Phospholipase D-Sp-256-A409C and its application

Abstract

The present invention discloses a phospholipase D‑Sp‑256‑A409C and its application, belonging to the technical field of functional enzymes. The amino acid sequence of the phospholipase D‑Sp‑256‑A409C is shown in SEQ ID NO.3, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.4. Application of the phospholipase D‑Sp‑256‑A409C in the preparation of DHA phosphatidylethanolamine. The present invention also provides a method for preparing DHA phosphatidylethanolamine. The present invention performs mutation modification on phospholipase D‑Sp‑256, and screens out phospholipase D‑Sp‑256‑A409C, which has a substrate conversion rate of up to 91.2% for synthesizing DHA phosphatidylethanolamine, and a transesterification activity of up to 115.0 μg / h / mg, which can meet the requirements of industrial production and application, and has broad application prospects.

Description

Technical Field

[0001] The invention relates to phospholipase D-Sp-256-A409C and application thereof, belonging to the technical field of functional enzymes. Background Art

[0002] Phospholipids are a class of phosphorus-containing lipids widely found in nature. According to their polar head groups, they can be divided into phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), etc. Among them, PE has multiple physiological activities and has positive effects on brain function, neural development, anti-tumor activity, lipid metabolism, etc. At present, DHA phosphatidylethanolamine (DHAPE, DHA refers to docosahexaenoic acid) has attracted more and more attention due to its advantages in oxidative stability, bioavailability and nutritional function. The preparation of high-purity DHAPE has broad market prospects.

[0003] Phospholipase D (PLD), also known as phosphatidylcholine hydrolase, is an efficient phospholipid modification catalyst that can selectively synthesize various rare phospholipids, such as DHAPE. Phospholipases D with different structures have different substrate spectra, catalytic efficiency, stability, product inhibition and other characteristics. So far, there have been no reports on the efficient synthesis of DHAPE using phospholipase D. Summary of the invention

[0004] In view of the above-mentioned prior art, the present invention provides a phospholipase D-Sp-256-A409C and application thereof, belonging to the technical field of functional enzymes.

[0005] The present invention is achieved through the following technical solutions:

[0006] A phospholipase D-Sp-256-A409C, whose amino acid sequence is shown in SEQ ID NO.3.

[0007] The nucleotide sequence of the gene encoding the phospholipase D-Sp-256-A409C is shown in SEQ ID NO.4.

[0008] The phospholipase D-Sp-256-A409C is used in preparing DHA phosphatidylethanolamine.

[0009] In specific applications, DHA phosphatidylcholine (DHAPC) and ethanolamine hydrochloride are used as substrates to synthesize DHA phosphatidylethanolamine under the catalysis of phospholipase D-Sp-256-A409C.

[0010] Furthermore, the specific process of synthesizing DHA phosphatidylethanolamine is as follows: 20 mg DHA phosphatidylcholine is dissolved in 1 mL of organic solvent cyclopentyl methyl ether; 130 mg ethanolamine hydrochloride and 10-50 μg of phospholipase D-Sp-256-A409C are dissolved in 1 mL of buffer; the two are mixed evenly and reacted in a 40°C water bath and a shaking table at 200 rpm for 8 h; the buffer is a pH 6.0, 20 mmol / L citric acid-sodium citrate buffer containing 50 mmol / L calcium chloride.

[0011] The DHA phosphatidylcholine is derived from squid eggs, which contain a high content of polyunsaturated fatty acid phospholipids and a high content of DHA. DHA phosphatidylcholine is usually extracted from squid eggs using an organic solvent extraction method (a conventional technical means in the prior art).

[0012] The present invention also provides a method for preparing DHA phosphatidylethanolamine, which is as follows: using DHA phosphatidylcholine and ethanolamine hydrochloride as substrates, and synthesizing DHA phosphatidylethanolamine under the catalytic action of phospholipase D-Sp-256-A409C.

[0013] Furthermore, the specific process is as follows: 20 mg DHA phosphatidylcholine is dissolved in 1 mL of organic solvent cyclopentyl methyl ether; 130 mg ethanolamine hydrochloride and 10-50 μg of phospholipase D-Sp-256-A409C are dissolved in 1 mL of buffer; the two are mixed evenly and reacted in a 40°C water bath and a shaking table at 200 rpm for 8 h; the buffer is a pH 6.0, 20 mmol / L citric acid-sodium citrate buffer containing 50 mmol / L calcium chloride.

[0014] The present invention has found through research that phospholipase D-Sp-256 can be used to synthesize DHA phosphatidylethanolamine, but the substrate conversion rate is only 26.9%, which is not ideal. Therefore, the present invention has carried out mutation modification on phospholipase D-Sp-256, and screened out phospholipase D-Sp-256-A409C, whose substrate conversion rate for synthesizing DHA phosphatidylethanolamine is as high as 91.2%, and the transesterification activity can reach 115.0 μg / h / mg, which is 5.0 times higher than that of phospholipase D-Sp-256, can meet the requirements of industrial production and application, and has broad application prospects.

[0015] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 :Schematic diagram of the catalytic pocket and key amino acids predicted by Swiss-Model.

[0017] Figure 2 :Schematic diagram of the catalytic pocket and key amino acids predicted by AlphaFold2.

[0018] Figure 3 : Schematic diagram of the 10 mutation sites.

[0019] Figure 4 : Schematic diagram of the relative enzyme activities of PLD-Sp-256 and its mutants, where WT represents PLD-Sp-256.

[0020] Figure 5 : Schematic diagram of substrate conversion rate in the synthesis of PE catalyzed by PLD-Sp-256 and its mutants.

[0021] Figure 6 : Schematic diagram of substrate conversion rate in the synthesis of DHAPE catalyzed by PLD-Sp-256 and its mutants.

[0022] Figure 7 : Schematic diagram of substrate conversion rate and transesterification activity of PLD-Sp-256 and mutants A409C, A409L, A409T, and A409F in catalyzing the synthesis of DHAPE, where WT represents PLD-Sp-256. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0024] The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods available in the prior art.

[0025] Example 1 Mining of Phospholipase D-Sp-256

[0026] The present invention found a gene from Streptomyces in the NCBI database. Streptomyces sp.2224.1 The phospholipase D gene fragment (sequence number: WP_256339225.1) has a total open reading frame length of 1746 bp and encodes a 581 amino acid sequence (the open reading frame includes a phospholipase D consisting of 539 amino acids, as well as protein tags such as 6×His tag and T7 tag).

[0027] The phospholipase D was named phospholipase D-Sp-256, abbreviated as PLD-Sp-256, and its amino acid sequence is shown in SEQ ID NO.1, and the predicted protein molecular weight is 70 kDa. After domain and phylogenetic tree analysis, it was found that PLD-Sp-256 belongs to the PLD superfamily enzyme and has two highly conserved HKD key catalytic activity centers.

[0028] According to the codon preference of the host Escherichia coli, the gene sequence encoding the phospholipase D was codon optimized for efficient expression in Escherichia coli. The nucleotide sequence of the codon-optimized gene is shown in SEQ ID NO.2.

[0029] The amino acid sequence of phospholipase D-Sp-256 is as follows, as shown in SEQ ID NO.1:

[0030] .

[0031] The nucleotide sequence of the gene after codon optimization (direction 5'-3') is as follows, as shown in SEQ ID NO.2:

[0032]

[0033] Example 2 Heterologous expression of phospholipase D-Sp-256

[0034] exist E. coli Phospholipase D-Sp-256 was heterologously expressed in BL21 (DE3) as follows:

[0035] (1) Obtaining gene fragments

[0036] The gene shown in SEQ ID NO.1 was artificially synthesized and used as a template for conventional PCR amplification to obtain a large number of gene fragments.

[0037] (2) Construction of recombinant expression vector

[0038] The gene fragment obtained by the above amplification was connected with the pET-28a cloning vector using seamless cloning technology, and the connection product was transferred into E. coli DH5α competent cells were plated on LB medium solid plates containing 50 μg / mL kanamycin and cultured in a 37°C incubator for 16 hours. Single clones were picked and transferred to LB liquid medium containing 50 μg / mL kanamycin, cultured at 37°C, 220 rpm shaking for 12 hours, sequenced after positive verification, and the recombinant plasmid was named pET-28a-PLD-Sp-256.

[0039] (3) Construction of recombinant engineered bacteria

[0040] Extract the recombinant plasmid with correct sequencing and transform it into the host E. coli In BL21 (DE3) competent cells, the constructed engineered bacteria grew on kanamycin sulfate resistance plates.

[0041] (4) Expression of phospholipase D-Sp-256

[0042] The recombinant engineered bacterial strain grown on the kanamycin sulfate resistance plate was picked and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate, and cultured at 37°C and 220 rpm for 12 hours. The inoculum was inoculated into ZYP-5052 medium containing 50 μg / mL kanamycin sulfate at a rate of 1%, and cultured at 20°C and 200 rpm for 48 hours to auto-induce the expression of phospholipase D-Sp-256.

[0043] (5) Purification of phospholipase D-Sp-256

[0044] The culture medium was centrifuged at 8000 g for 10 min at 4°C, the bacteria were collected, resuspended in Tirs-HCl buffer (50 mM, pH 8.0), ultrasonically disrupted for 30 min, and centrifuged at 12000 g for 15 min. The supernatant was the crude enzyme solution, which was freeze-dried to obtain the crude enzyme powder.

[0045] The crude enzyme solution was purified by affinity chromatography using a nickel column. The column was equilibrated with a 10 mM imidazole solution (10 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl), and then the weakly binding impurities were eluted with a 40 mM imidazole solution (40 mM imidazole, 500 mM NaCl, 50 mMTris-HCl). The target protein was eluted with an 80 mM imidazole solution (80 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl). The eluted components in this portion were collected to obtain the purified enzyme solution. The SDS-PAGE analysis showed that the molecular weight of the purified protein was approximately 70 KDa, which was consistent with the prediction.

[0046] Example 3 Transesterification reaction catalyzed by phospholipase D-Sp-256

[0047] Using a classic two-phase reaction system, phosphatidylcholine (lecithin extracted from soybeans) is used as a raw material to synthesize phosphatidylethanolamine with ethanolamine hydrochloride under the catalytic action of PLD-Sp-256; DHA phosphatidylcholine (extracted from squid eggs) is used as a raw material to synthesize DHA phosphatidylethanolamine with ethanolamine hydrochloride under the catalytic action of PLD-Sp-256; the specific process is as follows:

[0048] 10 mg of phosphatidylcholine was dissolved in 1 mL of organic solvent cyclopentyl methyl ether, and 130 mg of ethanolamine hydrochloride and 10 mg of crude enzyme powder (prepared in Example 2) were dissolved in 1 mL of buffer (pH 6.0, 20 mmol / L citric acid-sodium citrate buffer, containing 50 mmol / L calcium chloride); the two were mixed evenly and reacted in a water bath shaker at 40°C (speed 200 rpm, 8h).

[0049] 20 mg of DHA phosphatidylcholine was dissolved in 1 mL of organic solvent cyclopentyl methyl ether, and 130 mg of ethanolamine hydrochloride and 10 mg of crude enzyme powder (prepared in Example 2) were dissolved in 1 mL of buffer (pH 6.0, 20 mmol / L citric acid-sodium citrate buffer, containing 50 mmol / L calcium chloride); the two were mixed evenly and reacted in a water bath shaker at 40°C (speed 200 rpm, 8 h).

[0050] After the reaction, a certain amount of the reaction mixture was taken, centrifuged and layered, and the upper layer was taken for quantitative analysis and detected by high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). The results showed that the substrate conversion rate of PE catalyzed by PLD-Sp-256 was 62.8%, and the substrate conversion rate of DHAPE catalyzed by PLD-Sp-256 was 26.9%, both of which were not ideal. Under the same reaction conditions, the substrate conversion rate of PC was much greater than that of DHAPC. The reason is that DHAPC has a more complex fatty acid side chain. When PLD-Sp-256 catalyzes the reaction, PC is easier to enter the substrate active pocket of PLD-Sp-256 than DHAPC to bind to it and participate in the reaction.

[0051] The DHA phosphatidylcholine used in the present invention is extracted from squid eggs (it can also be purchased from the conventional market), and the specific operation is as follows: the squid eggs are thawed, spread flat in a glass dish, pre-frozen at -80°C for 4 hours, and vacuum-freeze for 48 hours. After freeze-drying, the sample is ground into powder with a grinder and stored in a -20°C refrigerator. Weigh 60-80 g of squid egg powder in a beaker, add a dichloromethane-methanol mixed solution (the volume ratio of dichloromethane to methanol is 2:1; the solid-liquid ratio is 1:6, unit g:mL), stir and mix well, and stand and soak for 12 hours; filter into a 1000 mL separatory funnel with qualitative filter paper, add 1 / 4 of the volume of the filtrate 0.9% sodium chloride solution, shake well and stand and separate; collect the lower layer solution into a 500 mL rotary evaporator, vacuum rotary evaporation under 45°C water bath conditions until the liquid is completely evaporated, and the orange solids remaining on the rotary evaporator are total lipids. Add dichloromethane until the solids are completely dissolved, transfer to a 50 ml centrifuge tube, add 30 mL of acetone (frozen at -20°C), shake quickly for 20 seconds, then centrifuge quickly at 8000 rpm for 2 min, and pour out the supernatant; repeat this operation once (add frozen acetone, centrifuge, and pour out the supernatant); DHA phosphatidylcholine is obtained as the substrate for the transesterification reaction.

[0052] Example 4 Mutation modification of phospholipase D-Sp-256

[0053] The above studies show that the substrate conversion rate of phospholipase D-Sp-256 in catalyzing the synthesis of PE and DHAPE is not ideal. Therefore, the present invention attempts to transform it through molecular biotechnology to improve its potential for synthesizing rare phospholipids.

[0054] The homology modeling of PLD-Sp-256 was performed using the online analysis tools Swiss-Model and AlphaFold2 to obtain the three-dimensional structure of PLD-Sp-256. The obtained three-dimensional structures were uploaded to the HotSpot Wizard 3.1 website to design a mutation library based on structural information and sequence alignment to identify the key amino acids related to the PLD catalytic reaction.

[0055] The schematic diagram of the catalytic pocket and key amino acids predicted by Swiss-Model is shown in the figure below. Figure 1 As shown, the catalytic pocket and key amino acids predicted by AlphaFold2 are shown in Figure 2 As shown in the figure, the results of the two predictions are quite different. Based on the prediction results of the two, a total of 10 mutation sites were selected for site-directed modification in the hope of improving the catalytic performance of the enzyme. The schematic diagram of these 10 mutation sites is shown in Figure 3 shown.

[0056] The specific method of site-directed modification is as follows: the amino acid at position 152 of PLD-Sp-256 is mutated from alanine to valine and leucine respectively; the amino acid at position 155 is mutated from leucine to cysteine, serine, valine and methionine respectively; the amino acid at position 159 is mutated from leucine to serine and cysteine ​​respectively; the amino acid at position 160 is mutated from valine to cysteine ​​and phenylalanine respectively; the amino acid at position 193 is mutated from serine to alanine; the amino acid at position 194 is mutated from leucine to cysteine, threonine, phenylalanine and methionine respectively; the amino acid at position 331 is mutated from leucine to alanine, phenylalanine and valine respectively; the amino acid at position 376 is mutated from leucine to alanine, phenylalanine, glycine, methionine and valine respectively; the amino acid at position 409 is mutated from alanine to threonine, leucine, cysteine ​​and phenylalanine respectively; the amino acid at position 410 is mutated from valine to alanine, cysteine ​​and leucine respectively. There are 30 mutants, temporarily named as A152V, A152L, L155C, L155S, L155V, L155M, L159S, L159C, V160C, V160F, S193A, L194C, L194T, L194F, L194M, L376A, L376F, L376G, L376M, L376V, L331A, L331F, L331V, A409T, A409L, A409C, A409F, V410A, V410C, V410L. Accordingly, the codons at the mutation sites on the coding gene are mutated to the codons of the corresponding amino acids.

[0057] Primers were designed using Snapgene with pET-28a-PLD-Sp-256 (the recombinant plasmid constructed in Example 2) as a template to construct the recombinant plasmids of the above 30 mutants. The engineering bacteria were constructed by conventional methods, and cultured, expressed and extracted conventionally (the method was the same as in Example 2) to obtain crude enzyme liquid and crude enzyme powder of each mutant.

[0058] Example 5 Comparison of the hydrolysis activity and transesterification activity of phospholipase D-Sp-256 and its mutants

[0059] The hydrolysis activity of crude enzyme solution of PLD-Sp-256 and its mutants was determined by enzyme-linked colorimetry. The determination method was as follows: 10 μL of citric acid buffer (0.1 mol / L, pH 6.0), 5 μL of calcium chloride solution (0.1 mol / L) and 15 μL of Trix X-100 solution (7.5%, volume percentage) were added to 100 μL of enzyme solution. The reaction was carried out at 37°C for 20 min, inactivated in a boiling water bath for 5 min, cooled to room temperature, 200 μL of colorimetric solution was added, and the reaction was carried out in a 37°C water bath for 3 hours. After the reaction was completed, the absorbance value at 500 nm was detected.

[0060] The color developing solution is composed of the following components: 1.0 M pH 8.0 Tris-HCl buffer, 100 mL; choline oxidase 50 U; peroxidase 100 U; 4-aminoantipyrine 100 mg; phenol 50 mg; Triton X-100 1.0 g.

[0061] Definition of enzyme activity: One unit is the amount of enzyme required to release 1 μmol of choline per minute.

[0062] The hydrolysis activity of PLD-Sp-256 was determined to be 0.018 U.

[0063] The relative enzyme activity of each mutant was calculated with the hydrolysis activity of PLD-Sp-256 as 100%. The relative enzyme activity of PLD-Sp-256 and its mutants is shown in the figure below. Figure 4 As shown, the hydrolysis activities of mutants A152V, A152L, S193A, L194C, L194T and L376M decreased significantly, while the hydrolysis activities of mutants V160F and L376V increased significantly, and the hydrolysis activities of the remaining mutants did not change significantly.

[0064] The substrate conversion efficiency of each mutant was measured (the method was the same as in Example 3). The schematic diagram of the substrate conversion rate of PLD-Sp-256 and its mutants in catalyzing the synthesis of PE is shown in Figure 5 As shown, the schematic diagram of substrate conversion rate of PLD-Sp-256 and its mutants catalyzing the synthesis of DHAPE is shown in Figure 6 shown.

[0065] Depend on Figure 5 It can be seen that there are 7 dominant mutants for PC transformation: L159C, V160C, L194M, L194F, A409C, A409L, and V410C. Among them, the substrate conversion rates of L159C, A409C, and V410C are 81.9%, 87.2%, and 86.8%, respectively, all of which can reach a conversion rate of more than 80%.

[0066] Depend on Figure 6 It can be seen that there are 12 advantageous mutants for DHAPC conversion: L155C, L155V, L159C, V160C, L194M, L194F, L376V, A409C, A409T, A409L, V410C, and V410L. Among them, the substrate conversion rate of A409L is 76.7%, which is 2.85 times that of the original enzyme PLD-Sp-256, and the substrate conversion rate of A409C is 66.3%, which is 2.46 times that of the original enzyme PLD-Sp-256. The beneficial mutations for PE synthesis are also more conducive to the synthesis of DHAPE.

[0067] Example 6 Protein purification and screening of 409-position mutants of phospholipase D-Sp-256

[0068] From the first round of screening in Example 5, it was found that the conversion rates of two mutants A409C and A409L at position 409 were greatly improved, and the other two mutants A409T and A409F at position 409 also had changes in substrate conversion rates. Therefore, the crude enzyme solutions of mutants A409C, A409L, A409T and A409F were purified (the method was the same as that in Example 2) to obtain pure enzyme solutions, and the protein concentration was determined by Coomassie Brilliant Blue.

[0069] The transesterification reaction was carried out using a classical two-phase reaction system (the method was the same as in Example 3). The enzyme addition amount of each mutant was controlled to be 5 μg, 10 μg, 20 μg and 50 μg, respectively. The reaction was carried out under the same reaction conditions (40°C, 8 h). After the reaction, the reaction product was detected by HPLC-ELSD. The substrate conversion rate and transesterification activity of PLD-Sp-256 and mutants A409C, A409L, A409T, and A409F catalyzing the synthesis of DHAPE are shown in the following figure. Figure 7 shown.

[0070] Depend on Figure 7It can be seen that when the enzyme addition amount was 10 μg, the substrate conversion rate of mutant A409C had reached 82.9%, and its transesterification activity was 115.0 μg / h / mg; while the highest transesterification activity of the original enzyme PLD-Sp-256 was only 22.6 μg / h / mg, and the transesterification activity of mutant A409C increased by 5.0 times.

[0071] When the amount of enzyme added gradually increased, the substrate conversion rate increased accordingly. However, when the amount of enzyme added was 50 μg, the substrate conversion rate of the original enzyme PLD-Sp-256 could only reach 35.7%, while the mutants at the 409 site could reach more than 80%, among which the conversion rate of mutant A409C could reach 91.2%, the conversion rate of mutant A409L could reach 94.7%, the conversion rate of mutant A409T was 87.0%, and the conversion rate of mutant A409F was 84.1%. In summary, mutant A409C had the best transesterification effect.

[0072] The mutant A409C was named as phospholipase D-Sp-256-A409C, abbreviated as PLD-Sp-256-A409C, and the mutant A409L was named as phospholipase D-Sp-256-A409L, abbreviated as PLD-Sp-256-A409L. The amino acid sequence of phospholipase D-Sp-256-A409C is shown in SEQ ID NO.3, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.4. The amino acid sequence of phospholipase D-Sp-256-A409L is shown in SEQ ID NO.5, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.6.

[0073] The amino acid sequence of phospholipase D-Sp-256-A409C is as follows, as shown in SEQ ID NO.3:

[0074] MAPHRLHRLIPASVALTTVCATLPASAAYAADTPPTPHLDSIERSLRETSPGLEGSVWERTDGNRLDAPADNPSGWLLQTPGCWGDAGCKDRAGTRRMLDKMTRNIADARHTVDVSSLAPFPNGGFEDAVVAGLKASVKAGHSPRVRILVGAAPLYHLNVVPSRYRDDLIGKLGPAAGKVTLNVASMTTSKTSLSWNHSKLLVVDGKTAVTGGINGWKDDYLDTDHPVSDVDMALSGPAARSAGKYLDTLWDWTCRNASDPAKVWLATSNGSSCMPSMEKDEAGATPAEPTGDVPVIAVGGLGVGIKESDPSSGYHPDLPTAPDTKCTVGLHDHTNGDRDYDTVNPEESALRSLIASARSHVEISQQDLNGTCPPLPRYDIRTYDTLAGKLAAGVKVRIVVSDPANRGCVGSGGYSQIKSLDEISDTLRTRLVALTGDNEKASRALCGNLQLASFRSSDAAKWADGKPYALHHKLVSVDDSAFYIGSKNLYPAWLQDFGYIVESPAAAKQLKTELLDPEWKYSRQAASTPAGCPAGATG。

[0075] The nucleotide sequence (in the direction 5'-3') of the coding gene for phospholipase D-Sp-256-A409C is shown below, as shown in SEQ ID NO. 4:

[0076]

[0077] The amino acid sequence of phospholipase D-Sp-256-A409L is as follows, as shown in SEQ ID NO.5:

[0078] .

[0079] The nucleotide sequence (direction 5'-3') of the gene encoding phospholipase D-Sp-256-A409L is as follows, as shown in SEQ ID NO.6:

[0080]

[0081] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. A phospholipase D-Sp-256-A409C, characterized in that: The amino acid sequence is shown in SEQ ID NO.

3.

2. The coding gene of phospholipase D-Sp-256-A409C according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

4.

3. Use of the phospholipase D-Sp-256-A409C according to claim 1 in the preparation of DHA phosphatidylethanolamine.

4. The use of phospholipase D-Sp-256-A409C according to claim 3 in the preparation of DHA phosphatidylethanolamine, characterized in that: DHA phosphatidylcholine and ethanolamine hydrochloride are used as substrates and DHA phosphatidylethanolamine is synthesized under the catalysis of phospholipase D-Sp-256-A409C.

5. Use of the phospholipase D-Sp-256-A409C according to claim 4 in the preparation of DHA phosphatidylethanolamine, characterized in that: The specific process of synthesizing DHA phosphatidylethanolamine is as follows: 20 mg DHA phosphatidylcholine is dissolved in 1 mL of organic solvent cyclopentyl methyl ether; 130 mg ethanolamine hydrochloride and 10-50 μg of phospholipase D-Sp-256-A409C are dissolved in 1 mL of buffer; the two are mixed evenly and reacted in a 40°C water bath and a shaking table at 200 rpm for 8 hours; the buffer is a pH 6.0, 20 mmol / L citric acid-sodium citrate buffer containing 50 mmol / L calcium chloride.

6. A method for preparing DHA phosphatidylethanolamine, characterized in that: DHA phosphatidylcholine and ethanolamine hydrochloride are used as substrates, and DHA phosphatidylethanolamine is synthesized under the catalytic action of the phospholipase D-Sp-256-A409C described in claim 1.

7. The method for preparing DHA phosphatidylethanolamine according to claim 6, characterized in that: 20 mg DHA phosphatidylcholine was dissolved in 1 mL of organic solvent cyclopentyl methyl ether; 130 mg ethanolamine hydrochloride and 10-50 μg phospholipase D-Sp-256-A409C were dissolved in 1 mL of buffer; the two were mixed evenly and reacted in a 40°C water bath and a shaking table at 200 rpm for 8 hours; the buffer was a pH 6.0, 20 mmol / L citric acid-sodium citrate buffer containing 50 mmol / L calcium chloride.

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