Method for preparing butanediamine and spermidine by enzyme method

Abstract

The invention discloses a method for preparing butanediamine and spermidine by an enzyme method, and belongs to the field of bioengineering. The novel arginine decarboxylase mutant is successfully provided, the mutant shows excellent catalytic performance under the alkaline condition, and the specific enzyme activity of the mutant reaches 24.9 U / g and is improved by 8.3 times compared with that of wild type enzyme. The invention develops a two-step synthesis process based on the constructed arginine decarboxylase mutant, and the two-step synthesis process is used for efficiently preparing spermidine. Experimental results show that the process successfully realizes the remarkable increase of the yield of spermidine, and the final yield reaches 200.4 mg / L.

Description

Technical Field

[0001] This invention relates to a method for the enzymatic preparation of butanediamine and spermidine, belonging to the field of bioengineering. Background Technology

[0002] Spermidine is a natural polyamine synthesized from putrescine (PUT), widely present in various organisms and playing important roles in multiple biological processes. As an autophagy inducer, spermidine not only helps extend lifespan but also protects cells from chronic diseases such as cardiovascular disease and neurodegenerative diseases. As a bioactive molecule with multi-target regulatory functions, spermidine from dietary sources can play a crucial role in maintaining gut microbiota balance and immune homeostasis by regulating gut microbiota metabolism and enhancing intestinal barrier function, thus being considered a potential nutritional intervention strategy. Despite the enormous market potential of spermidine, its production still faces certain challenges. Currently, spermidine production methods include chemical synthesis and microbial transformation. However, microbial production of spermidine is low-yield and costly. Industrially, spermidine production primarily uses 1,4-butanediamine and acrylonitrile as raw materials, first undergoing an addition reaction, followed by hydrogenation reduction of the cyano group to obtain spermidine. However, this reaction has a low conversion rate, leaving a large amount of butanediamine residue, which significantly affects the quality of the spermidine product. Furthermore, purification costs are high, hindering industrial production. Especially since spermidine is used as a nutritional supplement, its source must be non-toxic. Therefore, developing efficient biotechnology production processes is crucial for the commercial application of spermidine.

[0003] In vitro enzyme cascade reactions often use purified enzymes, cell lysates, cell-free extracts, or freeze-dried whole cells as catalysts, and then fine-tune the amount of each catalyst in the system to ultimately maximize the throughput and yield of the product. Summary of the Invention

[0004] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: The first objective of this invention is to provide an arginine decarboxylase mutant, wherein the arginine decarboxylase mutant is obtained by mutating histidine at position 730 of the arginine decarboxylase as shown in SEQ ID NO.1 to aspartic acid, glutamic acid at position 467 to lysine, and histidine at position 736 to glutamic acid.

[0005] A second object of the present invention is to provide a gene encoding the arginine decarboxylase mutant or a recombinant vector carrying the gene.

[0006] A third object of the present invention is to provide recombinant cells that express the mutant or carry the gene or the recombinant vector.

[0007] In one embodiment of the present invention, the recombinant cells are bacteria or fungi as host cells.

[0008] A fourth object of the present invention is to provide a recombinant enzyme catalyst containing the said arginine decarboxylase mutant, which is any one of the following forms: (1) Culture recombinant expression transformants containing the arginine decarboxylase mutant and isolate transformant cells containing the recombinant arginine decarboxylase mutant enzyme; (2) Cultivate recombinant expression transformants containing the arginine decarboxylase mutant, isolate transformant cells containing the recombinant arginine decarboxylase mutant enzyme, and break the transformant cells containing the recombinant arginine decarboxylase mutant enzyme to obtain cell lysate. (3) Cultivate recombinant expression transformants containing the arginine decarboxylase mutant, isolate transformant cells containing the recombinant arginine decarboxylase mutant enzyme, break the transformant cells containing the recombinant arginine decarboxylase mutant enzyme, obtain cell lysate, and freeze-dry the cell lysate containing the recombinant arginine decarboxylase mutant enzyme to obtain lyophilized enzyme powder.

[0009] The fifth objective of this invention is to provide an enzymatic method for preparing spermidine, wherein the method uses methionine and arginine as substrates and employs the recombinant enzyme catalyst to catalyze the conversion and synthesis of spermidine.

[0010] In one embodiment of the present invention, the specific steps of the method are as follows: (1) Using arginine as a substrate and the recombinant enzyme catalyst, a reaction solution containing butanediamine was prepared in a system containing guanidine butanease SpeB and arginine decarboxylase SpeA. (2) Add methionine and ATP to the reaction solution of step (1), and prepare spermidine under the catalysis of methionine adenosine transferase, S-adenosine methionine decarboxylase and spermidine synthase.

[0011] In one embodiment of the present invention, in step (1), arginine is 15-35 mmol / L, dithiothreitol is 1-10 mmol / L, EDTA is 1-5 mmol / L, pyridoxal phosphate (PLP) is 0.8-1.2 mmol / L, MgSO4 is 0.08-0.12 mmol / L, guanidine amino acid enzyme SpeB is 1-5 mg / mL, arginine decarboxylase SpeA is 1-5 mg / mL, and the recombinant enzyme catalyst is 1-5 mg / mL.

[0012] In one embodiment of the present invention, in step (2), methionine is 40-60 mmol / L, ATP is 5-15 mmol / L, and Mg... 2+ 15~30 mmol / L, methionine adenosine transferase 1~5 mg / mL, S-adenosylmethionine decarboxylase 1~5 mg / mL, spermidine synthase 1~5 mg / mL.

[0013] In one embodiment of the present invention, the amino acid sequence of guanidine amino acid enzyme SpeB is shown in SEQ ID NO.3, the amino acid sequence of arginine decarboxylase SpeA is shown in SEQ ID NO.4, the amino acid sequence of methionine adenosine transferase is shown in SEQ ID NO.5, the amino acid sequence of S-adenosylmethionine decarboxylase is shown in SEQ ID NO.6, and the amino acid sequence of spermidine synthase is shown in SEQ ID NO.7.

[0014] In one embodiment of the present invention, in step (1), the pH is 7.0~9.0, the temperature is 37~45℃, the reaction time is 6~12 h, and the rotation speed is 200-250 r / min; in step (2), the pH is 7.0-9.0, the temperature is 20~40℃, the reaction time is 6~12 h, and the rotation speed is 200-250 r / min.

[0015] A sixth object of the present invention is to provide the use of the recombinase catalyst, or the method, in the preparation of spermidine or products containing spermidine.

[0016] Beneficial effects (1) This invention successfully provides a novel arginine decarboxylase mutant adiA H730D-E467K-H736E This mutant exhibits superior catalytic performance under alkaline conditions (pH 7.0), with a specific enzyme activity of 24.9 U / g, which is 8.3 times higher than that of the wild-type enzyme (specific enzyme activity of only 3 U / g). This significant improvement not only optimizes the enzyme's catalytic efficiency but also greatly enhances its practicality in enzymatic catalytic systems, providing a stable and efficient enzyme catalyst for the efficient biosynthesis of butanediamine, and has significant potential for industrial applications.

[0017] (2) Based on the constructed arginine decarboxylase mutant adiA H730D-E467K-H736E This invention further develops a two-step synthesis process for the efficient preparation of spermidine. Experimental results show that this process successfully achieved a significant increase in spermidine yield, with a final yield of 200.4 mg / L. Attached Figure Description

[0018] Figure 1 : Catalytic reaction diagram for the preparation of spermidine. Detailed Implementation

[0019] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0020] 1. The culture medium involved in the following examples LB solid culture: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride and 2 g / L agar powder.

[0021] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride.

[0022] SOB medium: 5 g / L yeast extract, 20 g / L peptone, 0.5 g / L sodium chloride, 0.95 g / L MgCl2 and 0.186 g / L KCl.

[0023] 2. The biomaterials involved in the following examples The plasmids pETDuet-adiA, pETDuet-SpeB, and pETDuet-speA-speB are disclosed in the application document with publication number CN117535274A.

[0024] 3. The detection methods involved in the following embodiments (1) Method for detecting arginine decarboxylase activity: Prepare a 200 µL enzyme activity reaction system according to Table 1, and determine the arginine decarboxylase activity. The reaction system was incubated at 37°C for 10 min, and the reaction was terminated by adding 20 µL of 40% trichloroacetic acid. The reaction solution was then cooled in an ice-water bath. After centrifugation at 12000 r / min for 10 min, the supernatant was derivatized and used for HPLC determination of butanediamine yield.

[0025] Unit enzyme activity is defined as U / g = 1 mole butyl diamine / min / g protein.

[0026] The 15 mmol / L potassium sodium phosphate buffer solution was prepared as follows: the pH of 15 mmol / L KH2PO4 was adjusted to 7.0 using 15 mmol / L Na2HPO4.

[0027] Table 1. Reaction system for detecting arginine decarboxylase activity

[0028] (2) HPLC detection of spermidine 1) Sample derivatization and extraction The pre-column derivatization procedure for dansyl chloride is as follows: After centrifuging the fermentation broth or reaction solution at 12000 r / min for 10 min, take 500 μL of the supernatant as the sample. Add 500 μL of saturated NaHCO3 solution and internal standard (5 μL of 10 g / L heptamethylenediamine), mix thoroughly, and adjust the pH to 10 with saturated NaOH solution. Then add 1 mL of the derivatization reagent dansyl chloride (5 g / L, soluble in acetone). Incubate the mixture in a light-protected water bath at 60°C for 30 min, add 2 mL of anhydrous diethyl ether, and extract for 10 min. Collect the upper organic phase. Repeat the extraction operation twice, mix the two organic phases, and dry them with nitrogen to remove the diethyl ether. Dissolve the derivatized compound in 500 μL of acetonitrile solution, filter through a 0.22 μm filter membrane, and use for HPLC detection.

[0029] 2) HPLC chromatographic determination Chromatographic conditions: High-performance liquid chromatography (HPLC) separation of diamine dansyl chloride derivatives was performed on a C18 column at a separation temperature of 30°C and a UV detection wavelength of 254 nm. The injection volume was 10 μL. Mobile phase A was ultrapure water, and mobile phase B was HPLC-grade acetonitrile. Both mobile phases were filtered through a 0.22 μm filter before use. The gradient elution program was set as follows: 0–4 min, 55%–70% B; 4–6 min, 70% B; 6–11 min, 70% B; 11–12 min, 95% B; 12–13 min, 95% B; 13–16 min, 55% B. The total flow rate was set to 0.7 mL / min.

[0030] (3) HPLC detection of butanediamine 1) Sample derivatization and extraction The pre-column derivatization procedure for dansyl chloride was as follows: After centrifuging the fermentation broth at 12000 r / min for 10 min, 500 μL of the supernatant was collected as a sample. 500 μL of saturated NaHCO3 solution and an internal standard (5 μL of 10 g / L heptamethylenediamine) were added and mixed thoroughly. The pH was adjusted to 10 with saturated NaOH solution, followed by the addition of 1 mL of the derivatization reagent dansyl chloride (5 g / L, soluble in acetone). The mixture was incubated in a light-protected water bath at 60°C for 30 min. Extraction was performed for 10 min with 2 mL of anhydrous diethyl ether, and the upper organic phase was collected. This extraction was repeated twice. The two organic phases were mixed and dried using a nitrogen evaporator to remove the diethyl ether. The derivatized compound was dissolved in 500 μL of acetonitrile solution, filtered through a 0.22 μm filter membrane, and then analyzed by HPLC.

[0031] 2) HPLC chromatographic determination Chromatographic conditions: High-performance liquid chromatography (HPLC) separation of diamine dansyl chloride derivatives was performed on a C18 column at a separation temperature of 30°C and a UV detection wavelength of 254 nm. The injection volume was 10 μL. Mobile phase A was ultrapure water, and mobile phase B was HPLC-grade acetonitrile. Both mobile phases were filtered through a 0.22 μm filter before use. The gradient elution program was set as follows: 0–4 min, 55%–70% B; 4–6 min, 70% B; 6–11 min, 70% B; 11–12 min, 95% B; 12–13 min, 95% B; 13–16 min, 55% B. The total flow rate was set to 0.7 mL / min.

[0032] The raw materials used in the following examples: Methionine, adenosine-5'-triphosphate disodium salt (ATP), and MgCl2 were all purchased from Maclean's. Arginine: Beyotime.

[0033] Example 1: Preparation of Arginine Decarboxylase AdiA Mutant The amino acid sequence of the wild-type arginine decarboxylase AdiA is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2. Using the recombinant plasmid pETDuet-adiA as a template, primers containing the mutation site were designed, and full-plasmid PCR was performed on the pETDuet-adiA plasmid using the corresponding primers in Table 2 to construct the mutant recombinant expression plasmid. Through primers adiA -H730D-F and adiA -H730D-R was used to perform full-plasmid PCR on pETDuet-adiA. The PCR product was purified and then used. Dpn The bacteria were digested with enzyme I, and the digested product was then introduced into E. coli JM109. Screening was performed by colony PCR and Sanger sequencing to finally obtain adiA. H730D Mutant recombinant plasmid pETDuet-adiA H730D .

[0034] pass adiA -H736E-F / R adiA -E467K-F / R sequentially applied to pETDuet-adiA H730D Perform whole-plasmid PCR, and use the PCR products after purification. Dpn The bacteria were digested with enzyme I, and the digested product was then introduced into E. coli JM109. Screening was performed by colony PCR and Sanger sequencing to obtain the recombinant plasmid pETDuet-adiA containing H730D, E467K, and H736E mutants. H730D-E467K-H736E .

[0035] Table 2: Primer Sequence Listing

[0036] Example 2: Expression of mutant arginine decarboxylase and purification of AdiA pETDuet-adiA, pETDuet-SpeB, pETDuet-speA-speB, and the mutant plasmid constructed in Example 1 were transformed into Escherichia coli BL21(DE3) to obtain recombinant bacteria carrying guanidine amino acid enzyme SpeB, arginine decarboxylase AdiA, or arginine decarboxylase AdiA mutants, respectively.

[0037] The method for preparing recombinant plasmids expressing the methionine adenosine transferase (metK) gene, spermine synthase (speE) gene, and SAM decarboxylase (speD) gene is as follows: Using primers metK-F and metK-R, from E. coli E. coli The metK gene fragment was amplified from MG1655; using pETDuet-1 plasmid as a template, vector fragment 1 was amplified using primers pET-metK-F and pET-metK-R. Vector fragment 1 and gene fragment metK were ligated together with homologous recombinase to obtain the pET-metK plasmid.

[0038] Using primers speE-F and speE-R, from E. coli E. coli The speE gene fragment was amplified from MG1655; using the correctly sequenced pET-metK plasmid as a template, vector fragment 2 was amplified using primers pET-speE-F and pET-speE-R. Vector fragment 2 and gene fragment speE were ligated together with homologous recombinase to obtain plasmid pET-metK-speE.

[0039] Using primers speD-F and speD-R, from Bacillus subtilis Bacillus subtilis The gene speD was amplified in plasmid 168. Using pRSFDuet-1 plasmid as a template, vector fragment 3 was obtained by amplification using primers pRSF-speD-F and pRSF-speD-R. Vector fragment 3 and gene fragment speD were ligated together with homologous recombinase to obtain plasmid pRSF-speD.

[0040] The amino acid sequence of methionine adenosine transferase metK is shown in SEQ ID NO.5: MAKHLFTSESVSEGHPDKIADQISDAVLDAILEQDPKARVACETYVKTGMVLVGGEITTSAWVDIEEITRNTVREIGYVHSDMGFDANSCAVLSAIGKQSPDINQGVDRADPLEQGAGDQGLMFGYATNETDVLMPAPITYAHRLVQRQAEVRKNGTLPWLRPDAKSQVTFQYDDGKIVGIDAVVLSTQHSEEIDQKSLQEAVMEEIIKPILPAEWLTSATKFFINPTGRFVIGGPMGDCGLTGRKIIVDTYGGMARHGGGAFSGKDPSKVDRSAAYAARYVAKNIVAAGLADRCEIQVSYAIGVAEPTSIMVETFGTEKVPSEQLTLLVREFFDLRPYGLIQMLDLLHPIYKETAAYGHFGREHFPWEKTDKAQLLRDAAGLK The amino acid sequence of spermidine synthase speE is shown in SEQ ID NO.6: MAEKKQWHETLHDQFGQYFAVDNVLYHEKTDHQDLIIFENAAFGRVMALDGVVQTTERDEFIYHEMMTHVPLLAHGHAKHVLIIGGGDGAMLREVTRHKNVESITMVEIDAGVVSFCRQYLPNHNAGSYDDPRFKLVIDDGVNFVNQTSQTFDVIISDCTDPIGPGESLFTSAFYEGCKRCLNPGGIFVAQNGVCFLQQEEAIDSHRKLSHYFSDVGFYQAAIPTYYGGIMTFAWATDNDALRHLSTEIIQARFLASGLKCRYYNPAIHTAAFALPQYLQDALASQPS The amino acid sequence of SAM decarboxylase speD is shown in SEQ ID NO.7; MTVTIKELTNHNYIDHELSATLDSTDAFEGPEKLLEIWFFPHKKSITTEKTLRNIGMDRWIEILKLVKCEVLSMKKTKELDAFLLSESSLFVFDHKLTMKTCGTTTTLFCLEKLFQIVEQELSWAFRTTQGGKYKPFKVFYSRRCFLFPCKQAAIHQNWADEVDYLNKFFDNGKSYSVGRNDKSNHWNLYVTETDRST PKGKEYIEDDDETFEVLMTELDPECASKFVCGPEASTTALVEPNEDKGHNLGYQMTKNTRLDEIYVNSAQDSDLSFHHDAFAFTPCGYSSNMILAEKYY YTLHVTPEKGWSYASFESNIPVFDISQGKQDNLDVLLHILNVFQPREFSMTFFTKNYQNQSFQKLLSINESLPDYIKLDKIVYDLDDYHLFYMKLQKKI Table 3 Primers required for constructing the spermidine pathway

[0041] All recombinant bacteria were first inoculated into 20 mL of LB liquid medium at 50 μL and cultured overnight at 37°C with shaking at 250 r / min as seed culture. Then, the seed culture was inoculated at 1% (v / v) in 500 mL of SOB medium and cultured at 37°C for 3 h. IPTG was then added to a final concentration of 0.1 mmol / L, and the culture was incubated overnight at 30°C to induce protein expression. The overnight cultured cells were collected by refrigerated centrifugation at 8000 rpm for 5 min, resuspended in 50 mL of Tris-HCl buffer, and then sonicated. The supernatant was purified by affinity chromatography using a Ni-NTA Superflow resin column.

[0042] Pure enzyme solutions of wild-type AdiA, guanidine amino acid enzyme SpeB, various mutants, methionine adenosine transferase metK, spermidine synthase speE, and SAM decarboxylase speD were prepared.

[0043] According to the reaction system in Table 1, that is, to the reaction system containing dithiothreitol, EDTA, pyridoxal phosphate (PLP), MgSO4, arginine, and guanidine amino acid enzyme SpeB pure enzyme solution in potassium sodium phosphate buffer, add arginine decarboxylase AdiA or mutant pure enzyme solution, and the final concentration is shown in Table 1.

[0044] The results showed that the enzyme activity detection results of each mutant were different for WT and adiA. H730D adiA H730D-E467K-H736E The enzyme activities were 3 U / g, 9.3 U / g, and 24.9 U / g, respectively.

[0045] Example 3: Production of spermidine catalyzed by multi-enzyme cascade reaction A two-step enzymatic method was used to prepare spermidine. The first step utilized the purified guanidinediamine enzyme SpeB, arginine decarboxylase SpeA, and arginine decarboxylase AdiA (wild-type or mutant) from Example 2 to prepare butanediamine using arginine as a substrate. The second step used methionine adenosyltransferase, S-adenosylmethionine decarboxylase, and spermidine synthase to enzymatically prepare spermidine using methionine as a substrate. Figure 1 The specific steps are as follows: (1) Enzymatic preparation of butanediamine (10 mL catalytic system) The reaction system included arginine 25 mmol / L, dithiothreitol 5 mmol / L, EDTA 3.5 mmol / L, pyridoxal phosphate (PLP) 1 mmol / L, MgSO4 0.1 mmol / L, guanidine aminotransferase SpeB 2 mg / mL, arginine decarboxylase SpeA 2 mg / mL, and arginine decarboxylase AdiA or its mutant adiA. H730D-E467K-H736E 2 mg / mL; the reaction conditions were: initial pH 7.0, 42℃, 250 rpm for 2 h, to obtain a reaction solution containing butanediamine.

[0046] (2) Enzymatic preparation of spermidine (10 mL catalytic system) The reaction system includes 8 mL of the reaction solution containing butanediamine prepared in step (1), 50 mmol / L methionine, 10 mmol / L ATP, and Mg. 2+ The reaction was carried out at 20 mmol / L, with methionine adenosine transferase at 2 mg / mL, S-adenosylmethionine decarboxylase at 3 mg / mL, and spermidine synthase at 2 mg / mL. The reaction conditions were 30℃ and 250 rpm for 2 h to catalyze the synthesis of spermidine.

[0047] Table 4. Spermine production

[0048] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An arginine decarboxylase mutant, characterized in that, The arginine decarboxylase mutant is obtained by mutating histidine at position 730 of the arginine decarboxylase shown in SEQ ID NO.1 to aspartic acid, glutamic acid at position 467 to lysine, and histidine at position 736 to glutamic acid.

2. The gene encoding the arginine decarboxylase mutant of claim 1 or a recombinant vector carrying the gene.

3. A recombinant cell expressing the mutant of claim 1 or carrying the gene of claim 2 or the recombinant vector, characterized in that, The recombinant cells use bacteria or fungi as host cells.

4. A recombinant enzyme catalyst containing the arginine decarboxylase mutant of claim 1, characterized in that, It is any of the following forms: (1) Culture recombinant expression transformants containing the arginine decarboxylase mutant and isolate transformant cells containing the recombinant arginine decarboxylase mutant enzyme; (2) Cultivate recombinant expression transformants containing the arginine decarboxylase mutant, isolate transformant cells containing the recombinant arginine decarboxylase mutant enzyme, and break the transformant cells containing the recombinant arginine decarboxylase mutant enzyme to obtain cell lysate. (3) Cultivate recombinant expression transformants containing the arginine decarboxylase mutant, isolate transformant cells containing the recombinant arginine decarboxylase mutant enzyme, break the transformant cells containing the recombinant arginine decarboxylase mutant enzyme, obtain cell lysate, and freeze-dry the cell lysate of the recombinant arginine decarboxylase mutant enzyme to obtain lyophilized enzyme powder.

5. A method for preparing spermidine by enzymatic process, characterized in that, The method involves using methionine and arginine as substrates and employing the recombinase catalyst described in claim 4 to catalytically convert and synthesize spermidine.

6. The method according to claim 5, characterized in that, The specific steps of the method are as follows: (1) Using arginine as a substrate and employing the recombinant enzyme catalyst described in claim 4, a reaction solution containing butanediamine is prepared in a system containing guanidine butanease SpeB and arginine decarboxylase SpeA. (2) Add methionine and ATP to the reaction solution of step (1), and prepare spermidine under the catalysis of methionine adenosine transferase, S-adenosine methionine decarboxylase and spermidine synthase.

7. The method according to claim 6, characterized in that, In step (1), arginine is 15-35 mmol / L, dithiothreitol is 1-10 mmol / L, EDTA is 1-5 mmol / L, pyridoxal phosphate (PLP) is 0.8-1.2 mmol / L, MgSO4 is 0.08-0.12 mmol / L, guanidine amino acid enzyme SpeB is 1-5 mg / mL, arginine decarboxylase SpeA is 1-5 mg / mL, and the recombinant enzyme catalyst described in claim 4 is 1-5 mg / mL; In step (2), methionine is 40-60 mmol / L, ATP is 5-15 mmol / L, and Mg... 2+ 15~30 mmol / L, methionine adenosine transferase 1~5 mg / mL, S-adenosylmethionine decarboxylase 1~5 mg / mL, spermidine synthase 1~5 mg / mL.

8. The method according to claim 6 or 7, characterized in that, The amino acid sequence of guanidine amino acid enzyme SpeB is shown in SEQ ID NO.3, the amino acid sequence of arginine decarboxylase SpeA is shown in SEQ ID NO.4, the amino acid sequence of methionine adenosine transferase is shown in SEQ ID NO.5, the amino acid sequence of S-adenosylmethionine decarboxylase is shown in SEQ ID NO.6, and the amino acid sequence of spermidine synthase is shown in SEQ ID NO.

7.

9. The method according to claim 7, characterized in that, In step (1), the pH is 7.0-9.0, the temperature is 37-45℃, the reaction time is 6-12 h, and the rotation speed is 200-250 r / min; in step (2), the pH is 7.0-9.0, the temperature is 20-40℃, the reaction time is 6-12 h, and the rotation speed is 200-250 r / min.

10. The use of the recombinase catalyst of claim 4, or the method of any one of claims 5 to 9, in the preparation of spermidine or products containing spermidine.

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