A high-spermidine synthase mutant and its application

Abstract

The present invention discloses a high-spermidine synthase mutant and its application, belonging to the technical field of enzyme engineering. In the present invention, the aspartic acid at the 361st position of the amino acid sequence shown in SEQ ID NO.1 is replaced with glutamic acid, glutamine or asparagine, so that the efficiency of the obtained mutant in catalyzing the reaction of propanediamine and putrescine is significantly improved compared with the wild type, and among them, the mutant D361E is improved by 6.85 times. Applying the mutant D361E constructed in the present invention to whole-cell catalysis for synthesizing spermidine, 65.3 g / L of spermidine can be obtained in a 1L whole-cell catalysis system after reacting for 14 h.

Description

Technical Field

[0001] The present invention relates to a high spermidine synthase mutant and its application, belonging to the technical field of enzyme engineering. Background Art

[0002] Spermidine is an aliphatic polyamine widely present in organisms. Spermidine carries a positive charge and easily interacts with negatively charged molecules such as DNA, RNA, and lipids in cells, participating in multiple cellular processes and playing an active role in the prevention and treatment of various diseases such as cardiovascular diseases, neurodegenerative diseases, Alzheimer's disease, and liver adipose tissue inflammation. Moreover, as a natural autophagy inducer, spermidine has certain anti-aging properties. Frank Madeo et al. (Spermidine in health and disease. Science 359eaan2788 (2018)) reviewed the important role of spermidine in aspects related to health and disease.

[0003] The biological synthesis pathway of spermidine requires multiple enzymes to catalyze reactions synergistically, with problems such as complex processes and low yields. Currently, there is no report on the single-enzyme catalyzed synthesis of spermidine at home and abroad. Tait, G.H. (The formation of homospermidine by an enzyme from Rhodopseudomonas viridis. Biochemical Society transactions 7(1):199-201 (1979)) identified a high spermidine synthase from Rhodopseudomonas viridis (same as Blastochloris viridis) that can catalyze the formation of high spermidine from putrescine, and found that propanediamine is a strong competitive inhibitor of this enzyme.

[0004] Sebastian Krossa et al. (Comprehensive Structural Characterization of the Bacterial Homospermidine Synthase - an Essential Enzyme of the Polyamine Metabolism. Scientific Reports 6:19501 (2016)) analyzed the crystal structure of the homospermidine synthase from Blastochloris viridis (hereinafter referred to as BvHSS) and proposed the catalytic reaction process of this enzyme. Since the structures and chemical properties of the 1,3 - diaminopropane and putrescine molecules are extremely similar, but BvHSS only specifically catalyzes the reaction of putrescine. According to the substrate specificity and three - dimensional structural characteristics of BvHSS, site - directed mutagenesis was carried out on the conserved residues of the active center of BvHSS, and it was found that the aspartic acid at position 361 is the key amino acid affecting the catalytic substrate specificity of this enzyme. The obtained single - point mutant D361E has the activity of catalyzing the reaction of 1,3 - diaminopropane and putrescine to generate spermidine. Summary of the Invention

[0005] The present invention provides a homospermidine synthase mutant and its application. This homospermidine synthase mutant can catalyze the reaction of 1,3 - diaminopropane and putrescine to prepare spermidine, and spermidine is a compound of important value in the pharmaceutical and health product industries.

[0006] The present invention provides a homospermidine synthase mutant, which mutates the aspartic acid at position 361 of the wild - type homospermidine synthase derived from Blastochloris viridis into glutamic acid, glutamine or asparagine.

[0007] In one embodiment, the homospermidine synthase mutant is D361E and has the amino acid sequence shown in SEQ ID NO.2.

[0008] In one embodiment, the homospermidine synthase mutant is D361Q and has the amino acid sequence shown in SEQ ID NO.4.

[0009] In one embodiment, the homospermidine synthase mutant is D361N and has the amino acid sequence shown in SEQ ID NO.5.

[0010] In one embodiment, the amino acid sequence of the wild - type homospermidine synthase is as shown in SEQ ID NO.1.

[0011] The present invention provides a gene encoding the homospermidine synthase mutant.

[0012] In one embodiment, the nucleotide sequence of the gene encoding the mutant D361E is as shown in SEQ ID NO.3; the nucleotide sequence encoding the mutant D361Q is as shown in SEQ ID NO.6; the nucleotide sequence encoding the mutant D361N is as shown in SEQ ID NO.7.

[0013] The present invention also provides an expression vector containing the encoding gene.

[0014] The present invention also provides a genetically engineered bacterium expressing the mutant.

[0015] In one embodiment, the genetically engineered bacterium uses Escherichia coli BL21(DE3) as the host and pRSFDuet-1 as the expression vector to express the mutant.

[0016] The present invention also provides a cell catalyst containing the genetically engineered bacterium.

[0017] In one embodiment, the cell catalyst is prepared by the following method: inoculating the genetically engineered bacterium into a fermentation medium and culturing until OD 600 reaches 0.5 - 0.7 and adding IPTG for induction.

[0018] In one embodiment, the fermentation medium uses glucose as the carbon source and yeast powder as the nitrogen source.

[0019] In one embodiment, the fermentation medium contains: peptone, sodium chloride, glucose, yeast powder, disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate.

[0020] In one embodiment, the induction is carried out at 15 - 20 °C.

[0021] In one embodiment, the addition amount of IPTG is 0.4 - 0.8 mM in terms of the final concentration.

[0022] The present invention also provides the application of the high - spermidine synthase mutant or the cell catalyst in catalyzing the preparation of spermidine from propanediamine and putrescine.

[0023] In one embodiment, the application is to use the high - spermidine synthase mutant cells as a catalyst in the presence of cofactors to catalyze the substrates propanediamine and putrescine to react and generate spermidine.

[0024] In one embodiment, the cofactor is NAD + .

[0025] In one embodiment, the cell concentration of the cell catalyst in the reaction system is ≥20 g / L.

[0026] In one embodiment, the concentration ratio of propanediamine to putrescine is (1-2):1.

[0027] In one embodiment, the reaction temperature is 35-45 °C.

[0028] Beneficial effects:

[0029] In the present invention, the high spermidine synthase BvHSS derived from Blastochloris viridis was rationally designed and modified. Propanediamine is a strong competitive inhibitor of BvHSS. After modification, a high spermidine synthase mutant that can catalyze the reaction of propanediamine and putrescine to produce spermidine was obtained. The specific enzyme activity of mutant D361E is 28.7 U / mg, and whole-cell catalysis can produce 65.3 g / L of spermidine. Description of the drawings

[0030] Figure 1 It is a polyamine molecule reaction that mutant D361E can catalyze.

[0031] Figure 2 It is the SDS-PAGE protein electrophoresis result diagram of the wild type of BvHSS and mutant D361E.

[0032] Figure 3 It is the optimal temperature and optimal pH for the enzymatic catalysis reaction of mutant D361E.

[0033] Figure 4 It is the optimization of the best growth conditions of mutant D361E strain and the optimization of the best conditions for induction expression.

[0034] Figure 5 It is the optimization of the best conditions for whole-cell conversion of mutant D361E and the amount of spermidine produced by conversion in a 5 L bioreactor. Specific embodiments

[0035] The experimental methods in the present invention are all conventional methods unless otherwise specified. For gene cloning operations, specific reference can be made to "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al.

[0036] The recombinant Escherichia coli carrying the high spermidine synthase gene involved in the present invention uses the vector pRSFDuet-1 and the host is Escherichia coli BL21(DE3).

[0037] Reagents used in the downstream catalytic process: Propanediamine and putrescine were purchased from Aladdin (Shanghai, China); cofactor NAD +Purchased from Sangon Biotech (Shanghai, China); other common reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. In the text of this application, the three-letter or single-letter expressions of amino acids adopt the amino acid codes specified by IUPAC (Eur. J. Biochem., 138: 9-37, 1984).

[0038] Standard detection system for the enzyme activity of norspermidine synthase: appropriate amount of enzyme solution, 1 mM substrate, 0.06 mM NAD + , the total system is 1 mL, and the reaction medium is 50 mM potassium phosphate buffer at pH 9.0. React at 50 °C for 30 min. The amount of enzyme required to catalyze the conversion of the substrate to produce 1 μmol of spermidine within one minute is defined as one enzyme activity unit (U).

[0039] Table 1 Design of saturated mutation primers

[0040]

[0041]

[0042]

[0043] Example 1 Construction of recombinant bacteria of BvHSS wild type and mutants

[0044] Entrusted Yixin Biotech Co., Ltd. (Shanghai) to provide codon optimization and gene synthesis services. The wild type of BvHSS (NCBI accession number: L77975.1) was synthesized on the pRSFDuet-1 plasmid and carried a His-tag sequence (located at the N-terminus of the protein) for convenient protein purification. The target gene was placed between the restriction enzyme sites BamH I and EcoR I. The pRSFDuet-1-BvHSS-WT recombinant plasmid was obtained and transformed into Escherichia coli BL21(DE3) competent cells.

[0045] Using the pRSFDuet-1-BvHSS-WT recombinant plasmid as a template, 19 pairs of upstream and downstream primers containing mutation sites (Table 1) were designed, and site-directed saturation mutagenesis of aspartic acid at position 361 was performed by whole plasmid PCR.

[0046] Among them, the reaction system of PCR is as follows:

[0047] Table 2 PCR reaction system

[0048]

[0049] PCR program: 1) Pre-denaturation at 94 °C for 2 min; 2) Denaturation at 98 °C for 10 s, annealing at 58 °C for 10 s, extension at 72 °C for 30 s, 33 cycles; 3) Extension at 72 °C for 5 min.

[0050] After verification by agarose gel electrophoresis, the PCR products were digested with restriction endonuclease DpnⅠ.

[0051] The digestion system is: PCR product 7μL, Buffer 2μL, DpnⅠ1μL, ddH 2 O 10 μL. Incubate in 37°C water bath for 30 min.

[0052] Finally, the digestion product was transformed into Escherichia coli BL21 (DE3) competent cells using the heat shock method and cultured overnight in a 37°C incubator. Single colonies grown on Kana resistance plates were selected, and colony PCR was used to verify that the transformants contained the target gene. The plasmids of the corresponding transformants were extracted and sent to Yixin Biotechnology (Shanghai) Co., Ltd. for sequence determination, and the corresponding 19 single-point mutant recombinant bacteria were preserved.

[0053] Example 2 Microbial culture and preparation of crude enzyme solution

[0054] (1) Cultivation of microorganisms

[0055] LB liquid medium (basic fermentation medium) composition: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L, dissolved in deionized water and fixed to volume, LB solid medium was additionally added with agar powder 20g / L, sterilized at 121°C for 20min, and set aside.

[0056] E. coli BL21 (DE3) containing the relevant gene was inoculated into 5 mL LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 200 rpm and 37°C. 1 mL of the culture was transferred to 50 mL fresh LB medium containing 50 μg / mL kana and cultured at 200 rpm and 37°C until OD 600 Add isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.2 mM to induce protein expression, and culture at 200 rpm and 20°C for 24 h to obtain the bacterial concentration OD 600 The fermentation liquid is about 1.8.

[0057] (2) Preparation of crude enzyme solution

[0058] After the cultivation in step (1) was completed, the fermentation broth was centrifuged at 4°C and 8000 rpm for 10 min to collect the cells. Approximately 0.4 g of cells were obtained from 50 mL of the fermentation broth, and they were resuspended in 15 mL of 50 mM potassium phosphate buffer (pH 7.0). The cells were disrupted by an ultrasonic cell disruptor on ice. The supernatant was collected by centrifugation at 4°C and 8000 rpm for 10 min, and the resulting supernatant was the crude enzyme solution of the corresponding enzyme. After measurement, the enzyme activity of the crude enzyme solution was 36.71 U / mL.

[0059] Example 3 Isolation, Purification and Enzyme Activity Determination of Enzyme

[0060] (1) Isolation and Purification of Enzyme

[0061] Use BeaverBeads TM Purify the crude enzyme solution with His-tag Protein Purification nickel ion chelating magnetic beads. According to the steps described in the product manual, first perform a pretreatment operation on the magnetic beads, wash the magnetic beads twice repeatedly with Binding Buffer (20 mM phosphate buffer containing 5 mM imidazole and 500 mM NaCl), then mix the crude enzyme solution with the magnetic beads to bind the target protein to the magnetic beads. Then, elute the impurity proteins with Washing Buffer (20 mM phosphate buffer containing 50 mM imidazole and 500 mM NaCl), and finally elute the target protein with Elution Buffer (20 mM phosphate buffer containing 200 mM imidazole and 500 mM NaCl) and desalt the purified protein with a desalting column.

[0062] (2) Enzyme Catalytic Reaction System and Enzyme Activity Determination Method

[0063] The enzyme-catalyzed reaction was carried out in a 50 mM potassium phosphate buffer solution with a total volume of 1 mL and pH = 8.8, which contained 1 mM each of the substrates propylenediamine and putrescine, 0.6 mM of the coenzyme NAD + and an appropriate amount of enzyme solution. The reaction was carried out at 37°C for 30 min, and 50 μL of trichloroacetic acid was added to terminate the reaction. Derive polyamines with dansyl chloride. Select 1,7-heptanediamine (used at a concentration of 0.1 mg / mL) as the internal standard reference substance. Add 1 mL of saturated sodium bicarbonate solution, 100 μL of sodium hydroxide solution (1 mol / L) and 1 mL of dansyl chloride derivative reagent (10 mg / mL) to the samples respectively. Derive in a 60°C dark water bath for 30 min, add 1 mL of ether for extraction. After the solution was layered, absorb the upper organic phase, blow dry the ether completely with a nitrogen blowing instrument in a 40°C water bath, add 1 mL of acetonitrile to dissolve in the test tube, dilute by a certain multiple, filter with a 0.22 μm organic filter membrane and then perform HPLC liquid phase detection.

[0064] For the liquid-phase detection of polyamines, a C18 chromatographic column (column length 250 mm, column inner diameter 4.6 mm, column packing particle size 5 μm) is selected. The ultraviolet detection wavelength is 254 nm, the injection volume is 10 μL, the column temperature is 30 °C, mobile phase A is 90% acetonitrile plus 10% 0.01 mol / L ammonium acetate solution containing 0.1% formic acid; mobile phase B is 10% acetonitrile plus 90% 0.01 mol / L ammonium acetate solution containing 0.1% formic acid. The flow rate is set at 0.8 mL / min, and the gradient elution program is as follows: 0 min, 60% A - 40% B; 22 min, 85% A - 15% B; 25 min, 100% A - 0% B; 32 min, 100% A - 0% B; 32.01 min, 60% A - 40% B; 37 min, 60% A - 40% B.

[0065] (3) Determination of the optimum pH and optimum temperature of the enzyme

[0066] The activity of D361E was measured at different temperatures (30 °C - 60 °C). As shown in Figure 3 Figure A, between 30 - 55 °C, the enzyme activity of the mutant D361E increased with the increase of temperature, reached about 20 U / mg between 45 - 50 °C, and remained relatively stable. When the temperature was higher than 50 °C, the enzyme activity decreased sharply. Therefore, the optimum reaction temperature of the mutant D361E was determined to be 50 °C.

[0067] To determine the optimum pH for the catalytic reaction of D361E, buffer solutions NaH 2 PO 4 -Na 2 HPO 4 (pH 6.0 - 8.0, 100 mM), Tris-HCl (pH 7.0 - 9.0, 100 mM), and Gly-NaOH (pH 8.5 - 10.0, 100 mM) were prepared respectively. 1 mM propylenediamine and 1 mM putrescine, 0.6 mM coenzyme NAD + and an appropriate amount of enzyme solution were added to each buffer system, and the enzyme activity was measured. The results showed that when the mutant D361E was at pH 8 - 9.5, the specific enzyme activity was above 15 U / mg. When pH = 9, the enzyme activity of D361E reached 20 U / mg, which was the optimum reaction pH value.

[0068] Under the optimum catalytic reaction conditions of D361E, when the concentration of one of the two substrates, propylenediamine and putrescine, was fixed at 1 mM, the concentration of the other substrate was sequentially set from low to high, and the catalytic activity of D361E was measured. The kinetic parameters K m and k cat, statistical analysis was performed using the Graphpad Prism 8 software package, and the Km values of D361E for propanediamine and putrescine were both 0.49, and the enzyme catalytic efficiency Kcat / Km value was 44.9 mM -1 S -1 , which was 7 times higher than the catalytic efficiency of the wild-type BvHSS.

[0069] Example 4 Optimization of the culture conditions of recombinant Escherichia coli D361E

[0070] (1) Optimization of the culture medium components

[0071] To obtain the optimal growth medium for the recombinant strain, the components of its culture medium were optimized from three aspects: carbon source, nitrogen source, and inorganic salts. Based on the fermentation basal medium, glucose, glycerol, sucrose, maltose, soluble starch, lactose, and fructose at 20 g / L were used as carbon sources respectively; peptone, beef extract, yeast powder, potassium nitrate, ammonium sulfate, ammonium chloride, and urea at 15 g / L were used as nitrogen sources respectively; disodium hydrogen phosphate, sodium chloride, sodium sulfate, potassium chloride, potassium dihydrogen phosphate, potassium carbonate, magnesium chloride, and magnesium sulfate at 10 g / L were used as inorganic salts for the optimization of the culture medium components. The recombinant expression strain D361E was inoculated at an inoculum size of 5%, and cultured at 37 °C for 1.5 - 2.5 h until its OD 600 reached about 0.6, and then IPTG with a final concentration of 0.4 mM was added, and induced at 15 °C for 24 h. Since different culture medium compositions may affect the expression of the recombinant enzyme and thus affect the production of spermidine, the OD value of the recombinant strain after induction and the corresponding yield of spermidine catalyzed were measured. The total volume of the reaction system for the whole-cell conversion of recombinant D361E to synthesize spermidine was 5 mL, containing 5 mM propanediamine and 5 mM putrescine at the final concentration, cofactor NAD + 0.6 mM, and the whole cells of recombinant D361E at 20 g / L. The reaction pH = 9, and the reaction was carried out at a constant temperature of 50 °C for 30 min. After the reaction, samples were prepared by dansyl chloride derivatization method and the yield of spermidine was detected by liquid phase.

[0072] The experimental results are as shown in Figure 4 A - C. The optimal carbon source for the growth of D361E strain is glucose, and the optimal nitrogen source is yeast powder. Although inorganic salts affected the OD value of cell growth to a certain extent, they did not have a significant effect on the amount of spermidine catalyzed by the enzyme. Therefore, the inorganic salts in the culture medium components were selected to be a mixture of disodium hydrogen phosphate, potassium carbonate, magnesium sulfate, and sodium chloride.

[0073] (2) Optimization of the induction conditions

[0074] Based on the optimized medium components, by final concentration: peptone 10 g / L, sodium chloride 10 g / L, glucose 25 g / L, yeast powder 25 g / L, disodium hydrogen phosphate 6 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate 3 g / L as the fermentation medium, inoculate the strain expressing D361E at an inoculum size of 5%, and culture at 37 °C until the OD of D361E cells 600 reaches 0.6, then add the inducer IPTG at a final concentration of 0.4 mM, and induce expression at 15, 20, 25, 30, and 35 °C for 24 h respectively. Measure the OD of the strain after induction 600 value and the yield of spermidine produced by the whole-cell catalysis of propanediamine and putrescine. The total volume of the reaction system for the recombinant D361E whole-cell transformation to synthesize spermidine is 5 mL. By final concentration, each of the substrates propanediamine and putrescine is 5 mM, the cofactor NAD + is 0.6 mM, the whole cells of recombinant D361E are 20 g / L, the reaction pH = 9, react at a constant temperature of 50 °C for 30 min. After the reaction, prepare samples by dansyl chloride derivatization method and detect the yield of spermidine by liquid phase. The results are as Figure 4 shown in D. The yield of spermidine at an induction temperature of 15 - 20 °C can reach 1.33 - 1.35 g / L.

[0075] Then, use peptone 10 g / L, sodium chloride 10 g / L, glucose 25 g / L, yeast powder 25 g / L, disodium hydrogen phosphate 6 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate 3 g / L as the fermentation medium, inoculate the strain expressing D361E at an inoculum size of 5%, and culture until the OD of D361E cells 600 reaches 0.6, and then optimize the inducer concentration by adding IPTG at final concentrations of 0.2, 0.4, 0.6, 0.8, 1, and 1.2 mM respectively. Place the bacterial solutions added with different concentrations of inducer at 20 °C for induction expression for 24 h, and measure the OD of the strain after induction 600 value and the yield of spermidine produced by the whole-cell catalysis of propanediamine and putrescine. The total volume of the reaction system for the recombinant D361E whole-cell transformation to synthesize spermidine is 5 mL. By final concentration, each of the substrates propanediamine and putrescine is 5 mM, the cofactor NAD + is 0.6 mM, the whole cells of recombinant D361E are 20 g / L, the reaction pH = 9, react at a constant temperature of 50 °C for 30 min. After the reaction, prepare samples by dansyl chloride derivatization method and detect the yield of spermidine by liquid phase. The results are as Figure 4 shown in E. The optimal IPTG addition amount is 0.6 mM. Under the optimal conditions, the maximum cell density of D361E can reach 12.03 after 24 hours of induction expression. The whole cells catalyze the reaction of excess equimolar substrates (propanediamine and putrescine) for 30 min, and the yield of spermidine can be 1.36 g·L -1 .

[0076] Example 5 Whole-cell catalysis of recombinant Escherichia coli D361E for the production of spermidine from propanediamine and putrescine

[0077] (1) Optimization of the addition amounts of two substrates

[0078] Since in the products of the reaction of D361E catalyzing propanediamine and putrescine, in addition to spermidine, norspermidine is also produced. To promote the enzymatic production of spermidine to a greater extent, different concentration ratios of the two substrates were added to the whole-cell catalysis system respectively, and the addition amounts of the two substrates most favorable for the production of spermidine were determined. The wet whole-cell biomass of D361E was obtained under the optimal growth medium of D361E (10 g / L peptone, 10 g / L sodium chloride, 25 g / L glucose, 25 g / L yeast extract, 6 g / L disodium hydrogen phosphate, 2 g / L potassium dihydrogen phosphate, 3 g / L magnesium sulfate) and the optimal induction conditions (induction temperature 20 °C, IPTG addition amount 0.6 mM, induction time 24 h). The whole-cell catalysis reactions were carried out with the same weight of wet cells respectively to optimize the optimal addition amounts of propanediamine and putrescine. The whole-cell conversion system was: 20 g / L wet cells, coenzyme NAD + 0.6 mM, the addition amount of putrescine was fixed at 3 mM, and propanediamine with final concentrations of 1.5, 3, 4.5, 6, 7.5, and 9 mM was added respectively, and the reaction was carried out at 37 °C for 30 min. The yields of spermidine generated at different addition ratios of the two substrates were detected. The results are as Figure 5 shown in A. When the concentration ratio of propanediamine and putrescine added to the system was 1.5, the yield of spermidine could reach 1.52 g·L -1 .

[0079] (2) Determination of the optimal temperature for whole-cell conversion of D361E

[0080] Based on the aforementioned optimized parameter conditions, the wet whole-cell biomass of D361E was obtained under the optimal growth medium of D361E (10 g / L peptone, 10 g / L sodium chloride, 25 g / L glucose, 25 g / L yeast extract, 6 g / L disodium hydrogen phosphate, 2 g / L potassium dihydrogen phosphate, 3 g / L magnesium sulfate) and the optimal induction conditions (induction temperature 20 °C, IPTG addition amount 0.6 mM, induction time 24 h). Seven completely identical whole-cell conversion systems were prepared: 20 g / L wet cells, coenzyme NAD + 0.6 mM, 4.5 mM propanediamine, 3 mM putrescine, and the reactions were carried out at 7 different temperature conditions from 20 to 50 °C for 30 min respectively. The whole-cell conversion efficiencies corresponding to different temperatures were detected. As Figure 5 shown in B, 40 °C was selected as the optimal temperature for whole-cell conversion, and at this time the yield of spermidine could reach 1.66 g / L.

[0081] (3) Optimize the addition amount of whole cells in the whole-cell catalyzed reaction of D361E

[0082] Based on the above condition optimization, the wet whole cells of D361E were obtained under the optimal growth medium of D361E (10 g / L peptone, 10 g / L sodium chloride, 25 g / L glucose, 25 g / L yeast extract, 6 g / L disodium hydrogen phosphate, 2 g / L potassium dihydrogen phosphate, 3 g / L magnesium sulfate) and the optimal induction conditions (induction temperature 20 °C, addition amount of inducer IPTG 0.6 mM, induction time 24 h). Prepare the whole-cell reaction system: in Tris-HCl buffer with pH 9, containing coenzyme NAD + 0.6 mM, propanediamine 4.5 mM, putrescine 3 mM. The addition amounts of wet whole cells were set to be 10 - 60 g / L respectively, with a gradient of every 10 g of cell mass. The 6 reaction systems were placed at 40 °C for reaction for 30 min, and the content of spermidine generated by the catalytic reaction was detected by liquid phase. As Figure 5 shown in C, when the wet cell concentration is greater than 40 g / L, the content of spermidine in the product no longer increases significantly, and at this time, the content of spermidine in the product can reach 2.0 g / L.

[0083] Example 6: Whole-cell catalysis of propanediamine and putrescine to produce spermidine by recombinant Escherichia coli D361E in a 5 L bioreactor

[0084] Under the optimal growth medium of D361E (10 g / L peptone, 10 g / L sodium chloride, 25 g / L glucose, 25 g / L yeast extract, 6 g / L disodium hydrogen phosphate, 2 g / L potassium dihydrogen phosphate, 3 g / L magnesium sulfate) and the optimal induction conditions (induction OD 600 about 0.6, induction temperature 20 °C, addition amount of inducer IPTG 0.6 mM, induction time 24 h), the wet whole cells of D361E were obtained. The whole-cell transformation experiment was carried out in a 5 L bioreactor. The total volume of the initial transformation system was 1 L, pH was 9, the transformation temperature was 40 °C, 40 g / L of cells were added according to the final concentration, the stirring speed was 400 rpm, the aeration rate was 1.5 L / min, the initial feeding amount of propanediamine was 30 g / L, the initial feeding amount of putrescine was 20 g / L, and then 10 g / L of propanediamine and 10 g / L of putrescine were fed every 2 h respectively, with a total of four feedings. The total feeding amounts of propanediamine and putrescine were 60 g / L and 50 g / L respectively. The results are as Figure 5 shown in D, the yield of spermidine gradually increased with the passage of time. After 14 h, the yield of spermidine reached the maximum value, which was 65.3 g / L.

[0085] Comparative Example 1:

[0086] The specific implementation method is the same as that of Example 1. The enzyme solution was prepared and purified according to the methods of Examples 2 to 3, and the enzyme activity was measured using propylenediamine and putrescine as substrates. The results showed that compared with the wild type, the catalytic abilities of mutants D361Q, D361N, D361S, D361L, D361I, D361W, D361K, D361Y, D361T, D361V and D361A were all significantly improved; however, mutants D361G, D361P, D361F, D361R, D361H, D361C and D361M were inactivated.

[0087] Table 3 Specific enzyme activities of different mutants

[0088]

[0089] Among them, nd indicates that no enzyme activity was detected.

[0090] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A high - spermidine synthase mutant, characterized in that, the aspartic acid at the 361st position of the high - spermidine synthase shown in SEQ ID NO.1 is mutated to glutamic acid, glutamine or asparagine.

2. A gene encoding the high - spermidine synthase mutant according to claim 1.

3. An expression vector containing the gene according to claim 2.

4. A genetically engineered bacterium expressing the mutant according to claim 1.

5. A recombinant Escherichia coli, characterized in that, using Escherichia coli BL21(DE3) as the host and pRSFDuet - 1 as the expression vector to express the mutant according to claim 1.

6. A cell catalyst containing the recombinant Escherichia coli according to claim 5.

7. The cell catalyst according to claim 6, characterized in that, The cell catalyst is prepared by the following method: inoculating the recombinant Escherichia coli into a fermentation medium and culturing until OD 600 reaches 0.5 - 0.7, then adding IPTG for induction.

8. A method for producing spermidine by whole - cell catalysis, characterized in that, using the recombinant Escherichia coli according to claim 5 or the cell catalyst according to any one of claims 6 - 7 to catalyze the preparation of spermidine from propanediamine and putrescine.

9. The method according to claim 8, characterized in that, The cofactor NAD is also added to the catalytic reaction + , and the concentration ratio of propylenediamine to putrescine is (1-2):1, and the reaction temperature is 35-45 °C.

10. Use of the high - spermidine synthase mutant according to claim 1, or the recombinant Escherichia coli according to claim 5, or the cell catalyst according to any one of claims 6 - 7, or the method according to any one of claims 8 - 9 in the production of spermidine or products containing spermidine.

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