Application of a spermidine synthase in the preparation of spermidine

Abstract

This invention discloses the application of a spermidine synthase in the preparation of spermidine, wherein propylene diamine and putrescine are used as substrates in the preparation of spermidine; the amino acid sequence of the spermidine synthase is shown in SEQ ID NO:1. The spermidine synthase used in this invention synthesizes spermidine using propylene diamine and putrescine as precursors. This reaction process is simple, has low reaction cost, and produces a high yield of spermidine, showing great promise for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of enzyme catalysis technology, specifically relating to the application of a spermidine synthase in the preparation of spermidine. Background Technology

[0002] Spermidine, also known as spermidine or N-(3-aminopropyl)1,4-butanediamine, is an aliphatic polyamine widely found in living organisms, with the chemical formula C7H. 19 N3, the structural formula is:

[0003]

[0004] Spermidine is present in cells, tissues, and organs, and can bind to negatively charged molecules such as DNA, RNA, and proteins, playing an important role in various stages of cellular life activities. Furthermore, spermidine can induce autophagy, helping the body recycle and reuse damaged organelles, cell membranes, proteins, and other cellular components, thereby influencing the overall cellular life processes. Spermidine also possesses various biological activities, including improving blood pressure, promoting nerve regeneration, and enhancing immune responses. Tobias Eisenberg et al. (Induction of autophagy by spermidine promotes longevity. Nat Cell Biol11, 1305–1314 (2009)) and Yu-Qing Ni et al. (New Insights into the Roles and Mechanisms of Spermidine in Aging and Age-Related Diseases. Aging Dis. 2021; 12(8):1948-1963) also detailed the important roles and possible mechanisms of action of spermidine in health and disease-related aspects.

[0005] However, spermidine is scarce in nature, making it difficult to extract in large quantities from natural resources, and its chemical synthesis involves challenging purification steps. There are two main biosynthetic pathways for spermidine: one is the Sam pathway, which involves the condensation of the precursor putrescine with decarboxylated S-adenosylmethionine (Sam). The other is the Asa pathway, where L-aspartic acid β-semialdehyde (Asa) is transferred as an aminopropyl donor to putrescine to produce the intermediate carboxysemidine, which is then decarboxylated to produce spermidine. Both pathways are costly and have low yields, making industrial-scale production difficult. Therefore, a low-cost, high-yield synthetic method for spermidine is needed. Summary of the Invention

[0006] To address the aforementioned technical problems in the prior art, this invention provides an application of spermidine synthase in the preparation of spermidine. The novel spermidine synthase screened in this invention uses propylenediamine and putrescine as precursors to catalyze the in vitro biosynthesis of spermidine.

[0007] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0008] The first aspect of the present invention provides the application of spermidine synthase in the preparation of spermidine, wherein propylenediamine and putrescine are used as substrates in the preparation of spermidine; the amino acid sequence of the spermidine synthase is shown in SEQ ID NO: 1.

[0009] A second aspect of the present invention provides a method for preparing spermidine, the method comprising: contacting and reacting a spermidine synthase with a substrate to obtain spermidine; wherein the substrate is propylenediamine and putrescine, and the amino acid sequence of the spermidine synthase is shown in SEQ ID NO: 1.

[0010] In some embodiments of the present invention, the reaction further includes the use of a coenzyme and a metal ion, wherein the coenzyme is NAD+. + The metal ion is K + or Ca 2+ .

[0011] In some embodiments of the present invention, the amount of spermidine synthase added is based on the mass of the corresponding wet bacterial cells, and its concentration in the reaction system is 10~100g / L, preferably 40~60g / L, more preferably 40 or 60g / L.

[0012] In some embodiments of the present invention, the molar concentration of putrescine in the reaction system is 1~100mM, preferably 1~80mM, more preferably 6mM or 60mM.

[0013] In some embodiments of the present invention, the molar ratio of putrescine to 1,3-propanediamine is 1:0.5~2, preferably 1:1.

[0014] In some embodiments of the present invention, the molar ratio of putrescine to the coenzyme is 1 to 2000:1, preferably 10 to 1000:1, for example 100:1.

[0015] In some embodiments of the present invention, the molar ratio of putrescine to metal ions is 1~100:50, preferably 6~60:50.

[0016] In some embodiments of the present invention, the pH value of the reaction is 8.0-10, preferably 8.0-9.0.

[0017] In some embodiments of the present invention, the reaction temperature is 30-50°C, preferably 40-48°C.

[0018] In some embodiments of the present invention, the reaction time is 30-90 min, preferably 50-60 min.

[0019] A third aspect of the present invention provides a composition comprising propylenediamine, putrescine, and spermidine synthase; the amino acid sequence of the spermidine synthase is shown in SEQ ID NO: 1.

[0020] In some embodiments of the present invention, the composition further includes a coenzyme and a metal ion, wherein the coenzyme is NAD+. + The metal ion is K + or Ca 2+ .

[0021] A fourth aspect of the present invention provides a reaction end product system, the reaction end product system comprising:

[0022] Propylene diamine;

[0023] putrescine;

[0024] Spermine synthase, the amino acid sequence of which is shown in SEQ ID NO: 1; and

[0025] Spermine.

[0026] In some embodiments of the present invention, the reaction end product system further includes a coenzyme and metal ions; preferably, the coenzyme is NAD. + The metal ion is K + or Ca 2+ .

[0027] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0028] The reagents and raw materials used in this invention are all commercially available.

[0029] The positive and progressive effects of this invention are as follows:

[0030] The method for synthesizing spermidine provided by this invention has a simple reaction process, low reaction cost, and high spermidine yield, and has great potential for industrial application. Detailed Implementation

[0031] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0032] 1. Information on some of the reagents used in this invention is as follows:

[0033] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0034] TB liquid culture medium: tryptone 10 g / L, yeast extract 18 g / L, K2HPO4 12.54 g / L, KH2PO4 2.31 g / L, glycerol 0.4% (v / v).

[0035] Propylene diamine and putrescine (butanediamine) were purchased from Maclean's; cofactor NAD+ + Purchased from Aladdin; other commonly used reagents were purchased from Sinopharm Chemical Reagent Co., Ltd., and plasmid pET28a was purchased from BioWind.

[0036] 2. Spermine synthase activity standard detection system: 1 mM putrescine (butanediamine), 1 mM 1,3-propanediamine, 0.6 mM NAD + The reaction mixture consisted of 50 mM KCl and an appropriate amount of enzyme solution, with a total volume of 1 mL. The reaction buffer was a 50 mM Tris-HCl buffer at pH 8.5. The reaction temperature was 45 °C, and the reaction time was 1 h. The enzyme activity unit (U) refers to the amount of enzyme required per minute to catalyze the conversion of the substrate into 1 μmol of spermidine.

[0037] 3. HPLC detection method:

[0038] Dansyl chloride was used to derive polyamines, with 1,7-heptanediamine selected as the internal standard for detection. Chromatographic column: C18 column (5 μm, 250 mm × 4.6 mm), UV detection wavelength 254 nm, injection volume 10 μL, column temperature 30 °C; mobile phase A: 90% acetonitrile plus 10% ammonium acetate solution containing 0.1% formic acid (0.01 mol / L); mobile phase B: 10% acetonitrile plus 90% ammonium acetate solution containing 0.1% formic acid (0.01 mol / L). Flow rate: 0.8 mL / min. Detection was performed according to the elution program shown in Table 1 below.

[0039] Table 1 Gradient elution program

[0040]

[0041] Example 1 Screening of spermidine synthase

[0042] 1.1 Obtaining the spermidine synthase gene

[0043] Will come from Pseudomonas frederiksbergensis The amino acid sequence of spermidine synthase of the strain (numbered HSS01, NCBI accession number: WP_123358861.1) was obtained by protein sequence BLAST on the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and then phylogenetic analysis was performed using the Uniport website (https: / / www.uniprot.org / align). Based on the analysis results, 9 sequences with high homology to HSS01 were selected and numbered HSS02-10, as shown in Table 2 below.

[0044] Table 2. Sources and NCBI Registry Numbers of Spermine Synthase

[0045]

[0046] 1.2 Construction of spermidine synthase expression strain

[0047] The HSS01-10 sequence was codon-optimized for *E. coli*, and then constructed into the pET28a plasmid to obtain the pET28a-HSS01-10 plasmid (synthesized by Beijing Qingke Biotechnology Co., Ltd.). The synthesized pET28a-HSS01-10 plasmid was transformed into *E. coli* BL21(DE3) competent cells to obtain BL21(DE3) / pET28a-HSS01-10 strains. The specific steps are as follows:

[0048] (1) Preparation of Escherichia coli BL21(DE3) competent cells: The cells were prepared using the rapid competent cell preparation kit from Sangon Biotech (Shanghai) Co., Ltd.

[0049] (2) Plasmid transformation: Take 1 μL (about 100~500 ng) of pET28a-HSS01-10 plasmid and transform it into BL21(DE3) competent cells respectively. Incubate on ice for 30 min. Take out the centrifuge tube and heat shock it in a 42℃ water bath for 60-90 s. Take it out immediately and then incubate on ice for 3-5 min. Then add 800 μL of LB liquid medium to each tube. After shaking culture at 37℃ for 45 min, take out the cultured bacterial solution, centrifuge, remove part of the supernatant, and leave about 100 μL of supernatant to resuspend the bacterial cells. Take the resuspended bacterial solution and spread it on LB plate containing 50 μg / mL kanamycin using a disposable spreader. Invert the plate and incubate at 37℃ overnight. When a single colony grows, the BL21(DE3) / HSS01-10 strains are obtained respectively.

[0050] 1.3 Preparation of crude spermidine synthase solution

[0051] (1) Preparation of fermentation broth: BL21(DE3) / HSS01-10 strains were inoculated into 5 mL LB tubes containing 50 μg / mL kanamycin and cultured at 37℃ and 250 rpm for 5-6 h; 2 mL of the culture broth was transferred to 100 mL TB liquid medium containing 50 μg / mL kanamycin and cultured at 37℃ and 250 rpm for 3-4 h (OD200). 600 (Approximately 0.8~1.2), add 0.1 mM MIPTG to induce protein expression, and culture at 250 rpm and 25℃ for 24 h to obtain fermentation broth expressing HSS01-10.

[0052] (2) Preparation of crude enzyme solution: The fermentation broth obtained above was centrifuged at 10,000 rpm for 10 min at 4 °C to obtain whole-cell wet bacterial cells expressing HSS01-10. The wet bacterial cells were collected, and 50 mM potassium phosphate buffer (pH 7.0) was added to homogenize the wet bacterial cells at a ratio of 1:5 (g / mL) to obtain cell lysate. The cell lysate was centrifuged at 10,000 rpm for 10 min at 4 °C to collect the supernatant, which is the crude enzyme solution.

[0053] (3) Determination of enzyme activity

[0054] The crude enzyme solution of HSS01-10 was tested according to the spermidine synthase activity assay method. The results are shown in Table 3 below:

[0055] Table 3 Results of spermidine synthase activity assay

[0056]

[0057] The enzyme activity test results showed that HSS02 had the highest activity, reaching 180.45 U / mL. HSS02 originated from... Pseudomonas syringae The strain, with NCBI accession number KNH28933.1, was selected for subsequent research on spermidine synthesis.

[0058] Example 2 Synthesis of spermidine

[0059] 2.1 Synthesis of spermidine catalyzed by crude enzyme solution

[0060] An enzyme-catalyzed synthesis system for spermidine using putrescine and 1,3-propanediamine as substrates was prepared. The reaction system consisted of 6 mM putrescine, 6 mM 1,3-propanediamine, and 0.6 mM NAD. +The reaction mixture consisted of 50 mM KCl, 2 mL HSS02 crude enzyme solution (equivalent to 0.4 g of wet bacterial cells), and a total volume of 10 mL. The reaction buffer was 50 mM Tris-HCl at pH 8.5. The prepared reaction mixture was incubated at 45 °C for 1 h. After the reaction was completed, the concentration of spermidine was determined by HPLC, and the concentration was 194.8 mg / L.

[0061] 2.2 Whole-cell catalytic synthesis of spermidine

[0062] A whole-cell synthesis reaction system for spermidine was prepared. The reaction system consisted of 60 mM putrescine, 60 mM 1,3-propanediamine, and 0.6 mM NAD. + The total volume of the reaction mixture was 20 mL, consisting of whole-cell wet bacterial cells of 50 mM KCl and 60 g / L BL21(DE3) / HSS02, with a reaction buffer of 50 mM Tris-HCl at pH 8.5. The mixture was incubated at 45 °C for 12 h. After the reaction, the concentration of spermidine was determined by HPLC to be 3.2 g / L.

[0063] The partial sequences used in this invention are as follows:

[0064] SEQ ID NO: 1 (HSS02)

[0065]

[0066] MSDLEHVIQPFNARLVMIGSGCIGKGVLPLLLRHLEIEPQRLLILSPDDDGRLLAQEFGVSHQAEQLDQSNYKAVLDPLLSAGDFLLNLSVGVSSLSLIQFAQRKGVLYLDTCIEPWE GGYVDPDKSLSERSNYALREAALQLRGESAQGLSTAMLTHGANPGLVSHLVKQALVNLAEDLGDTTPLPASREEWAQLASRLNIRCIHIAERDSQYSSQHKIADEFVNTWSVDGFISEG SQPAELGWGSHEKALPEDGYYHAFGCQAGIYLQRPGASTRVRTWTPSTGPTHGLLVTHNEALSIANYLTLGQGRQPTYRPTVHYAYRPCDDALLSMYELAERNWKPQSGERLLDDEIL DGSDELGVLLMGHARNSYWYGSRLSIQQARALCPYNSATSLQVTAAVLAGVIWAIRNPQRGIVEPDELPFREILQICLPYLGTVEGIYSDWTPLTGRHSLMPEQTDESDPWQFVNVRIT

[0067] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. Use of a spermidine synthetase in the preparation of spermidine, wherein, When preparing the spermidine, propylene diamine and putrescine are used as substrates; the amino acid sequence of the spermidine synthase is shown in SEQ ID NO:

1.

2. A method of preparing spermidine, characterized by, The method includes: contacting and reacting spermidine synthase with a substrate to obtain spermidine; the substrate is propylenediamine and putrescine, and the amino acid sequence of the spermidine synthase is shown in SEQ ID NO:

1.

3. The method of claim 2, wherein, The reaction also includes the use of a coenzyme, which is NAD + , and a metal ion, which is K + or Ca 2+ .

4. The method of claim 3, wherein, The amount of spermidine synthase added is based on the mass of the corresponding wet bacterial cells, and its concentration in the reaction system is 10~100g / L; And / or, the molar concentration of putrescine in the reaction system is 1~100mM, preferably 1~80mM; And / or, the molar ratio of putrescine and 1,3-propanediamine added is 1:0.5~2; And / or, the molar ratio of putrescine to the coenzyme is 1~2000:1, preferably 10~1000:1; And / or, the molar ratio of putrescine to metal ions is 1~100:50, preferably 6~60:

50.

5. The method according to any one of claims 2-4, characterized in that, The pH value of the reaction is 8.0-10, preferably 8.0-9.0; And / or, the reaction temperature is 30-50°C, preferably 40-48°C; And / or, the reaction time is 30-90 min, preferably 50-60 min.

6. A composition, characterized in that, The composition comprises propylenediamine, putrescine, and spermidine synthase; the amino acid sequence of the spermidine synthase is shown in SEQ ID NO:

1.

7. The composition according to claim 6, characterized in that, The composition also includes a coenzyme and a metal ion, the coenzyme being NAD + , the metal ion being K + or Ca 2+ .

8. A reaction end product system, characterized in that, The reaction end product system includes: Propylene diamine; putrescine; Spermine synthase, the amino acid sequence of which is shown in SEQ ID NO: 1; and Spermine.

9. The reaction end product system as described in claim 8, characterized in that, The reaction end product system also includes a coenzyme, which is NAD + , and a metal ion, which is K + or Ca 2+ .

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