine products prepared in Example 2 and Comparative Examples 1 to 5 were tested, and their conversion rates and purities are shown in Table 1.

[0090] Table 1

[0091] project Conversion rate purity Yield Example 2 ≥99％ ≥98％ 95-98％ Comparative Example 1 ≥99％ ≈60％ 70～75％ Comparative Example 2 ≥99％ ≈50％ 75～80％ Comparative Example 3 ≈65％ ≥98％ 95-98％ Comparative Example 4 ≥99％ ≥98％ ≈55％ Comparative Example 5 ≈65％ ≥98％ ≈48％

[0092] As can be seen from Table 1, the conversion rate, purity and yield of agmatine prepared by the method of the present invention are higher than those of Comparative Examples 1 to 5. In addition, the cost of preparing agmatine by the method of the present invention is very low. A nickel column is used as the conversion site. From the beginning of use to failure, a nickel column can convert more than ten tons of arginine. The use cost of the nickel column is only about 20 yuan / day. The method of the present invention greatly reduces the production cost of agmatine.

[0093] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application. SEQUENCE LISTING <110> Xintai Jiahe Biotechnology Co., Ltd., Shantou Jiahe Biotechnology Co., Ltd. <120> A kind of preparation method of agmatine <130> 2021 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 1914 <212> DNA <213> Artificial sequence <400> 1 atgaatgatt ggtctattga tgatgctcgt gctgggtaca atgttatactca ctggagccaa 60 ggttttg ggattagcga tcaggtgag gtgacagtgt cgccggatcc taaaaatcct 120 gatacaaga ttggcttaa tgagctagcc aaagacatgg ttaaagcggg ggtcgcttta 180 cctgtactag ttcgttttcc gcagatcctg caccacagag tgaacagttt gtgccaagca 240 ttcgaccaag cgatacagaa gtatgagtat caggcggact atttgttggt ttacccgatt 300 aaagttaacc aaaacaac tgtggtggaa gagatcctag cgagtcaggc atcaaagaa 360 gttccgcaac tgggtttaga agcggcagt aagcctgaat tgatggctgt gctcgcgatg 420 gcacaaaag ccagttcagt gattgttttgt aatggttata aagataacga atatattcgc 480 ttagcactga ttggtgaaa gttaggccac aaggtctata tcgttttaga gaaactgtct 540 gagcttaaga tggtattagc tgaatctaaa cgtctggggcg tgacaccgcg tttaggtcta 600 cgtgcgcgtt tagcgttcca aggcaaggc aagtggcagg cgagtggcgg 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gtgatgctgg atattacctg tgactctgac ggtattgttg atcaatatgt cgatggtcaa 1560 gggattgaaa ccacgctacc agtaccagca tggagcgctg aaagtccata tttgattggt 1620 ttcttccttg tgggagctta tcaagaaatt ttgggtgata tgcacaatct ttttggtgat 1680 accaactccg cggtagttcg gattgaagag aatggtgtca cgaatattga atctgtgcta 1740 gcgggtgata cggttgctga cgtattgcgt tacgttaact tagatgcggt ggcctttatg 1800 cgcacttacg aagagctagt gaatttacat attgaagaag atgaacgcgc acaaatttta 1860 gaagaactac aagttggcct taaaggttac acttatttag aagatttttc ttaa 1914 <210> 2 <211> 36 <212> DNA <213> Artificial sequence <400> 2 ggccgcggcg atgatgatga tgatgatgat gattaa 36 <210> 3 <211> 1956 <212> DNA <213> Artificial sequence <400> 3 gcatatgaat gattggtcta ttgatgatgc tcgtgctggg tacaatgtta ctcactggag 60 ccaaggtttt tatgggatta gcgatcaagg tgaggtgaca gtgtcgccgg atcctaaaaa 120 tcctgaatac aagattggct taaatgagct agccaaagac atggttaaag cggggtcgc 180 tttacctgta ctagttcgtt ttccgcagat cctgcaccac agagtgaaca gtttgtgcca 240 agcattcgac caagcgatac agaagtatga gtatcaggcg gactatttgt tggtttaccc 300 gattaaagtt aaccaacaac aaactgtggt ggaagagatc ctagcgagtc aggcatcaaa 360 agaagttccg caactgggtt tagaagcggg cagataagcct gaattgatgg ctgtgctcgc 420 gatggcacaa aaagccagtt cagtgattgt ttgtaatggt tataaagata acgaatatat 480 tcgcttagca ctgattggtg aaaagttagg ccacaaggtc tatatcgttt tagagaaact 540 gtctgagctt aagatggtat taggctgaatc taaacgtctg ggcgtgacac cgcgtttagg 600 tctacgtgcg cgtttagcgt tccaaggcaa aggcaagtgg caggcgagtg gcggtgaaaa 660 gtcgaaattt ggtttatctg cagcgcaaat tttaaccgtg gttgaccagt taaaacaaaa 720 cgatatgctg gattcgttgc aattgctgca tttccattta ggttcgcaaa ttgccaatat 780 tcgtgatatt cgccaaggtg taagcgaagc gggtcgtttc tactgtgaat tacgtgagtt 840 gggagcaagc gttaattgct tcgacgtggg tggtggttta gcggtggatt acgatggtac 900 tcgcagccaa agtaataatt caatgaatta tggcctcacc gaatacgcca acaatatcgt 960 taatgtattg actgatatct gtaatgaata tgcgcagcct atgccacgta ttatttcaga 1020 gtctggacgt tatttaacgg cacatcacgc cgtattgatc acggatgtga tcggtacaga 1080 ggcttaccaa cctgaaaata ttcaaccgcc agcggaagag tcaccccagt tattgcacaa 1140 catgtggcat tcatggtctg agatcagcgg ccgtgctgat caacgcgctt tgattgagat 1200 ttatcatgat agccaaagtg acttacaaga agcgcagtca ctgtttgctt taggtcagtt 1260 aagtttggct gaacgtgcgt gggctgagca ggccaatcta cgtgtttgtc atgaagttca 1320 agggttgttg agcactaaaa accgttatca cagaccgatt attgacgaac tcaatgaaaa 1380 gttggctgat aagttcttcg taaacttctc gctattccag tcattaccag atgcttgggg 1440 tattgatcaa gtgttccctg tattgccgct ttcgggatta gataaagcgc cagagcgccg 1500 cgctgtgatg ctggatatta cctgtgactc tgacggtatt gttgatcaat atgtcgatgg 1560 tcaagggatt gaaaccacgc taccagtacc agcatggagc gctgaaagtc catatttgat 1620 tggtttcttc cttgtgggag cttatcaaga aattttgggt gatatgcaca atctttttgg 1680 tgataccaac tccgcggtag ttcggattga agagaatggt gtcacgaata ttgaatctgt 1740 gctagcgggt gatacggttg ctgacgtatt gcgttacgtt aacttagatg cggtggcctt 1800 tatgcgcact tacgaagagc tagtgaattt acatattgaa gaagatgaac gcgcacaaat 1860 tttagaagaa ctacaagttg gccttaaagg ttacacttat ttagaagatt tttctggccg 1920 cggcgatgat gatgatgatg atgatgatta agtacc 1956 <210> 4 <211> 366 <212> DNA <213> Artificial Sequence <400> 4 atatgagcac tctttccact atccctacag tgttatggct tgaacaatca cgaaacaata 60 attggtacgt acgatctttc agccgactca aacatcaaat cttacaaatg tagtctttga 120 aagtattaca tatgtaagat ttaaatgcaa ccgttttttc ggaaggaaat gatgacctcg 180 tttccaccgg aattagcttg gtaccagcta ttgtaacata atcggtacgg gggtgaaaaa 240 gctaacggaa aagggagcgg aaaagaatga tgtaagcgtg aaaaattttt tatcttatca 300 cttgacattg gaagggagat tctttataat aagaatgtgg aattgtgagc ggataacaat 360 ttcaac 366 <210> 5 <211> 3461 <212> DNA <213> Artificial sequence <400> 5 ccatcctcca aagttggaga gtgagtttta tgtcgcaaat attaatgttt ctggtgaacc 60 ttatcaaatt ttcgttgatt taatagaaac atagcggtaa aattagcagt aacttaatag 120 aacggaaatg aaaaaagcca ctctcatatg agcactcttt ccactatccc tacagtgtta 180 tggcttgaac aatcacgaaa caataattgg tacgtacgat ctttcagccg actcaaacat 240 caaatcttac aaatgtagtc tttgaaagta ttacatatgt aagatttaaa tgcaaccgtt 300 ttttcggaag gaaatgatga cctcgtttcc accggaatta gcttggtacc agctattgta 360 acataatcgg tacgggggtg aaaaagctaa cggaaaaggg agcggaaaag aatgatgtaa 420 480 tgtggaattg tgagcggata acaatttcaa ctcaactgtt tactaaaaat cagtttcatc 540 aagcaatgaa acacgccaaa gtaaacaatt taagtaccat tacttatgag caagtattgt 600 ctatttttaa tagttatcta ttatttaacg ggaggaaata attctatgag tcgctttttt 660 aaatttggaa agttacacgt tactaaaggg aatggagata aattattaga tatactactg 720 acagcttcca agaaggtaaa gaggtcccta gcgcctacgg ggaatttgta tcgggattga 780 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttatccctt ttttgcggca 840 ttttgccttc ctgttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 900 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 960 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 1020 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct 1080 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 1140 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 1200 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat 1260 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 1320 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta 1380 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 1440 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 1500 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 1560 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 1620 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata 1680 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 1740 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 1800 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 1860 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 1920 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg 1980 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 2040 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 2100 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 2160 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 2220 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 2280 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 2340 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 2400 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 2460 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc 2520 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc 2580 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat 2640 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt 2700 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt 2760 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat 2820 tacgccaagc ttgcatgcct gcaggtcgac tctagaggat ccccgggtac cgagctcgaa 2880 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa 2940 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga 3000 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct 3060 ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc 3120 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg 3180 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat 3240 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg 3300 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt 3360 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaatac cctgataaat gcttcaata t <210> 6 <211> 5378 <212> DNA <213> The snowstorm <400> 6 ccatcctcca aagttggaga gtgagtttta tgtcgcaaat attaatgttt ctggtgaacc ttatcaaatt ttcgttgatt father atagcggtaa aattagcagt aacttaatag aacggaatg aaaaaagcca ctctcatatg agcactcttt ccactatccc tacagtgtta 240. tggcttgaac aatcacgaaa caataattgg tacgtacgat ctttcagccg actcaaacat caaatcttac aaatgtagtc tttgaaagta ttacatatgt aagatttaaa tgcaaccgtt ttttcggaag gaaatgatga cctcgtttcc accggaatta gcttggtacc agctattgta acataatcgg tacgggggtg aaaaagctaa cggaaaaggg agcggaaaag aatgatgtaa gcgtgaaaaa ttttttatct tatcacttga cattggaagg gagattcttt father tgtggaattg tgagcggata acaatttcaa ctcaactgtt tactaaaaat cagtttcatc aagcaatgaa acacgccaaa gtaaacaatt taagtaccat tacttatgag caagtattgt 600 ctatttttaa tagttatcta tattttaacg ggaggaata attctatgag tcgctttttt 660 aaatttggaa agttacacgt tactaaaggg atggagata attattaga tatactactg 720 acagcttcca agaaggtaaa gaggtcccta gcgcctacgg ggaatttgta tcgggattga 780 aaaaggaaga gtatgagtat tcacattc cgtgtcgccc ttattcctt tttgcggca 840 ttttgccttc ctgtttttgc tcaccagaa acgctggtga aagtaaaga tgctgaagat 900 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 960 agttttcgcc ccgaagaacg tttccaatg atgagcactt ttaagttct gctatgtggc 1020 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct 1080 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 1140 gtaagagaat tatgcagtgc tgccataacc atgagtgata acacctgcggc caacttactt 1200 ctgacaacga tcggaggacc gaaggagcta accgcttt tgcacacat gggggatcat 1260 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 1320 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta 1380 cttactctag cttccccggca acaattaata gactggatgg aggcggataa agttgcagga 1440 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 1500 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 1560 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 1620 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata 1680 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatcctttt 1740 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 1800 gtagaaaaga tcaaaggatc ttcttgagat ccttttttc tgcgcgtaat ctgctgcttg 1860 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 1920 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg 1980 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 2040 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 2100 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 2160 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 2220 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 2280 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 2340 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 2400 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 2460 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc 2520 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc 2580 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat 2640 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt 2700 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt 2760 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat 2820 tacgccaagc ttgcatatga atgattggtc tattgatgat gctcgtgctg ggtacaatgt 2880 tactcactgg agccaaggtt tttatgggat tagcgatcaa ggtgaggtga cagtgtcgcc 2940 ggatcctaaa aatcctgaat acaagattgg cttaaatgag ctagccaaag acatggttaa 3000 agcgggggtc gctttacctg tactagttcg ttttccgcag atcctgcacc acagagtgaa 3060 cagtttgtgc caagcattcg accaagcgat acagaagtat gagtatcagg cggactattt 3120 gttggtttac ccgattaaag ttaaccaaca acaaactgtg gtggaagaga tcctagcgag 3180 tcaggcatca aaagaagttc cgcaactggg tttagaagcg ggcagtaagc ctgaattgat 3240 ggctgtgctc gcgatggcac aaaaagccag ttcagtgatt gtttgtaatg gttataaaga 3300 taacgaatat attcgcttag cactgattgg tgaaaagtta ggccacaagg tctatatcgt 3360 tttagagaaa ctgtctgagc ttaagatggt attagctgaa tctaaacgtc tgggcgtgac 3420 accgcgttta ggtctacgtg cgcgtttagc gttccaaggc aaaggcaagt ggcaggcgag 3480 tggcggtgaa aagtcgaaat ttggtttatc tgcagcgcaa attttaaccg tggttgacca 3540 gttaaaacaa aacgatatgc tggattcgtt gcaattgctg catttccatt taggttcgca 3600 aattgccaat attcgtgata ttcgccaagg tgtaagcgaa gcgggtcgtt tctactgtga 3660 attacgtgag ttgggagcaa gcgttaattg cttcgacgtg ggtggtggtt tagcggtgga 3720 ttacgatggt actcgcagcc aaagtaataa ttcaatgaat tatggcctca ccgaatacgc 3780 caacaatatc gttaatgtat tgactgatat ctgtaatgaa tatgcgcagc ctatgccacg 3840 tattatttca gagtctggac gttatttaac ggcacatcac gccgtattga tcacggatgt 3900 gatcggtaca gaggcttacc aacctgaaaa tattcaaccg ccagcggaag agtcacccca 3960 gttattgcac aacatgtggc attcatggtc tgagatcagc ggccgtgctg atcaacgcgc 4020 tttgattgag atttatcatg atagccaaag tgacttacaa gaagcgcagt cactgtttgc 4080 tttaggtcag ttaagtttgg ctgaacgtgc gtgggctgag caggccaatc tacgtgtttg 4140 tcatgaagtt caagggttgt tgagcactaa aaaccgttat cacagaccga ttattgacga 4200 actcaatgaa aagttggctg ataagttctt cgtaaacttc tcgctattcc agtcattacc 4260 agatgcttgg ggtattgatc aagtgttccc tgtattgccg cttcgggat tagataaagc 4320 gccagagcgc cgcgctgtga tgctggatat tacctgtgac tctgacggta ttgttgatca 4380 atatgcgat ggtcaaggga ttgaaaccac gctaccagta ccagcatgga gcgctgaaag 4440 tccatatttg attggtttct tccttgtggg agcttatcaa gaaattttgg gtgatatgca 4500 caatcttttt ggtgatacca actccgcggt agttcggatt gaagagaatg gtgtcacgaa 4560 tattgaatct gtgctagcgg gtgatacggt tgctgacgta ttgcgttacg ttaacttaga 4620 tgcggtggcc tttatgcgca cttacgaaga gctagtgaat ttacatattg aagaagatga 4680 acgcgcacaa atttagaag aactacaagt tggccttaaa ggttacactt atttagaaga 4740 ttttctggc cgcggcgatg atgatgatga tgatgatgat taagtaccga gctcgaattc 4800 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg 4860 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg 4920 cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct 4980 tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga 5040 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc 5100 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg 5160 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct 5220 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg 5280 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc 5340 gctcatgaga caataaccct gataaatgct tcaataat 5378

Claims

1. A method for preparing agmatine, characterized in that: The following steps are involved: (1) The pH value of the fermentation broth containing arginine decarboxylase is adjusted to 4.4, and then the broth is decolorized by activated carbon and filtered through a ceramic membrane to obtain a filtrate. The filtrate is subjected to affinity chromatography using a nickel column, and then the arginine slurry is added to the nickel column, and then PLP is added in batches to control the pH value of the liquid in the nickel column to be 4.8-5.2, and the liquid flowing out of the nickel column is collected, which is the agmatine conversion liquid; the pore size of the ceramic membrane is 100 nm, and the pressure is 0.5 MPa; 50 mM PBS is used as a binding buffer when the filtrate is subjected to affinity chromatography using a nickel column; the flow rate of the arginine slurry is 50 cm / h; the amount of the PLP added is 0.03-0.04 wt% of the arginine slurry; The fermentation broth of the arginine decarboxylase is prepared by the following method: 1) Preparation of arginine decarboxylase producing bacteria The nucleotide sequence composition of the SpeA gene as shown in SEQ ID NO.1 is codon optimized, and a Gly-Arg-Gly-8His sequence is added to the end of the optimized SpeA gene as shown in SEQ ID NO.2; the optimized SpeA gene, i.e., the target gene, is obtained as shown in nucleotides 5 to 1924 in SEQ ID NO.3; The Pg3 promoter was optimized to obtain an optimized Pg3 promoter as shown in SEQ ID NO.4, the plasmid pHT304 was double-digested with NdeⅠ and HincⅡ, and the optimized Pg3 promoter was integrated into the double-digested plasmid pHT304 to obtain the plasmid pHT304-Pg3 as shown in SEQ ID NO.5; The plasmid pHT304-Pg3 was double-digested with SphⅠ and SacⅠ, and the target gene was integrated into the double-digested plasmid pHT304-Pg3 to obtain a recombinant expression vector as shown in SEQ ID NO.6; 2) Construction of arginine decarboxylase producing bacteria The recombinant expression vector is introduced into Bacillus subtilis to obtain transformants; Spread the transformant on the LB plate, and after a single colony grows, inoculate it on the LB plate containing 50 μg / ml AMP. After a single colony grows on the resistance plate, pick out the single colony grown on the AMP plate and take it as a positive transformant; The positive transformant was inoculated into the fermentation medium; when the OD was 20%, the temperature was lowered to 28°C, and 0.2 mM IPTG was added to induce fermentation; the induction culture time was ensured to be no less than 14 hours, and the fermentation was terminated when the OD increased slowly or not for a long time; the fermentation liquid obtained after the culture was subjected to bacteria breaking treatment, centrifuged at 4000 r / min for 15 minutes, and the supernatant was collected to obtain the fermentation liquid containing arginine decarboxylase; The fermentation medium is composed of: 12 g / L peptone, 8 g / L yeast extract, 3 g / L sodium chloride, 2.5 g / L ammonium sulfate, 4 g / L potassium dihydrogen phosphate trihydrate, 0.3 g / L ammonium ferric citrate, 2.1 g / L citric acid, 10 g / L glycerol, 0.5 g / L magnesium sulfate heptahydrate, 100 ppm kanamycin sulfate, and 0.5 g / L defoamer. Before inoculation, the pH of the fermentation medium is adjusted to 6.9-7.0 with ammonia water. The fermentation culture temperature is 37° C. and the pH is 6.9-7.

0. The preparation method of the arginine slurry is as follows: L-arginine is added to pure water, 30wt% concentrated hydrochloric acid is added to adjust the pH to 5.5-6.0, and the temperature is lowered to 38° C. to obtain the arginine slurry; the concentration of the arginine slurry is 300g / L; (2) The agmatine conversion liquid is subjected to membrane, first decolorization, electrodialysis, first concentration, and first separation to obtain agmatine crude product; the crude product is then redissolved, second decolorized, second concentrated, stirred for crystal growth, and second separated to obtain agmatine fine product, and finally dried to obtain solid agmatine; the pH of the electrodialysis is controlled at 5.7-6.1, and the temperature is controlled below 40°C; the electrodialysis is stopped when the conductivity of the dilute chamber is ≤1000μs / cm, and the dilute water is released; the first concentration is to The fresh water released by electrodialysis is concentrated to 400-450g / L by rotary evaporation; the re-dissolution is as follows: the crude product is re-dissolved at 150g / L, and the re-dissolution temperature is 60°C; the stirring crystallization is as follows: crystallization at room temperature under stirring; during the first separation, the crude product is washed with pure water of 0.5-1 times the volume of the crude product, and the washing liquid is refluxed to the first decolorization for repeated use; during the second separation, the fine product obtained by stirring crystallization is washed with pure water of 1-2 times the volume of the fine product, and the washing liquid is refluxed to the first decolorization for repeated use; The membrane-passing is to filter the agmatine conversion liquid through a layer of diatomaceous earth and a layer of activated carbon in sequence; the amount of the activated carbon accounts for 0.5wt% of the agmatine conversion liquid, and the amount of the diatomaceous earth accounts for 0.5wt% of the agmatine conversion liquid; The first decolorization is as follows: decolorization with activated carbon at 75°C for 40 minutes; the amount of activated carbon used is 1.5wt% of the agmatine conversion solution; the second decolorization is as follows: adding 5-10wt‰ of activated carbon to the heavy solution, decolorizing at 60°C for 30 minutes to obtain a decolorized solution, and filtering the decolorized solution to make the transmittance of the filtrate reach more than 98%; the second concentration is to concentrate the filtrate by rotary evaporation to 400-450g / L.