CN115820591A - High spermidine synthetase mutant and application thereof - Google Patents

Abstract

The invention discloses a high spermidine synthetase mutant and application thereof, belonging to the technical field of enzyme engineering. According to the invention, the amino acid sequence shown in SEQ ID NO.1, wherein the 361 st aspartic acid is replaced by glutamic acid, glutamine or asparagine, so that the efficiency of the obtained mutant for catalyzing the reaction of the propane diamine and putrescine is obviously improved compared with the wild type, wherein the mutant D361E is improved by 6.85 times. The mutant D361E constructed by the invention is applied to the synthesis of spermidine by whole-cell catalysis, and the spermidine with 65.3g/L can be obtained by 1L of whole-cell catalytic system reaction for 14 h.

Description

High spermidine synthetase mutant and application thereof

Technical Field

The invention relates to a high spermidine synthetase mutant and application thereof, belonging to the technical field of enzyme engineering.

Background

Spermidine is an aliphatic polyamine that is widely found in the body. Spermidine has positive charge, is easy to interact with molecules such as DNA, RNA and lipid with negative charge in cells, participates in multiple cell processes, and plays an active role in preventing and treating cardiovascular diseases, neurodegenerative diseases, senile dementia, liver adipose tissue inflammation and other diseases. And spermidine, as a natural autophagy inducer, has certain anti-aging properties. Frank Madeo et al (Spermidine in health and disease. Science 359eaan2788 (2018)) reviewed the important role of Spermidine in health and disease related aspects.

The method for synthesizing spermidine by a biological method needs a plurality of enzymes to perform concerted catalytic reaction, and has the problems of complex process, low yield and the like. At present, no report of single enzyme catalysis synthesis of spermidine exists at home and abroad. Tait, G.H. (The formation of homoSperidine by an enzyme from Rhodopseudomonas viridis.Biochemical Society transactions 7 (1): 199-201 (1979)) identified a homo-spermidine synthase from Rhodopseudomonas viridis (as well as Blastochloris viridis) that is capable of catalyzing putrescine to produce homo-spermidine, and it was found that propylenediamine is a strong competitive inhibitor of this enzyme.

Sebastian Krossa et al (Comprehensive Structural Characterization of the Bacterial Homoperrimine Synthase-an Essential Enzyme of the Polyamine metabolism. Scientific Reports6:19501 (2016)) analyzed the crystal structure of the high-order spermine Synthase (hereinafter referred to as BvHSS) from Blastochloris viridis and proposed the catalytic reaction process of the Enzyme. Because the structure and chemical properties of the molecules of the propylenediamine and the putrescine are very similar, but the BvHSS only carries out specific catalytic reaction on the putrescine. According to the substrate specificity and the three-dimensional structure characteristics of BvHSS, the invention carries out site-directed mutagenesis on conserved residues of the BvHSS active center, finds that the 361 st aspartic acid is a key amino acid influencing the substrate specificity of the enzyme, and obtains the single-point mutant D361E which has the activity of catalyzing the reaction of propylene diamine and putrescine to generate spermidine.

Disclosure of Invention

The invention provides a high spermidine synthetase mutant and application thereof, the high spermidine synthetase mutant can catalyze propylene diamine and putrescine to react to prepare spermidine, and the spermidine is a compound with important value in the pharmaceutical and health care product industries.

The invention provides a high spermidine synthetase mutant, which is obtained by mutating the 361 st aspartic acid of wild type high spermidine synthetase derived from Blastochloris viridis into glutamic acid, glutamine or asparagine.

In one embodiment, the high spermidine synthetase mutant is D361E, having the amino acid sequence shown in SEQ ID No. 2.

In one embodiment, the high spermidine synthetase mutant is D361Q, having the amino acid sequence shown in SEQ ID No. 4.

In one embodiment, the high spermidine synthetase mutant is D361N, having the amino acid sequence shown in SEQ ID No. 5.

In one embodiment, the amino acid sequence of said wild-type high spermidine synthetase is as shown in SEQ ID No. 1.

The invention provides a gene for coding the high spermidine synthetase mutant.

In one embodiment, the nucleotide sequence of the gene encoding the mutant D361E is set forth in SEQ ID No. 3.

The invention also provides an expression vector containing the coding gene.

The invention also provides a genetic engineering bacterium for expressing the mutant.

In one embodiment, the genetically engineered bacterium expresses the mutant with escherichia coli BL21 (DE 3) as a host and pRSFDuet-1 as an expression vector.

The invention also provides a cell catalyst containing the genetic engineering bacteria.

In one embodiment, the cell catalyst is prepared as follows: inoculating the genetically engineered bacteria into a fermentation medium, and culturing to OD ₆₀₀ IPTG is added for induction at 0.5-0.7.

In one embodiment, the fermentation medium comprises glucose as a carbon source and yeast powder as a nitrogen source.

In one embodiment, the fermentation medium comprises: peptone, sodium chloride, glucose, yeast powder, disodium hydrogen phosphate, potassium dihydrogen phosphate and magnesium sulfate.

In one embodiment, the induction is at 15 to 20 ℃.

In one embodiment, the IPTG is added in an amount of 0.4 to 0.8mM final concentration.

The invention also provides application of the high spermidine synthetase mutant or the cell catalyst in preparation of spermidine by catalyzing propane diamine and putrescine.

In one embodiment, the application is to catalyze the reaction of substrates of propylene diamine and putrescine to generate spermidine by taking a high spermidine synthetase mutant cell as a catalyst in the presence of a cofactor.

In one embodiment, the cofactor is NAD ⁺ 。

In one embodiment, the cell catalyst has a cell concentration in the reaction system of 20g/L or more.

In one embodiment, the concentration ratio of propylenediamine to putrescine is (1-2): 1.

in one embodiment, the reaction temperature is 35 to 45 ℃.

Has the beneficial effects that:

the invention modifies the high spermidine synthetase BvHSS from Blastochloris viridis through rational design, propylene diamine is a strong competitive inhibitor of BvHSS, and the modified high spermidine synthetase mutant which can catalyze the reaction of propylene diamine and putrescine to generate spermidine is obtained. The specific enzyme activity of the mutant D361E is 28.7U/mg, and 65.3g/L spermidine can be generated by whole-cell catalysis.

Drawings

FIG. 1 shows the polyamine molecular reaction catalyzed by mutant D361E.

FIG. 2 is a diagram showing the results of SDS-PAGE of the wild type BvHSS and the mutant D361E.

FIG. 3 shows the optimal temperature and pH for the reaction catalyzed by mutant D361E.

FIG. 4 shows the optimization of the optimal growth conditions and the optimal conditions for induced expression for mutant D361E strain.

FIG. 5 shows the optimization of the optimal conditions for whole-cell transformation of mutant D361E and the amount of spermidine produced in its 5L bioreactor.

Detailed Description

The experimental methods in the present invention are conventional methods unless otherwise specified, and the gene cloning procedures can be specifically described in molecular cloning protocols, compiled by J. Sambruka et al.

The invention relates to a recombinant Escherichia coli with high spermidine synthetase gene, wherein the vector is pRSFDuet-1, and the host is Escherichia coli BL21 (DE 3).

Reagents used in the downstream catalytic process: propylenediamine and putrescine were purchased from alatin (shanghai, china); cofactor NAD ⁺ Purchased from bio-engineering (shanghai, china); other commonly used reagents are available from the national pharmaceutical group chemical agents, ltd. The three-letter or one-letter expression of amino acids used in the present application uses the amino acid code specified by IUPAC (eur.j. Biochem., 138.

The enzyme activity standard detection system of the high spermidine synthetase comprises the following steps: appropriate amount of enzyme solution, 1mM substrate, 0.06mM NAD ⁺ The total volume was 1mL, and the reaction medium was 50mM potassium phosphate buffer, pH 9.0. Reacting at 50 ℃ for 30min, and defining the enzyme quantity required for catalyzing substrate conversion to generate 1 mu mol of spermidine in one minute as one enzyme activity unit (U).

TABLE 1 saturated mutant primer design

Example 1 construction of BvHSS wild type and mutant recombinant bacteria

Entrusted also with the provision of codon optimization and gene synthesis services by Biotechnology (Shanghai) Ltd, bvHSS wild type (NCBI accession number: L77975.1) was synthesized on pRSFDuet-1 plasmid with His tag sequence (at the N-terminus of the protein) for convenient protein purification, and the target gene was placed between the cleavage sites BamH I and EcoR I. The pRSFDuet-1-BvHSS-WT recombinant plasmid was obtained and transformed into E.coli BL21 (DE 3) competent cells.

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

Wherein, the reaction system of PCR is:

TABLE 2PCR reaction System

PCR procedure: 1) Pre-denaturation at 94 ℃ for 2min; 2) Denaturation at 98 ℃ for 10s, annealing at 58 ℃ for 10s, extension at 72 ℃ for 30s, and circulating for 33 times; 3) Extension at 72 ℃ for 5min.

After the PCR product was verified by agarose gel electrophoresis, the PCR product was digested with restriction enzyme Dpn I.

The digestion system is as follows: PCR product 7. Mu. L, buffer 2. Mu. L, dpn I1. Mu. L, ddH ₂ O10. Mu.L. Water bath at 37 deg.c for 30min.

And finally, transforming the digestion product into escherichia coli BL21 (DE 3) competent cells by adopting a heat shock method, carrying out overnight culture in an incubator at 37 ℃, selecting a single colony growing on a Kana resistance plate, verifying that the transformant contains a target gene through colony PCR, extracting plasmids of corresponding transformants, sending the plasmids to Gaixi Biotech (Shanghai) limited company for sequence determination, and respectively storing corresponding 19 single-point mutant recombinant bacteria.

Example 2 microbial culture and preparation of crude enzyme solution

(1) Cultivation of microorganisms

LB liquid medium (fermentation basal medium) composition: 10g/L of peptone, 5g/L of yeast powder and 10g/L of sodium chloride, dissolving with deionized water, fixing the volume, additionally adding 20g/L of agar powder into LB solid culture medium, and sterilizing at 121 ℃ for 20min for later use.

Coli BL21 (DE 3) containing the gene of interest was inoculated into a vaccine containing 50. Mu.g/mL kanamycinThe culture was carried out overnight at 37 ℃ and 200rpm in 5mL LB liquid medium containing the same. 1mL of the culture was transferred to 50mL of fresh LB medium containing 50. Mu.g/mL Kana, and cultured at 37 ℃ at 200rpm to OD ₆₀₀ About 0.6. Adding isopropyl-beta-D-thiogalactoside (IPTG) with final concentration of 0.2mM to induce protein expression, culturing at 200rpm and 20 deg.C for 24 hr to obtain thallus concentration OD ₆₀₀ About 1.8 fermentation broth.

(2) Preparation of crude enzyme solution

The fermentation broth after the end of the culture in step (1) was centrifuged at 8000rpm at 4 ℃ for 10min to collect cells, 50mL of the fermentation broth was used to obtain about 0.4g of cells, which were resuspended in 15mL of 50mM potassium phosphate buffer (pH 7.0), and the cells were disrupted by an ultrasonic cell disrupter on ice. Centrifuging at 4 deg.C and 8000rpm for 10min, collecting supernatant, and determining the activity of crude enzyme solution to be 36.71U/mL.

Example 3 isolation and purification of enzyme and determination of enzyme Activity

(1) Separation and purification of enzyme

Using BeaverBeads ^TM The crude enzyme solution was purified using His-tag Protein Purification nickel ion chelate magnetic beads. According to the procedure described in the product specification, the magnetic beads were first subjected to a pretreatment operation, the magnetic beads were washed twice with a Binding Buffer (20 mM phosphate Buffer solution containing 5mM imidazole and 500mM NaCl), the crude enzyme solution was mixed with the magnetic beads to bind the target protein to the magnetic beads, the Washing Buffer (20 mM phosphate Buffer solution containing 50mM imidazole and 500mM NaCl) was then used to elute the foreign protein, and finally the elusion Buffer (20 mM phosphate Buffer solution containing 200mM imidazole and 500mM NaCl) was used to elute the target protein and the desalting column was used to desalt the pure protein.

(2) Enzyme catalytic reaction system and enzyme activity determination method

The enzyme-catalyzed reaction was carried out in a total volume of 1mL of 50mM potassium phosphate buffer pH =8.8 containing 1mM each of the substrates propylenediamine and putrescine, coenzyme NAD ⁺ 0.6mM and a proper amount of enzyme solution. The reaction was carried out at 37 ℃ for 30min and stopped by adding 50. Mu.L of trichloroacetic acid. Derivatizing polyamines with dansyl chlorideSelecting 1,7-heptanediamine (with the concentration of 0.1 mg/mL) as an internal standard, respectively adding 1mL of saturated sodium bicarbonate solution, 100 mu L of sodium hydroxide solution (1 mol/L) and 1mL of dansyl chloride derivative reagent (10 mg/mL) into a sample, deriving for 30min in a dark water bath at 60 ℃, adding 1mL of diethyl ether for extraction, absorbing an upper organic phase after the solution is layered, blowing off the diethyl ether by a nitrogen blow dryer in the water bath at 40 ℃, adding 1mL of acetonitrile into a test tube for dissolving, diluting by a certain multiple, filtering by a 0.22 mu m organic filter membrane, and performing HPLC liquid phase detection.

Selecting a C18 chromatographic column (the column length is 250mm, the column inner diameter is 4.6mm, and the column packing particle diameter is 5 mu m) for liquid phase detection of polyamine, wherein the ultraviolet detection wavelength is 254nm, the sample injection amount is 10 mu L, the column temperature is 30 ℃, and a mobile phase A is 90% acetonitrile and 10% 0.01mol/L ammonium acetate solution containing 0.1% formic acid; the mobile phase B was 10% acetonitrile plus 90% 0.01mol/L ammonium acetate solution containing 0.1% formic acid. The flow rate was set at 0.8mL/min and the gradient elution program was: 0min,60% of A-40% of B;22min,85% A-15% B;25min,100% A-0% B;32min,100% A-0% B;32.01min,60% A-40% B;37min,60% A-40% B.

(3) Determination of optimum pH and optimum temperature of enzyme

The activity of D361E at different temperatures (30-60 ℃) was measured, and as shown in FIG. 3A, the activity of the mutant D361E increased with increasing temperature between 30-55 ℃ and reached about 20U/mg between 45-50 ℃ and remained relatively stable, with the temperature above 50 ℃ and the activity of the enzyme decreased sharply. Thus, the optimum reaction temperature for mutant D361E was determined to be 50 ℃.

Determining the optimum pH value of D361E catalytic reaction, and respectively preparing buffer solution 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). Add propylene diamine and putrescine to a final concentration of 1mM, 0.6mM coenzyme NAD under each buffer system ⁺ And a proper amount of enzyme solution, and determining the enzyme activity, wherein the results show that the specific enzyme activity of the mutant D361E is above 15U/mg at the pH of 8-9.5, and the enzyme activity of the D361E reaches 20U/mg at the pH =9, which is the optimum reaction pH value.

Under the optimal catalytic reaction condition of D361E, fixing propane diamine and humic acidThe concentration of one of two substrates of amine is 1mM, the concentration of the other substrate is sequentially set from low to high, the catalytic activity of D361E is measured, and the kinetic parameter K is used _m And k _cat Statistical analysis was performed using the Graphpad Prism 8 software package to obtain D361E with Km values of 0.49 for both propylenediamine and putrescine and an enzyme catalytic efficiency Kcat/Km value of 44.9mM ^-1 S ^-1 And the catalytic efficiency is improved by 7 times compared with that of wild BvHSS.

Example 4 optimization of culture conditions for recombinant E.coli D361E

(1) Optimization of media composition

In order to obtain an optimal growth medium for the recombinant strain, the medium composition was optimized in 3 aspects of carbon source, nitrogen source and inorganic salts, respectively. On the basis of a fermentation basal culture medium, respectively taking 20g/L of glucose, glycerol, sucrose, maltose, soluble starch, lactose and fructose as carbon sources; taking peptone, beef extract, yeast powder, potassium nitrate, ammonium sulfate, ammonium chloride and urea of 15g/L as nitrogen sources; optimization of medium components was carried out with 10g/L disodium hydrogen phosphate, sodium chloride, sodium sulfate, potassium chloride, potassium dihydrogen phosphate, potassium carbonate, magnesium chloride and magnesium sulfate as inorganic salts. Inoculating recombinant expression strain D361E with 5% of inoculum size, culturing at 37 deg.C for 1.5-2.5 hr to make its OD ₆₀₀ Reaching about 0.6, then adding IPTG with the final concentration of 0.4mM, inducing and expressing for 24h at 15 ℃, determining the OD value of the recombinant strain after induction and the yield of the corresponding catalytic generated spermidine because different culture medium compositions may influence the expression of recombinase and further influence the production of spermidine, and transforming the recombinant D361E whole cells into 5mL of the total volume of a reaction system for synthesizing spermidine, which contains 5mM propylene diamine and 5mM putrescine according to the final concentration and the cofactor NAD ⁺ 0.6mM, 20g/L of recombinant D361E whole cells, reaction pH =9, reaction at 50 ℃ for 30min, preparation of a sample by dansyl chloride derivatization after the end of the reaction and detection of the production of spermidine by liquid phase.

As shown in FIGS. 4A-C, the optimum carbon source for the growth of the D361E strain is glucose, the optimum nitrogen source is yeast powder, and inorganic salts influence the OD value of the growth of the bacteria to a certain extent, but have no significant influence on the amount of spermidine generated by enzyme catalysis, so that disodium hydrogen phosphate, potassium carbonate, magnesium sulfate and sodium chloride are selected and mixed to be used as the inorganic salts in the components of the culture medium.

(2) Optimization of Induction conditions

Based on the optimized medium components, according to the final concentration: 10g/L peptone, 10g/L sodium chloride, 25g/L glucose, 25g/L yeast powder, 6g/L disodium hydrogen phosphate, 2g/L potassium dihydrogen phosphate and 3g/L magnesium sulfate are used as fermentation culture media, a strain for expressing D361E is inoculated according to the inoculation amount of 5%, and the strain is cultured at 37 ℃ until OD (origin-destination) of D361E bacteria body ₆₀₀ At 0.6, the final concentration of 0.4mM inducer IPTG was added, and expression was induced at 15, 20, 25, 30, and 35 ℃ for 24 hours, respectively. Determination of OD of the Strain after Induction ₆₀₀ Value and the yield of spermidine produced by whole-cell catalysis of propylenediamine and putrescine. The total volume of a reaction system for synthesizing spermidine by transforming recombinant D361E whole cells is 5mL, and according to a final concentration meter, 5mM of each of substrate propylenediamine and putrescine and cofactor NAD ⁺ 0.6mM, 20g/L of recombinant D361E whole cells, reaction pH =9, reaction at 50 ℃ for 30min, preparation of a sample by dansyl chloride derivatization after the end of the reaction and detection of the production of spermidine by liquid phase. As shown in FIG. 4D, the spermidine yield at 15-20 ℃ was 1.33-1.35 g/L.

Then inoculating and expressing D361E strain with 5% inoculation amount by taking peptone 10g/L, sodium chloride 10g/L, glucose 25g/L, yeast powder 25g/L, disodium hydrogen phosphate 6g/L, potassium dihydrogen phosphate 2g/L and magnesium sulfate 3g/L as fermentation medium, and culturing until D361E thallus OD ₆₀₀ At 0.6, IPTG was added to the mixture at final concentrations of 0.2, 0.4, 0.6, 0.8, 1 and 1.2mM, respectively, to optimize the concentration of the inducer. Placing the bacterial liquid added with different concentrations of inducer at 20 deg.C for inducing expression for 24h, and measuring OD of strain after induction ₆₀₀ And the yield of spermidine by whole-cell catalysis of propylene diamine and putrescine. The total volume of a reaction system for synthesizing spermidine by transforming recombinant D361E whole cells is 5mL, and according to a final concentration meter, 5mM of each of substrate propylenediamine and putrescine and cofactor NAD ⁺ 0.6mM, 20g/L of recombinant D361E whole cells, reaction pH =9, constant temperature reaction at 50 ℃ for 30min, preparation of samples by dansyl chloride derivatization after the end of the reaction and passage through a liquid phaseAnd detecting the production of spermidine. As shown in FIG. 4E, the optimum IPTG addition was 0.6mM, the maximum cell density reached by induced expression of D361E for 24 hours was 12.03 under the optimum conditions, and the yield of spermidine was 1.36 g.L. ^-1 。

Example 5 recombinant E.coli D361E Whole cell catalysis of propylenediamine and putrescine to spermidine

(1) Optimizing the addition of two substrates

Because the products of the reaction of the propane diamine and the putrescine catalyzed by the D361E have high spermidine generation besides spermidine, in order to promote enzyme catalysis to generate spermidine to a greater extent, two substrates with different concentration ratios are respectively added into a whole-cell catalytic system, and the addition amount of the two substrates which are most beneficial to spermidine generation is determined. The wet cells of D361E were obtained in an optimal growth medium (10 g/L peptone, 10g/L sodium chloride, 25g/L glucose, 25g/L yeast powder, 6g/L disodium hydrogenphosphate, 2g/L potassium dihydrogenphosphate, 3g/L magnesium sulfate) and optimal induction conditions (induction temperature 20 ℃, addition of 0.6mM inducer IPTG, and induction time 24 hours) for D361E. Wet cells with the same weight are respectively used for whole cell catalytic reaction, and the optimal addition amount of the propylene diamine and the putrescine is optimized. The whole cell transformation system is as follows: wet cell 20g/L, coenzyme NAD ⁺ 0.6mM, the amount of putrescine added was fixed to 3mM, and propylenediamine was added to the reaction mixture at final concentrations of 1.5, 3, 4.5, 6, 7.5 and 9mM, respectively, and the reaction was carried out at 37 ℃ for 30min. And detecting the yield of spermidine generated when the two substrates are added at different ratios. As shown in FIG. 5A, when the concentration ratio of propylenediamine to putrescine added to the system was 1.5, the yield of spermidine was 1.52 g.L ^-1 。

(2) Determination of optimal temperature for transformation of D361E whole cells

Based on the optimized parameter conditions, the whole cell wet bacteria of D361E is obtained under the optimal growth medium (10 g/L of peptone, 10g/L of sodium chloride, 25g/L of glucose, 25g/L of yeast powder, 6g/L of disodium hydrogen phosphate, 2g/L of potassium dihydrogen phosphate and 3g/L of magnesium sulfate) and optimal induction conditions (the induction temperature is 20 ℃, the addition amount of an inducer IPTG is 0.6mM, and the induction time is 24 h) of D361EAnd (3) a body. 7 groups of identical whole-cell transformation systems were prepared: wet cells 20g/L, coenzyme NAD ⁺ 0.6mM, 4.5mM of propylene diamine and 3mM of putrescine, and reacting for 30min at 7 different temperatures of 20-50 ℃. And detecting the whole cell transformation efficiency corresponding to different temperatures. As shown in FIG. 5B, 40 ℃ was selected as the optimum whole cell transformation temperature, and the production of spermidine reached 1.66g/L.

(3) Optimizing the whole-cell addition amount of D361E whole-cell catalytic reaction

Based on the optimization of the conditions, the whole-cell wet thalli of D361E is obtained under the optimal growth culture medium (10 g/L of peptone, 10g/L of sodium chloride, 25g/L of glucose, 25g/L of yeast powder, 6g/L of disodium hydrogen phosphate, 2g/L of monopotassium phosphate and 3g/L of magnesium sulfate) and the optimal induction conditions (the induction temperature is 20 ℃, the addition amount of an inducer IPTG is 0.6mM, and the induction time is 24 h). Preparing a whole-cell reaction system: in Tris-HCl buffer solution with pH 9, coenzyme NAD is contained ⁺ 0.6mM, 4.5mM of propylene diamine and 3mM of putrescine, wherein the addition amount of the whole-cell wet thalli is respectively set to be 10-60g/L, and the thalli amount of each 10g is set as a gradient. And (3) placing the 6 reaction systems at 40 ℃ for reacting for 30min, and detecting the content of spermidine generated by catalytic reaction in a liquid phase. As shown in FIG. 5C, when the wet cell concentration is more than 40g/L, the spermidine content in the product does not rise significantly any more, and the spermidine content in the product can reach 2.0g/L.

Example 6 recombinant E.coli D361E was used to catalyze whole cells of propane diamine and putrescine to produce spermidine in a 5L bioreactor

Optimal growth medium (peptone 10g/L, sodium chloride 10g/L, glucose 25g/L, yeast powder 25g/L, disodium hydrogen phosphate 6g/L, potassium dihydrogen phosphate 2g/L, magnesium sulfate 3 g/L) and optimal induction conditions (OD of induction) at D361E ₆₀₀ About 0.6, induction temperature of 20 ℃, addition amount of inducer IPTG of 0.6mM and induction time of 24 h) to obtain D361E whole-cell wet thalli. Performing whole cell transformation experiment in a 5L bioreactor, wherein the total volume of an initial transformation system is 1L, the pH is 9, the transformation temperature is 40 ℃, adding thallus according to a final concentration meter, stirring the thallus at a rotating speed of 400rpm, ventilating the thallus at a rate of 1.5L/min, initially feeding propylene diamine at a rate of 30g/L and putrescine at a rate of 20g/L, and then respectively feeding propylene diamine at a rate of 10g/L and putrescine at a rate of 10g/L every 2hg/L, feeding four times in total, and feeding 60g/L of propylene diamine and 50g/L of putrescine. As shown in FIG. 5D, the production of spermidine gradually increased with time, and after 14h, the production of spermidine reached a maximum of 65.3g/L.

Comparative example 1:

in a specific implementation mode, as in example 1, enzyme solutions are prepared and purified according to the methods of examples 2 to 3, and propylenediamine and putrescine are used as substrates to determine enzyme activity, and the results show that compared with the wild type, the catalytic capacities of mutants D361Q, D N, D S, D L, D361I, D361W, D361K, D Y, D53361 64 zxft 53361V and D361A are both remarkably improved; however, mutants D361G, D P, D361F, D361 1R, D361H, D C and D361M were inactivated.

TABLE 3 specific enzyme Activity of different mutants

Where nd indicates that no enzyme activity was detected.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. A homo-spermidine synthetase mutant characterized by having a mutation of aspartic acid at position 361 of a wild-type homo-spermidine synthetase derived from Blastochloris viridis into glutamic acid, glutamine or asparagine.

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

3. An expression vector comprising the gene of claim 2.

4. A genetically engineered bacterium expressing the mutant of claim 1.

5. A recombinant Escherichia coli, wherein the mutant of claim 1 is expressed using Escherichia coli BL21 (DE 3) as a host and pRSFDuet-1 as an expression vector.

6. A cell catalyst comprising the genetically engineered bacterium of claim 5.

7. The cell catalyst according to claim 6, wherein the cell catalyst is prepared by the following method: inoculating the genetically engineered bacteria into a fermentation medium, and culturing to OD ₆₀₀ IPTG is added for induction at 0.5-0.7.

8. A method for producing spermidine through whole-cell catalysis, which is characterized in that the recombinant Escherichia coli of claim 5 or the cell catalyst of any one of claims 6 to 7 is used for catalyzing propane diamine and putrescine to prepare spermidine.

9. The method of claim 8, wherein the catalytic reaction further comprises addition of a cofactor NAD ⁺ The concentration ratio of the propanediamine to the putrescine is (1-2): 1, the reaction temperature is 35-45 ℃.

10. Use of the high spermidine synthase mutant as described in claim 1, or the recombinant escherichia coli as described in claim 5, or the cellular catalyst as described in any one of claims 6 to 7, or the method as described in any one of claims 8 to 9 for the production of spermidine or a product containing spermidine.

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