A phospholipase mutant and its preparation and application

Abstract

The present invention belongs to the field of enzyme genetic engineering technology and specifically relates to a phospholipase D mutant, its preparation, and use. The phospholipase D gene from Streptomyces antibioticus was molecularly modified to mutate histidine at position 443 to asparagine and serine at position 199 to histidine, resulting in the phospholipase D mutant of the present invention, the amino acid sequence of which is shown in SEQ ID NO. 3. The catalytic activity of this mutant is significantly enhanced compared to that of wild-type phospholipase D. The phospholipase D mutant obtained by the present invention is suitable for catalyzing the synthesis of glycerophospholipids and also provides a reference for the synthesis of other functional phospholipids.

Description

Technical field:

[0001] The present invention belongs to the technical field of enzyme genetic engineering, and in particular relates to a phospholipase D mutant and its preparation and application. Background technology:

[0002] Phospholipids are a class of lipid molecules containing phosphate functional groups. They are essential components of biological membranes and primarily include glycerophospholipids and sphingomyelins. Glycerophospholipids can be classified into phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidic acid (PA), based on the polar head groups. Phospholipids participate in cell signaling, various enzymatic reactions, and energy metabolism, representing an evolutionary and crucial step in the emergence of life. Due to their numerous nutritional and bioactive properties, phospholipids are widely used in the food, pharmaceutical, and cosmetic industries. PS is a naturally occurring glycerophospholipid with numerous physiological functions, such as alleviating mood and enhancing memory. Currently, PS has been approved by the National Health Commission of my country as a new resource food. Methods for preparing PS include physical extraction, chemical synthesis, and enzymatic methods. However, the first two methods are unsuitable for large-scale production of PS due to their complex processes, low yields, high toxicity, difficulty in purification, and potential environmental pollution. The enzymatic method uses phospholipase D (PLD) to catalyze the transesterification reaction between PC and L-serine to synthesize PS. The process is simple, with mild reaction conditions, eliminating the need for harsh conditions such as high temperature and pressure. The synthesis produces no hazardous waste, conforming to the requirements of green chemistry and sustainable development, and boasting high product safety. Therefore, the enzymatic synthesis of PS has broad application prospects.

[0003] Phospholipase D (PLD, EC.3.1.4.4) is a common enzyme found in abundance in plants, animals, and microorganisms. Hanahan and Chaikoff first discovered PLD in carrot and cabbage leaves in 1947. Subsequently, PLD has been discovered and extracted from various plant organs, including roots, leaves, and seeds. However, the extraction process for plant-derived PLD is complex. Furthermore, PLD in animals is primarily distributed in tissues such as the brain and liver, and most of it is a membrane-bound protein, making large-scale industrial production difficult. In comparison, microbial phospholipase D (e.g., from Streptomyces, Corynebacterium, Escherichia, Pseudomonas, Bacillus, and Salmonella) offers advantages such as low production cost, short culture cycles, high phosphatidyl transfer reaction activity, and excellent substrate specificity. Among them, PLD, derived from Streptomyces, has been widely studied due to its strong transesterification ability. However, the transesterification activity of PLD is currently insufficient for industrial production applications, and various methods are needed to improve its activity.

[0004] Protein engineering, based on the relationship between protein structure and biological function, uses chemical, physical, and molecular biological methods to modify or synthesize genes to modify existing proteins or create new ones to meet human needs in production and daily life. Protein engineering is generally categorized into irrational and rational design. Irrational design involves mutation and screening of the entire sequence using techniques such as error-prone PCR, DNA recombination, and staggered extension, as well as nonhomologous random recombination strategies. This approach requires neither knowledge of the target protein's three-dimensional structure nor relevant structure-function information, but is relatively tedious. Rational design, on the other hand, requires a deep understanding of protein structure and function. Advances in techniques such as molecular dynamics simulations and quantum chemical calculations have enabled precise design of protein mutations or structural changes. Compared to irrational design, rational design requires less effort, reduces ineffective mutations, and can yield mutants with superior performance in a shorter time.

[0005] Bacillus expression systems (such as Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis) are safe, efficient, and highly promising genetic engineering expression systems and have been widely used in various fields. Compared to commonly used Escherichia coli expression systems, they offer a unique advantage: they can directly secrete target proteins into the extracellular space, further reducing the cost and workload of target protein isolation and purification. With the advancement of molecular biology techniques and in-depth research on Bacillus, numerous industrial enzymes have been expressed in Bacillus expression systems and successfully produced on a large scale.

[0006] Therefore, in the present invention, the phospholipase D gene from Streptomyces antibioticus is molecularly modified to obtain a phospholipase D mutant gene with improved enzyme activity, and its expression in Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis is achieved. Summary of the invention:

[0007] The purpose of the present invention is to address the existing technical problems, molecularly modify phospholipase D (PLD) to obtain a mutant with improved transesterification activity, and realize its expression in Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis systems.

[0008] The technical route for achieving the purpose of the present invention is summarized as follows:

[0009] Wild-type phospholipase D (PLD) from Streptomyces antibioticus TCCC 21059 was obtained through molecular biological methods. A recombinant vector was constructed through enzyme digestion and ligation, and the wild-type PLD encoding gene, pld, was sequenced (SEQ ID NO. 2). Molecular dynamics simulations and other methods were used to rationally design the PLD mutant H443N / S199H and its encoding gene, pldm. This mutant, which mutates histidine at position 443 to asparagine and serine at position 199 to histidine, increases its activity to 3.14 times that of the wild-type. The H443N / S199H mutant was efficiently prepared and applied using Bacillus subtilis WB600, Bacillus amyloliquefaciens CGMCC No. 11218, and Bacillus licheniformis 2709.

[0010] One of the technical solutions provided by the present invention is a PLD mutant, which is obtained by causing H443N and S199H mutations to the wild-type PLD shown in SEQ ID NO.1, and the mutant is named H443N / S199H;

[0011] Furthermore, the PLD mutant of the present invention is H443N / S199H, and the amino acid sequence is shown in SEQ ID NO.3;

[0012] Furthermore, the nucleotide sequence of the gene pldm encoding the H443N / S199H mutant is shown in SEQ ID NO.4.

[0013] The second technical solution provided by the present invention is a recombinant vector or recombinant strain comprising the gene encoding the above-mentioned PLD mutant;

[0014] Furthermore, the expression vector used in the recombinant vector is the Escherichia coli-Bacillus subtilis shuttle plasmid pBSA43;

[0015] Furthermore, the hosts used by the recombinant strain are Bacillus subtilis WB600, Bacillus amyloliquefaciens CGMCC No.11218 and Bacillus licheniformis 2709.

[0016] The third technical solution provided by the present invention is the application of the above-mentioned recombinant vector or recombinant strain, especially in the production of the PLD mutant shown in SEQ ID NO.3.

[0017] A fourth technical solution provided by the present invention is the use of the PLD mutant shown in SEQ ID NO. 3, particularly in the preparation of glycerol-type phospholipids and their derivatives, and more particularly in the preparation of phosphatidylserine (PS);

[0018] Furthermore, the application forms of the PLD mutant include but are not limited to enzyme liquid, enzyme powder, emulsion, gel, and immobilized enzyme.

[0019] Beneficial effects:

[0020] 1. The invention uses overlapping PCR technology to perform site-directed mutagenesis on wild-type PLD. The mutant H443N / S199H has a specific activity that is 3.14 times that of wild-type PLD, and catalyzes the conversion of phosphatidylcholine and serine to phosphatidylserine at a yield that is 2.01 times that of wild-type PLD.

[0021] 2. The present invention uses the Bacillus subtilis expression system, the Bacillus amyloliquefaciens expression system, and the Bacillus licheniformis expression system to achieve efficient expression of PLD mutants in different ways.

[0022] The following definitions are used in the present invention:

[0023] 1. Nomenclature of amino acid and DNA sequences

[0024] The generally accepted IUPAC nomenclature for amino acid residues is used, using the three-letter / one-letter code format. The generally accepted IUPAC nomenclature for DNA nucleic acid sequences is used.

[0025] 2. Identification of Phospholipase Mutants

[0026] The term "amino acid substituted at the original amino acid position" is used to indicate the mutated amino acid in a mutant. For example, H443N indicates that the amino acid at position 443 is substituted from the wild-type His to Asn; S199H indicates that the amino acid at position 199 is substituted from the wild-type Ser to His. The position numbers correspond to the amino acid sequence numbering of the wild-type phospholipase in SEQ ID NO. 1.

[0027] In the present invention, the lowercase italic pld represents the encoding gene of the wild-type phospholipase, and the lowercase italic pldm represents the encoding gene of the mutant H443N / S199H, and the information is shown in the following table.

[0028]

[0029] In the present invention, the wild-type PLD sequence is shown in SEQ ID NO.1:

[0030] ADTPPTPHLDAIERSLRDTSPGLEGSVWQRTDGNRLDAPDGDPAGWLLQTPGCWGDAGCKDRAGTRRLLDKMTRNIADARHTVDISSLAPFPNGGFEDAVVDGLKASVAAGHSPRVRILVGAAPIYH LNVVPSRYRDELIGKLGAAAGKVTLNVASMTTSKTSLSWNHSKLLVVDGKTAITGGINGWKDDYLDTAHPVSDVDMALSGPAARSAGKYLDTLWDWTCRNASDPAKVWLATSNGASCMPSMEQDEAG SAPAEPTGDVPVIAVGGLGVGIKESDPSSGYHPDLPTAPDTKCTVGLHDNTNADRDYDTVNPEENALRSLIASARSHVEISQQDLNATCPPLPRYDIRTYDTLAGKLAAGVKVRIVVSDPANRGAVG SGGYSQIKSLDEISDTLRTRLVALTGDNEKASRALCGNLQLASFRSSDAAKWADGKPYALHHKLVSVDDSAFYIGSKNLYPAWLQDFGYIVESPAAAQQLKTELLDPEWKYSQQAAATPAGCPARQAG

[0031] In the present invention, the sequence of the PLD mutant H443N / S199H is shown in SEQ ID NO.3:

[0032] ADTPPTPHLDAIERSLRDTSPGLEGSVWQRTDGNRLDAPDGDPAGWLLQTPGCWGDAGCKDRAGTRRLLDKMTRNIADARHTVDISSLAPFPNGGFEDAVVDGLKASVAAGHSPRVRILVGAAPIYH LNVVPSRYRDELIGKLGAAAGKVTLNVASMTTSKTSLSWNHSKLLVVDGKTAITGGINGWKDDYLDTAHPVHDVDMALSGPAARSAGKYLDTLWDWTCRNASDPAKVWLATSNGASCMPSMEQDEAG SAPAEPTGDVPVIAVGGLGVGIKESDPSSGYHPDLPTAPDTKCTVGLHDNTNADRDYDTVNPEENALRSLIASARSHVEISQQDLNATCPPLPRYDIRTYDTLAGKLAAGVKVRIVVSDPANRGAVG SGGYSQIKSLDEISDTLRTRLVALTGDNEKASRALCGNLQLASFRSSDAAKWADGKPYALHNKLVSVDDSAFYIGSKNLYPAWLQDFGYIVESPAAAQQLKTELLDPEWKYSQQAAATPAGCPARQAG Description of the drawings:

[0033] Figure 1 This is the electrophoresis diagram of the PCR amplification of the wild-type PLD gene of the present invention, wherein: M is DNA Marker, 1 is the PLD gene;

[0034] Figure 2 : is the enzyme digestion verification diagram of the recombinant plasmid pBSA43-pldm of the present invention, wherein: M is DNA Marker, 1 is the double enzyme digestion electrophoresis diagram of the recombinant plasmid pBSA43-pldm;

[0035] Figure 3 This is an SDS-PAGE image of the wild-type PLD mutant H443N / S199H after purification of the present invention, wherein: M is a protein marker, 1 is a purified sample of the wild-type PLD, and 2 is a purified sample of the mutant H443N / S199H. Specific implementation method:

[0036] The technical content of the present invention will be further described below in conjunction with the embodiments, but the present invention is not limited to these embodiments, and the protection scope of the present invention cannot be limited by the following embodiments.

[0037] The culture medium and solution used in the embodiments of the present invention are as follows:

[0038] LB medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, and the rest is water.

[0039] LBS medium: 91.1 g sorbitol, 10 g NaCl, 5 g yeast extract, 10 g tryptone, dissolved in 1 L water.

[0040] The solid medium of the above medium was supplemented with 2% agar.

[0041] 20 mM phosphate buffer: 38 mL 0.2 M NaH2PO4, 62 mL 0.2 M Na2HPO4, pH 7.0.

[0042] SP salt solution (1 L): K2HPO4 18.34 g, KH2PO4 6.0 g, (NH4)2SO4 2.0 g, sodium citrate 1.0 g, MgSO4·7H2O 0.2 g, add 800 mL of water to dissolve, and continue to add water to make up to 1 L after complete dissolution.

[0043] SPI medium (200 mL): 195.2 mL of SP salt solution, 0.8 mL of 5% casein hydrolysate, 2 mL of 10% yeast extract, and 2 mL of 5% glucose solution. Mix well and dispense 5 mL of each into sterilized empty test tubes. Store at 4°C.

[0044] SPII medium: 292.8 mL of SP salt solution, 1.2 mL of 5% casein hydrolysate, 3 mL of 10% yeast extract, 3 mL of 5% glucose solution, 1.5 mL of 100 mM calcium chloride, and 1.5 mL of 50 mM magnesium chloride. Mix well and dispense 2 mL of each into sterilized empty test tubes. Store at 4°C.

[0045] Wash Buffer (mM): Weigh 29.25 g of NaCl, 2.42 g of Tris, and 3.5 g of imidazole and dissolve them in ultrapure water to a volume of 1 L. Filter through a 0.22 μm microporous membrane to remove impurities and store in a refrigerator at 4°C away from light.

[0046] Elution Buffer (mM): Weigh 29.25 g of NaCl, 2.42 g of Tris, and 13.6 g of imidazole and dissolve them in ultrapure water to a volume of 1 L. Filter through a 0.22 μm microporous membrane to remove impurities and store in a refrigerator at 4°C away from light.

[0047] Lysis Buffer (mM): Weigh 29.25 g of NaCl, 2.42 g of Tris, and 1.4 g of imidazole and fully dissolve them in ultrapure water to a volume of 1 L. Filter through a 0.22 μm microporous membrane to remove impurities and store in a refrigerator at 4°C away from light.

[0048] The present invention will be further explained below through specific examples.

[0049] Example 1: Obtaining the wild-type PLD gene

[0050] 1. The wild-type PLD gene was derived from the laboratory-preserved strain TCCC 21059 of Streptomyces antibioticus. The genome was extracted using the Bacterial DNA Kit D3350-02 from OMEGA, USA, according to the manufacturer's instructions.

[0051] (1) Strain activation: Use an inoculating loop to dip the antibiotic Streptomyces spore solution from the glycerol tube, inoculate it onto a solid culture medium plate, draw three lines, and culture at 28°C for 5-6 days;

[0052] (2) Transfer: Pick a single colony from the culture plate and inoculate it into 5 mL of liquid LB medium. Incubate at 220 rpm and 37°C for 12 h.

[0053] (3) Collecting bacteria: Take an appropriate amount of culture medium and dispense it into a 1.5 mL EP tube. Centrifuge at 12000 rpm for 2 min and discard the supernatant.

[0054] (4) Add 250 μL of ddH2O to resuspend the cells, add 50 μL of 50 mg / mL lysozyme, and incubate at 37°C for 20 min;

[0055] (5) Add 100 μL BTL Buffer and 20 μL Proteinase K and vortex;

[0056] (6) 55°C water bath for 40-50 min, shaking and mixing every 20-30 min;

[0057] (7) Add 5 μL of RNase, mix by inversion several times, and let stand at room temperature for 5 min;

[0058] (8) Centrifuge at 12000 rpm for 2 min, remove the undigested portion, and transfer the supernatant to a new 1.5 mL EP tube;

[0059] (9) Add 220 μL BDL Buffer, vortex to mix, and place in a 65°C water bath for 10 min;

[0060] (10) Add 220 μL of anhydrous ethanol and mix thoroughly by pipetting;

[0061] (11) Transfer the liquid in the EP tube to the adsorption column, let it stand for 1 min, centrifuge at 12000 rpm for 1 min, and pour the filtrate back into the recovery column. Repeat twice and discard the waste liquid.

[0062] (12) Add 500 μL HBC Buffer, centrifuge at 12000 rpm for 1 min, and discard the filtrate;

[0063] (13) Add 700 μL DNA Wash Buffer, centrifuge at 12000 rpm for 1 min, and discard the filtrate;

[0064] (14) Add 500 μL DNA Wash Buffer, centrifuge at 12000 rpm for 1 min, and discard the filtrate;

[0065] (15) Centrifuge at 12000 rpm for 2 min, discard the waste liquid tube, place the recovery column in a new EP tube, place in a metal bath at 55°C for 10 min, and air dry;

[0066] (16) Add 40 μL of 55°C ddH2O for elution, discard the recovery column, and store the genome at -20°C.

[0067] 2. Amplification of the wild-type PLD gene

[0068] Using the genome obtained in step 1 as a template, the wild-type PLD encoding gene pld was amplified.

[0069] A pair of primers were designed upstream and downstream of the ORF frame to introduce restriction enzyme sites BamHI and NotI, respectively.

[0070] The sequences of the primers for the wild-type PLD encoding gene pld (upstream primer PLD-F, downstream primer PLD-R) are as follows:

[0071] PLD-F:CG CGGATCC GCAGATACGCCGCCGA (the underlined part is the BamHI restriction site)

[0072] PLD-R:AAGGAAAAAA GCGGCCGC TTAGTGGTGGTGGTGGTGGTGACC AGCTTGGCGAGCG (the underlined part is the NotI restriction site);

[0073] PCR amplification was performed using the genome of Streptomyces antibioticus as a template. The reaction system was 50 μL and consisted of:

[0074] PrimeSTARMax 25 μL Upstream primer PLD-F (20 μmol / L) 2μL Downstream primer PLD-R (20 μmol / L) 2μL Genome 2μL <![CDATA[ddH2O]]> 19 μL Total volume 50μL

[0075] Note: The above reagents are from Takara Biotechnology Co., Ltd.

[0076] The amplification program was set as follows: pre-denaturation: 98°C for 30 s; denaturation: 98°C for 10 s; annealing: 54°C for 20 s; extension: 72°C for 8 s; 30 cycles of the above reaction; and extension: 72°C for 10 min.

[0077] The PCR product was subjected to agarose gel electrophoresis, and the wild-type PLD gene band was approximately 1500 bp (see Figure 1 ), the PCR product was recovered using a DNA gel excision recovery kit and sent to a sequencing company for sequencing, obtaining the wild-type pld gene sequence (shown in SEQ ID NO. 2). The pBSA43 plasmid was extracted, and the vector plasmid pBSA43 and the target gene pld were double-digested with the restriction endonucleases BamHI and NotI. The pld recovered from the gel excision was ligated with the pBSA43 vector to obtain the recombinant plasmid pBSA43-pld, which was then transformed into Bacillus subtilis WB600 to obtain the recombinant strain WB600 / pBSA43-pld.

[0078] Example 2: Construction of phospholipase D mutants

[0079] 1. Overlap PCR: Based on the wild-type pld gene, overlapping PCR was performed to first construct the mutant H443N gene. The primers (upstream primer f1, downstream primer r1) and reaction system were as follows:

[0080] f 1:GCGCTTCATAATAAACTGGTG

[0081] r 1: CACCAGTTTATTATGAAGCGC

[0082] In the first step of overlapping PCR, PLD-F and r1 were used as upstream and downstream primers, and f1 and PLD-R were used as upstream and downstream primers, respectively. PCR reactions were performed using plasmid pBSA43-pld as a template to obtain upstream and downstream fragments, respectively.

[0083] The reaction system for upstream fragment amplification is:

[0084] Upstream primer PLD-F (20 μmol / L) 2μL Downstream primer r1 (20 μmol / L) 2μL pBSA43-pld 2μL PrimerStarMax 25 μL <![CDATA[ddH2O]]> 19 μL

[0085] The reaction system for downstream fragment amplification is:

[0086] Upstream primer f1 (20 μmol / L) 2μL Downstream primer PLD-R (20 μmol / L) 2μL pBSA43-pld 2μL PrimerStarMax 25 μL <![CDATA[ddH2O]]> 19 μL

[0087] The amplification program was as follows: pre-denaturation at 98°C for 30 min; 30 cycles of denaturation at 98°C for 10 s, annealing at 54°C for 20 s, and extension at 72°C for 7 s; and extension at 72°C for 10 min.

[0088] After gel excision and recovery of upstream and downstream fragments, overlapping PCR reaction was performed. The reaction system was:

[0089] Upstream fragment 2μL Downstream fragment 2μL PrimerStarMax 25 μL <![CDATA[ddH2O]]> 21 μL

[0090] The amplification program was as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 54°C for 20 s, and extension at 72°C for 8 s, for 5 cycles; and extension at 72°C for 10 min.

[0091] After the PCR was completed, 2 μL of upstream primer PLD-F and downstream primer PLD-R were added to the system respectively for PCR reaction. The amplification program was as follows: pre-denaturation at 98°C for 30 seconds; denaturation at 98°C for 10 seconds, annealing at 54°C for 20 seconds, extension at 72°C for 10 seconds, reaction for 30 cycles; extension at 72°C for 10 minutes.

[0092] Upstream primer PLD-F (20 μmol / L) 2μL Downstream primer PLD-R (20 μmol / L) 2μL template 2μL PrimerStarMax 25 μL <![CDATA[ddH2O]]> 19 μL

[0093] The PCR amplification products were subjected to agarose gel electrophoresis and recovered using a small amount of DNA recovery kit.

[0094] After the PCR reaction, the PCR product and the vector plasmid were double-digested with BamHI and NotI, and then purified and recovered. The PCR product was ligated with the vector plasmid pBSA43 double-digested with BamHI and NotI, and transformed into the cloning host E. coli JM109. The transformants were picked and the plasmid was extracted for PCR and double-enzyme digestion verification. If successful, sequencing verification was performed to obtain the pBSA43-H443N plasmid.

[0095] 2. Overlap PCR was performed using the mutant H443N gene as a template to construct the mutant pldm gene. The primers (upstream primer f2, downstream primer r2) and reaction system were as follows:

[0096] f2:TCCGGTTCATGATGTTGAC

[0097] r 2: GTCAACATCATGAACCGGA

[0098] In the first step of overlapping PCR, PLD-F and r2 were used as upstream and downstream primers, and f2 and PLD-R were used as upstream and downstream primers, respectively. PCR reactions were performed using plasmid pBSA43-H443N as a template to obtain upstream and downstream fragments, respectively.

[0099] The reaction system for upstream fragment amplification is:

[0100] Upstream primer PLD-F (20 μmol / L) 2μL Downstream primer r2 (20 μmol / L) 2μL pBSA43-H443N 2μL PrimerStarMax 25 μL <![CDATA[ddH2O]]> 19 μL

[0101] The reaction system for downstream fragment amplification is:

[0102] Upstream primer f2 (20 μmol / L) 2μL Downstream primer PLD-R (20 μmol / L) 2μL pBSA43-H443N 2μL PrimerStarMax 25 μL <![CDATA[ddH2O]]> 19 μL

[0103] The amplification program was as follows: pre-denaturation at 98°C for 30 min; 30 cycles of denaturation at 98°C for 10 s, annealing at 54°C for 20 s, and extension at 72°C for 5 s; and extension at 72°C for 10 min.

[0104] After gel excision and recovery of upstream and downstream fragments, overlapping PCR reaction was performed. The reaction system was:

[0105] Upstream fragment 2μL Downstream fragment 2μL PrimerStarMax 25 μL <![CDATA[ddH2O]]> 21 μL

[0106] The amplification program was as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 54°C for 20 s, and extension at 72°C for 8 s, for 5 cycles; and extension at 72°C for 10 min.

[0107] After the PCR was completed, 2 μL of upstream primer PLD-F and downstream primer PLD-R were added to the system respectively for PCR reaction. The amplification program was as follows: pre-denaturation at 98°C for 30 seconds; denaturation at 98°C for 10 seconds, annealing at 54°C for 20 seconds, extension at 72°C for 10 seconds, reaction for 30 cycles; extension at 72°C for 10 minutes.

[0108] Upstream primer PLD-F (20 μmol / L) 2μL Downstream primer PLD-R (20 μmol / L) 2μL template 2μL PrimerStarMax 25 μL <![CDATA[ddH2O]]> 19 μL

[0109] The PCR amplification products were subjected to agarose gel electrophoresis and recovered using a small amount of DNA recovery kit.

[0110] After the PCR reaction, the PCR product and the vector plasmid were double-digested with BamHI and NotI, and then purified and recovered. The PCR product was ligated with the vector plasmid pBSA43 that had been double-digested with BamHI and NotI, and transformed into the cloning host E. coli JM109. The product was spread on LB solid medium and incubated in a 37°C incubator for 12 hours to obtain transformants. The plasmid in the transformant was extracted for PCR and double-digestion verification, such as Figure 2 As shown, sequencing verification was successfully performed to obtain the mutant plasmid.

[0111] The plasmid was transformed into Bacillus subtilis WB600, spread on LB solid medium containing kanamycin resistance, and cultured in a 37°C incubator for 12 hours to obtain the mutant H443N / S199H, the corresponding coding gene of which is pldm. The plasmid containing the gene was named pBSA43-pldm, and the recombinant strain was named Bacillus subtilis recombinant strain WB600 / pBSA43-pldm.

[0112] Example 3: Expression and purification of wild-type PLD and mutant H443N / S199H

[0113] 1. Expression of wild-type PLD and mutant H443N / S199H: The Bacillus subtilis strain WB600 / pBSA43-pld expressing wild-type phospholipase D and the Bacillus subtilis transformant WB600 / pBSA43-pldm expressing H443N / S199H were activated in solid plate culture. A single colony was picked and inoculated into 5 mL of liquid LB medium (Kan resistance) and cultured with shaking at 37°C, 220 rpm for 8 h. The inoculum size was then transferred to 50 mL of liquid LB medium (Kan resistance) with a 2% inoculum size and cultured with shaking at 37°C, 220 rpm for 48 h. The supernatant was centrifuged to obtain a crude enzyme solution.

[0114] 2. Purification of wild-type PLD and mutant H443N / S199H: Wash the Ni in the chromatography column with ddH2O 2+ Resin, rinse away residual ethanol, and then add two column volumes of Lysis Buffer to balance the pH of the resin;

[0115] The crude enzyme solutions of wild-type PLD and mutant H443N / S199H prepared in step 1 were mixed with pre-treated Ni 2+ The resin was mixed and combined (100r / min) for 60min, and then poured into the chromatography column to make Ni 2+ After the resin is deposited in the column and the filtrate is exhausted, 10 mL of Wash Buffer is added to wash away weakly bound impurities.

[0116] After the Wash Buffer has drained away, add 10 mL of Elution Buffer to elute the target protein bound to the resin, and collect the filtrate containing the target protein.

[0117] The collected filtrate contained a high concentration of imidazole, which affected the subsequent experiments. Therefore, the imidazole was replaced with Tris-HCl (pH 7.0, 50 mM), and the purified enzyme solution was collected and analyzed by SDS-PAGE. The results are shown in Figure 2. Figure 3 As shown, a single band with a size of 54 kDa was obtained.

[0118] Enzyme activity assay: The transesterification activity of PS was determined by catalyzing the synthesis of PS using PC and L-serine as substrates. The reaction was carried out in a biphasic system: 30 mg of L-serine was dissolved in 1 mL of acetic acid-sodium acetate buffer (0.2 M, pH 5.5), and 25 mg of PC was dissolved in 1.5 mL of ethyl lactate. Then, 0.12 mL of 0.5 mg / mL pure enzyme solution and CaCl2 (a final concentration of 25 mM) were added. The reaction was incubated at 50°C, 200 rpm / min, and the mixture was shaken in a water shaker for 20 minutes. After completion of the reaction, the mixture was extracted with 3 mL of chloroform / methanol (2:1, v / v) solution.

[0119] The lower layer of solution was removed and centrifuged at 12,000 rpm / min for 1 minute. PS was then analyzed by high-performance liquid chromatography using an Agilent 1260 high-performance liquid chromatograph with a UV detector. The chromatographic column was a Venusil XBP Silica (5 μm, 2.1 × 150 mm), the mobile phase was acetonitrile / methanol / 85% phosphoric acid (95:5:0.8, v / v / v), the UV detection wavelength was 205 nm, the flow rate was set to 0.3 mL / min, the column temperature was maintained at 25°C, and the injection volume was 10 μL. PS standard solutions with concentrations of 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1.0 mg / mL, 1.5 mg / mL, and 2.0 mg / mL were prepared for the above-mentioned tests, and a standard curve was drawn.

[0120] Enzyme activity determination: The enzyme activities of the pure enzyme solutions of wild-type PLD and mutant H443N / S199H were measured using the enzyme activity assay method. Calculation of the specific activity revealed that the wild-type enzyme had a specific activity of 1.90 U / mg, while the H443N / S199H mutant had a specific activity of 5.98 U / mg, 3.14 times that of the wild-type. This resulted in the identification of the mutant H443N / S199H, which exhibited significantly enhanced enzyme activity.

[0121] Example 4: Construction of a recombinant strain expressing a PLD mutant of Bacillus amyloliquefaciens

[0122] 1. Preparation of competent cells of Bacillus amyloliquefaciens CGMCC No.11218:

[0123] (1) Activate the strain and culture at 37°C for 24 h;

[0124] (2) Pick a single colony and inoculate it into LBS medium and culture it in a 37°C incubator at 220 rpm for 12 h;

[0125] (3) Inoculate 100 mL of LBS medium with a 2% inoculum and culture at 220 rpm in a 37°C incubator for 2.5 h to obtain an OD 600 The pH value is 0.4-0.6, centrifuge and discard the supernatant;

[0126] (4) Resuspend the cells in 30 mL of washing buffer (0.5 M sorbitol, 0.5 M mannitol, 10% glycerol), centrifuge, discard the supernatant, and repeat the washing process three times.

[0127] (5) Resuspend the cells in 10 mL of buffer (0.5 M sorbitol, 0.5 M mannitol, 10% glycerol, 14% PEG6000) and aliquot the competent cells. Store at -80°C until use.

[0128] 2. Electrotransformation of Bacillus amyloliquefaciens: Clean the electroporation cuvette with 75% alcohol; mix 10 ng of recombinant plasmid (pBSA43-pldm) and 100 μL of competent medium, transfer to an electroporation cuvette, and incubate on ice for 2 min; electroporate at 2100-2500 V for 4-6 ms, then immediately add 1 mL of resuscitation solution (LB + 0.5 M sorbitol + 0.38 M mannitol) and incubate at 37°C, 220 rpm for 2-3 h. Plate the cells on a solid culture medium containing kanamycin resistance, select transformants, extract the plasmid, and verify by enzyme digestion to obtain the recombinant Bacillus amyloliquefaciens strain CGMCC No. 11218 / pBSA43-pldm containing pBSA43-pldm.

[0129] The wild-type PLD recombinant strain CGMCC No.11218 / pBSA43-pld was prepared by the same method.

[0130] Example 5: Expression and preparation of wild-type PLD and mutant H443N / S199H in Bacillus amyloliquefaciens

[0131] 1. Activate the recombinant Bacillus amyloliquefaciens strains CGMCC No.11218 / pBSA43-pldm and CGMCC No.11218 / pBSA43-pld respectively;

[0132] 2. Transfer the inoculum to 50 mL of LB medium (containing kanamycin resistance) at a 2% inoculum volume, incubate at 37°C, 220 rpm for 48 h, and centrifuge at 12,000 rpm for 10 min to collect the fermentation supernatant to obtain crude enzyme solutions of H443N / S199H and wild-type PLD, respectively.

[0133] 3. Enzyme Activity Assay: The transesterification activity of PLD was determined by measuring the amount of PS produced by PLD using PC and L-serine as substrates. The reaction was performed in a biphasic system: 30 mg of L-serine was dissolved in 1 mL of 0.2 M acetic acid-sodium acetate buffer (pH 5.5), while 25 mg of PC was dissolved in 1.5 mL of ethyl lactate. 500 μL of crude enzyme solution and CaCl2 were added to a final concentration of 25 mM. The mixture was shaken at 50°C and 200 rpm for 20 min. After completion of the reaction, the mixture was extracted with 3 mL of chloroform / methanol (2:1, v / v).

[0134] Under the above catalytic conditions, the amount of enzyme required to produce 1 μmol of PS per minute using PC and L-serine as substrates is defined as one unit of transesterase activity, expressed as U / mL. Results showed that in the Bacillus amyloliquefaciens expression system, the wild-type enzyme had an activity of 17.52 U / mL, while the H443N / S199H enzyme had an activity of 62.79 U / mL.

[0135] Example 6: Construction of a recombinant strain expressing a PLD mutant of Bacillus licheniformis

[0136] 1. Preparation of competent Bacillus licheniformis 2709:

[0137] (1) Activate the strain and culture at 37°C for 24 h;

[0138] (2) Pick a single colony and inoculate it into LBS medium and culture it in a 37°C incubator at 220 rpm for 12 h;

[0139] (3) Inoculate 100 mL of LBS medium with a 2% inoculum and culture at 220 rpm in a 37°C incubator for 2.5 h to obtain an OD 600 The concentration is 0.8-1.0, centrifuge and discard the supernatant;

[0140] (4) Resuspend the cells in 30 mL of washing buffer (0.5 M sorbitol, 0.5 M mannitol, 10% glycerol), centrifuge, discard the supernatant, and repeat the washing process three times.

[0141] (5) Resuspend the cells in 10 mL of buffer (0.5 M sorbitol, 0.5 M mannitol, 10% glycerol, 14% PEG6000) and aliquot the competent cells. Store at -80°C until use.

[0142] 2. Electrotransformation of Bacillus licheniformis: Clean the electroporation cuvette with 75% alcohol; mix 10 ng of recombinant plasmid (pBSA43-pldm) and 100 μL of competent medium, transfer to the electroporation cuvette, and incubate on ice for 2 min; electroporate at 2100-2500 V for 4-6 ms, then immediately add 1 mL of resuscitation solution (LB + 0.5 M sorbitol + 0.38 M mannitol) and resuscitate at 37°C, 220 rpm for 2-3 h. Spread the solution on a solid culture medium containing kanamycin resistance, select transformants, extract the plasmid, and verify by enzyme digestion to obtain Bacillus licheniformis BL / pBSA43-pldm containing pBSA43-pldm.

[0143] The wild-type PLD recombinant strain BL / pBSA43-pld was prepared using the same method.

[0144] Example 7: Expression and preparation of wild-type PLD and mutant H443N / S199H in Bacillus licheniformis

[0145] 1. Activate the recombinant Bacillus licheniformis strains BL / pBSA43-pldm and BL / pBSA43-pld respectively;

[0146] 2. Transfer the inoculum to 50 mL of LB medium (containing kanamycin resistance) at a 2% inoculum volume, incubate at 37°C, 220 rpm for 48 h, and centrifuge at 12,000 rpm for 10 min to collect the fermentation supernatant to obtain crude enzyme solutions of H443N / S199H and wild-type PLD, respectively.

[0147] 3. The activity of the PLD mutant H443N / S199H, obtained by fermentation with Bacillus licheniformis, was determined using the enzyme activity assay described in Example 5. Using PC and L-serine as substrates, one unit of enzyme activity (U / mL) was defined as the amount of enzyme required to produce 1 μmol of PS per minute. The results showed that in the Bacillus licheniformis expression system, the wild-type enzyme had an activity of 26.89 U / mL, while the H443N / S199H mutant had an activity of 93.05 U / mL.

[0148] Example 8: Preparation of phosphatidylserine

[0149] 60 mg of L-serine was dissolved in 1 mL of acetic acid-sodium acetate buffer (0.2 M, pH 5.5), and 30 mg of PC was dissolved in 3 mL of ethyl lactate. CaCl2 was added to a final concentration of 25 mM. 500 μL of the crude H443N / S199H enzyme solution / wild-type PLD enzyme solution prepared in Example 7 were taken and reacted in a water bath shaker at 40°C and 200 rpm / min for 3 h. After the reaction, PS was extracted with 3 mL of chloroform / methanol (2:1, v / v) solution. The PS yield was determined by high performance liquid chromatography. The yield of PS synthesized by wild-type PLD was 44.0%, while the yield of PS synthesized by mutant H443N / S199H was 71.8%, which was 1.63 times that of wild-type PLD (PS yield (molar %) = PS yield / initial PC amount × 100%).

[0150] The above-described embodiments merely represent several implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that those skilled in the art will appreciate that variations, combinations, and improvements to the above-described embodiments are possible without departing from the scope of the present invention, and all such improvements and modifications are intended to fall within the scope of protection of the appended claims.

Claims

1. A phospholipase D mutant, characterized in that The phospholipase D mutant is obtained by mutating the 443rd histidine in the wild-type phospholipase D amino acid sequence to asparagine, and the 199th serine to histidine. The amino acid sequence of the mutant is shown in SEQ ID No.

3.

2. A gene encoding a phospholipase D mutant, characterized in that: The gene encodes the phospholipase D mutant according to claim 1.

3. The coding gene according to claim 2, wherein The nucleotide sequence is shown in SEQ ID No.

4.

4. A recombinant vector or recombinant strain comprising the encoding gene according to claim 2.

5. The recombinant vector according to claim 4, wherein The expression vector used was pBSA43.

6. The recombinant strain according to claim 4, characterized in that The host bacteria used are Bacillus subtilis WB600, Bacillus amyloliquefaciens CGMCC No.11218 or Bacillus licheniformis 2709.

7. The use of the phospholipase D mutant according to claim 1, wherein The invention is used in the preparation of phosphatidylserine.

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