Phospholipase d mutant and use thereof

Abstract

This invention discloses a phospholipase D mutant and its applications, the amino acid sequence of which is shown in SEQ ID NO: 02. This invention significantly improves the synthesis yield of phosphatidylglycerol (PG), optimizes the enzyme's catalytic efficiency under organic solvent-free conditions, and can meet the production requirements of industrial-scale production.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering and biocatalysis technology, specifically relating to a phospholipase D mutant and its applications. Background Technology

[0002] Phospholipase D (PLD, EC 3.1.4.4), as an important biocatalyst, plays a crucial role in lipid modification and the synthesis of functional phospholipids. Its core catalytic function lies in its ability to specifically cleave the phosphodiester bond in phospholipid molecules and, under suitable conditions, catalyze transphosphatidylation, thereby replacing the polar head group of the phospholipid with a group from other alcohols or amines. This unique catalytic mechanism provides an efficient and green enzymatic synthetic route for the preparation of phospholipids—such as phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylinositol (PI)—which are scarce in nature but possess important physiological functions. Compared with traditional physical extraction and chemical synthesis methods, PLD-based enzymatic synthesis technology has significant advantages such as mild reaction conditions, environmental friendliness, high product specificity, and simplified process flow, making it the most important and cutting-edge technology in the current field of functional phospholipid preparation.

[0003] Phosphatidylglycerol (PG) exhibits broad application prospects in multiple fields due to its unique structure and physicochemical properties. In the pharmaceutical field, PG is one of the main components of pulmonary surfactant, effectively reducing alveolar surface tension and preventing alveolar collapse, thus possessing significant clinical value in the treatment of respiratory distress syndrome in premature infants. Furthermore, PG is widely studied as a targeted delivery carrier for anticancer drugs, utilizing its affinity for tumor cells to achieve precise drug release at the lesion site, thereby improving efficacy and reducing toxic side effects. In the cosmetics and personal care industry, PG, with its excellent biocompatibility, biodegradability, and film-forming properties, is extensively used as an emulsifier, moisturizer, and raw material for liposome preparation, significantly improving product texture and stability, and enhancing the skin penetration and absorption of active ingredients. These broad application needs urgently require the development of efficient, low-cost, and highly safe PG synthesis technologies.

[0004] However, the reaction systems currently used for PLD-catalyzed synthesis of PG are mainly water-organic solvent biphase systems. Although this system can achieve high PG yields, the problem of residual organic solvents directly affects the safety of the product as food and pharmaceuticals. Therefore, developing a phospholipase D mutant capable of efficiently catalyzing the synthesis of PG in a pure aqueous system has become a key technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to overcome the defects of the prior art and provide a phospholipase D mutant.

[0006] Another object of the present invention is to provide the application of the above-mentioned phospholipase D mutant.

[0007] The technical solution of the present invention is as follows:

[0008] A phospholipase D mutant, the amino acid sequence of which is shown in SEQ ID NO: 02.

[0009] A nucleic acid molecule that encodes the aforementioned phospholipase D mutant.

[0010] An expression vector loaded with the aforementioned nucleic acid molecules.

[0011] In a preferred embodiment of the invention, it is based on pBADKP (see CN109136207B).

[0012] A recombinant cell loaded with the above-mentioned expression vector.

[0013] In a preferred embodiment of the invention, it is constructed based on Escherichia coli.

[0014] A method for preparing phosphatidylglycerol in a pure aqueous system, using the phospholipase D mutant to catalyze the transphosphatidyl reaction of lecithin and glycerol, the transphosphatidyl reaction being carried out in a buffer system free of organic solvents.

[0015] In a preferred embodiment of the present invention, the buffer system uses water as a solvent and contains 10 mM EDTA, 0.05% sodium deoxycholate, 200 mM Tris HCl, 24 mM glycerol and 10 mM CaCl2, with a pH of 7.5.

[0016] The application of the phospholipase D mutant in the preparation of phosphatidylglycerol is carried out in a buffer system free of organic solvents.

[0017] In a preferred embodiment of the present invention, the buffer system uses water as a solvent and contains 10 mM EDTA, 0.05% sodium deoxycholate, 200 mM Tris HCl, 24 mM glycerol and 10 mM CaCl2, with a pH of 7.5.

[0018] The beneficial effects of this invention are:

[0019] 1. This invention significantly improves the synthesis yield of phosphatidylglycerol (PG), optimizes the catalytic efficiency of the enzyme under organic solvent-free conditions, and can meet the production requirements of industrial-scale production.

[0020] 2. This invention significantly reduces the activity of hydrolytic enzymes, effectively inhibits hydrolytic side reactions during PG synthesis, reduces the generation of by-products, and improves the selectivity of the reaction.

[0021] 3. This invention significantly improves the activity of transphosphatidyl acylase, enhances the enzyme's ability to transesterify substrates, and further promotes the green and efficient synthesis of PG.

[0022] 4. This invention completely avoids the use of organic solvents, improves the safety of the product PG as food and medicine, and meets the strict safety standards of the green chemistry and biopharmaceutical industry.

[0023] 5. This invention simplifies the downstream separation and purification steps, significantly reduces production costs and environmental impact, and provides higher economic value and technical feasibility for the industrial application of PG.

[0024] 6. This invention achieves highly efficient catalysis in a pure aqueous system, providing a green process route that can be referenced for the enzymatic synthesis of other rare phospholipids, and has broad prospects for industrialization. Attached Figure Description

[0025] Figure 1 This shows the yield of PG synthesized by the phospholipase D mutant in Example 3 of the present invention.

[0026] Figure 2 This demonstrates the hydrolytic activity of the phospholipase D mutant in Example 4 of the present invention.

[0027] Figure 3 This shows the enzyme reaction kinetic parameters of the phospholipase D mutant G381I in Example 5 of the present invention. Detailed Implementation

[0028] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0029] In the following examples, the wild-type strain E. coli TOP10 / pBADKP-PLD2 was constructed as shown in Chinese Invention Patent CN109136207B.

[0030] Example 1: Construction of a heterologous expression strain of phospholipase D mutant

[0031] (1) Wild-type strain E. coli TOP10 / pBADKP-PLD2 was cultured. This strain contains the phospholipase D gene (Genebank no. MH237968) from Streptomyces antibioticus. Referring to the complete nucleotide sequence of phospholipase D in the wild-type strain (as shown in SEQ ID NO: 01), the coding sequence of the phospholipase D gene was designed according to the codon preference of E. coli. Different upstream and downstream primers for the mutation were designed and generated by snapgene software. The mutants constructed in this embodiment are G381I, W187A, W187I, D190A, D190I, Y191A, Y191I, G381A, G383A, G383I, Y385A and Y385I.

[0032] SEQ ID NO: 01: ADTPPTPHLDAIERSLRDTSPGLEGSVWQRTDGNRLDAPDGDPAGWLLQTPGCWGDAGCKDRAGTRRLLDKMTRNIADARHTVDISSLAPFPNGGFEDAVVDGLKAVVAAGHSPRVRILVGAA PIYHLNVVPSRYRDELIGKLGAAAGKVTLNVASMTTSKTSLSWNHSKLLVVDGKTAITGGINGWKDDYLDTAHPVSDVDMALSGPAAASAGKYLDTLWDWTCRNASDPAKVWLATSNGASCMPSMEQDE AGSAPAEPTGDVPVIAVGGLGVGIKESDPSSGYHPDLPTAPDTKCTVGLHDNTNADRDYDTVNPEENALRSLIASARSHVEISQQDLNATCPPLPRYDIRTYDTLAGKLAAGVKVRIVVSDPANRGAVG SGGYSQIKSLDEISDTLRTRLVALTGDNEKASRALCGNLQLASFRSSDAAKWADGKPYALHHKLVSVDDSAFYIGSKNLYPAWLQDFGYIVESPAAAQQLKTELLDPEWKYSQQAAATPAGCPARQAG;

[0033] (2) Take 1.5 mL of the overnight culture and extract the plasmid pBADKP of the wild-type strain using the Plasmid Mini Kit I kit. Use this plasmid as a template and construct the mutant by PCR amplification of the template plasmid using the designed mutant primers. The PCR amplification system is shown in Table 1.

[0034] Table 1 PCR amplification system

[0035] upstream primer 1 Downstream primer 1 Takara PrimerSTAR Max DNA Polymerase 25 Template 0.5 <![CDATA[H2O]]> 22.5 Overall reaction system 50

[0036] The PCR amplification conditions were as follows: 98 ℃ for 5 min; 98 ℃ for 10 s; 55-65 ℃ (the specific temperature was set according to the primer Tm value) for 30 s; 72 ℃ for 2 min; 30 cycles; 72 ℃ for 10 min.

[0037] (3) The template plasmid was digested with 2 μL Dpn I enzyme and the system was placed at 37°C for 1.5 h. The digestion product was then transformed into E. coli competent cells E. coli top10 by heat shock and plated on LB solid plates containing kanamycin for culture. Single clones were selected for LB liquid culture and the target mutant strain was obtained after sequencing identification.

[0038] Example 2: Fermentation of wild-type phospholipase D and its mutant expression strains, and extraction of phospholipase D.

[0039] (1) Wild-type phospholipase D and different mutant recombinant Escherichia coli strains obtained in Example 1 were inoculated into 10 mL LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) and cultured at 37°C and 200 rpm for 12-16 h.

[0040] (2) At an inoculation rate of 1% (v / v), transfer to 20 mL of TB medium (yeast extract 24 g / L, peptone 12 g / L, potassium dihydrogen phosphate 2.31 g / L, dipotassium hydrogen phosphate 12.54 g / L, glycerol 4 ml / L), and incubate at 37°C with a shaker at 200 rpm for 5-6 h until the OD600 is 5-6.

[0041] (3) Collect the cells by centrifugation at 10°C and 5000 rpm for 5 min. Add 20 mL of TB adjustment medium (10 g / L peptone; 5 g / L yeast extract; 5 g / L glycerol; 0.1 M potassium phosphate buffer, pH 7.2) to the same volume as before centrifugation, resuspend the cells, and add 0.035%·OD600. -1 Arabicose and 0.4 M sodium chloride were used to induce expression for 16 h in a shaker at 18°C ​​and 200 rpm.

[0042] (4) Centrifuge the obtained fermentation broth at 10°C and 10,000 rpm for 5 min, resuspend the cells in an equal volume of PLD extraction buffer (10 mM EDTA, 0.05% sodium deoxycholate, 200 mM Tris-HCl, pH 7.5), and incubate at room temperature for 1 h, vortexing for 5 s every 20 min. Then centrifuge at 4°C and 12,000 rpm for 10 min, and the supernatant is the obtained crude PLD enzyme solution.

[0043] Example 3: Wild-type and mutant phospholipase D catalyzes the synthesis of phosphatidylglycerol from lecithin in a pure aqueous system.

[0044] 1. The reaction system of phospholipase D catalyzing the synthesis of phosphatidylglycerol from lecithin.

[0045] 24 mM glycerol and 10 mM CaCl2 were added to 2.5 mL of PLD extract (10 mM EDTA, 0.05% sodium deoxycholate, 200 mM Tris HCl, pH 7.5), followed by 2.5 mL of crude PLD enzyme solution (prepared in Example 2) to form the aqueous phase. 10 mM lecithin was weighed into 5 mL of the aqueous phase and reacted at 40 °C and 200 rpm in a shaker for 12 h. After the reaction, the aqueous phase was separated by passing the reaction system through a 0.22 μm organic filter membrane using a vacuum filtration device; the product PG was retained on the filter membrane.

[0046] 2. Phosphatidylglycerol detection

[0047] After the reaction was completed, the dried PG product was dissolved in the mobile phase, filtered through a 0.22 μm organic filter membrane, and detected by high performance liquid chromatography. The detection conditions are shown in Table 2.

[0048] Table 2 HPLC detection conditions

[0049] mobile phase hexane:isopropanol:1% acetic acid = 8:8:1 (v / v / v) Flow rate 1.5 mL / min Column temperature 30 ℃ UV detection wavelength 203 nm Injection volume 10 μL

[0050] The yields of wild-type and mutant phospholipase D catalyzing the synthesis of PG from lecithin are as follows: Figure 1 As shown, compared to the wild type, the mutant phospholipase D significantly improved the yield of PG production in a pure aqueous system. The PG yield of G381I was 1.47 times that of the wild type. This demonstrates that the phospholipase D mutant G381I designed in Example 1 can effectively improve the yield of phosphatidylglycerol production, and is more suitable for catalytic production of phosphatidylglycerol than the wild type. Furthermore, the phosphatidylglycerol produced in this reaction system does not use organic solvents, making it safer and more environmentally friendly.

[0051] The amino acid sequence of the phospholipase D mutant G381I is shown in SEQ ID NO: 02: ADTPPTPHLDAIERSLRDTSPGLEGSVWQRTDGNRLDAPDGDPAGWLLQTPGCWGDAGCKDRAGTRRLLDKMTRNIADARHTVDISSLAPFPNGGFEDAVVDGLKAVVAAGHSPRVRILVGAAPIYHLNVVPSRYRDELIGKLGAAAGKVTLNVASMTTSKTSLSWNHSKLLVVDGKTAITGGINGWKDDYLDTAHPVSDVDMALSGPAAASAGKYLDTLWDWTCRNASDPAKVWLATSNGASCMPSMEQD EAGSAPAEPTGDVPVIAVGGLGVGIKESDPSSGYHPDLPTAPDTKCTVGLHDNTNADRDYDTVNPEENALRSLIASARSHVEISQQDLNATCPPLPRYDIRTYDTLAGKLAAGVKVRIVVSDPANRGAV ISGGYSQIKSLDEISDTLRTRLVALTGDNEKASRALCGNLQLASFRSSDAAKWADGKPYALHHKLVSVDDSAFYIGSKNLYPAWLQDFGYIVESPAAAQQLKTELLDPEWKYSQQAAATPAGCPARQAG;

[0052] The nucleotide sequence of the phospholipase D mutant G381I is shown in SEQ ID NO: 03:

[0053]

[0054] The primer sequences for constructing the mutant G381I are shown in Table 3:

[0055] Table 3 Primer Sequences

[0056] Downstream primer G381I-R ctgagaataaccaccgctaataactgcaccacgatttgccg (SEQ ID NO: 04) Upstream primer G381I-F cggcaaatcgtggtgcagttattagcggtggttattctcag (SEQ ID NO: 05)

[0057] Example 4: Assay of phospholipase D hydrolase activity in wild-type and mutant strains

[0058] (1) Substrate solution: 76 mg phosphatidylcholine (PC, source: soybean, >98%, Aladdin Reagent, Shanghai, China) dissolved in 10 mL of 5% Triton X-100 (w / v) solution;

[0059] (2) Reaction solution: 0.1 mL of 0.1 M Tris-maleate-NaOH (pH 5.5) solution, 0.1 mL of 2.5% Triton X-100 solution and 0.05 mL of 0.01 M CaCl2 solution; 0.1 mL of enzyme sample is mixed with 0.25 mL of reaction solution;

[0060] (3) Incubate 0.15 mL of substrate solution and the above mixed solution in a 37°C water bath for 5 min;

[0061] (4) Add 0.1 mL of enzyme sample to the reaction solution and react in a 37°C water bath for 10 min;

[0062] (5) Immediately add 0.2 mL of stop solution (1 M Tris-HCl, pH 8.0, 0.5 M EDTA), and incubate in a 100°C metal bath for 15 min;

[0063] (6) Cool the reaction solution to room temperature, add 0.2 mL of colorimetric solution (0.1 M Tris-HCl (pH 8.0), 1.25 U choline oxidase, 1 U peroxidase, 1 µmol 4-aminoantipyrine and 1.6 µmol phenol), and incubate in a 37°C water bath for 1 h;

[0064] (7) Dilute the sample at a ratio of 1:8 and add 1% Triton X-100 to the reaction solution. Take 0.2 mL of the sample and measure the absorbance at 500 nm on an ELISA reader.

[0065] A standard curve was determined using choline chloride solutions of different concentrations instead of enzyme samples. The absorbance values ​​were within the range of 0-0.22, and the choline concentration was within the range of 0-0.25 µmol, showing a good linear correlation. Under these conditions, the amount of phospholipase D releasing 1 µmol of choline per minute was defined as one enzyme activity unit (U).

[0066] The results of the assay of wild-type and mutant phospholipase D hydrolase activity are as follows: Figure 2 The hydrolytic enzyme activity of the mutants was significantly reduced. The hydrolytic enzyme activity of W187A was 0.16 times that of the wild type, the hydrolytic enzyme activity of W187I was 0.15 times that of the wild type, the hydrolytic enzyme activity of D190A was 0.36 times that of the wild type, the hydrolytic enzyme activity of D190I was 0.32 times that of the wild type, the hydrolytic enzyme activity of Y191A was 0.24 times that of the wild type, the hydrolytic enzyme activity of Y191I was 0.31 times that of the wild type, the hydrolytic enzyme activity of G381A was 0.31 times that of the wild type, the hydrolytic enzyme activity of G381I was 0.28 times that of the wild type, the hydrolytic enzyme activity of G383A was 0.33 times that of the wild type, the hydrolytic enzyme activity of G383I was 0.31 times that of the wild type, the hydrolytic enzyme activity of Y385A was 0.27 times that of the wild type, and the hydrolytic enzyme activity of Y385I was 0.36 times that of the wild type. The hydrolytic activity of the phospholipase D mutant obtained in Example 1 is significantly reduced, which is beneficial to reducing the hydrolytic side reactions during the synthesis and production of rare phospholipid PG.

[0067] Example 5: Kinetics and transacylase activity assay of wild-type and mutant G381I phospholipase D

[0068] 1. Enzyme kinetics assay

[0069] Add 2.4 mM glycerol and 10 mM CaCl2 to 4 mL of PLD extraction buffer (10 mM EDTA, 0.05% sodium deoxycholate, 200 mM Tris HCl, pH 7.5), then add 1 mL of crude PLD enzyme solution to form the aqueous phase. Weigh lecithin at concentrations of 5, 10, 15, 20, 25, 30, 35, and 40 mM into 5 mL of the aqueous phase and react at 40 °C and 200 rpm for 8 h in a shaker. After the reaction, separate the aqueous phase using a vacuum filtration device through a 0.45 μm organic filter membrane; the product PG is retained on the filter membrane. Dissolve the PG product obtained after the reaction in the mobile phase, filter through a 0.22 μm organic filter membrane, and detect by high-performance liquid chromatography (HPLC). The enzyme kinetic parameters of wild-type and mutant G381I phospholipase D are shown in Table 4, and the Michaelis-Menten equation fitting curves are shown in... Figure 3 As shown.

[0070] 2. Assay for transacylase activity

[0071] 2.4 M glycerol and 40 mM CaCl2 were added to 4 mL of PLD extraction buffer (10 mM EDTA, 0.05% sodium deoxycholate, 200 mM Tris HCl, pH 7.5), followed by 1 mL of crude PLD enzyme solution, forming the aqueous phase. Lecithin at a concentration of 25 mM was added to 5 mL of the aqueous phase, and the mixture was reacted at 40 °C and 200 rpm for 8 h in a shaker. After the reaction, the aqueous phase was separated by vacuum filtration through a 0.45 μm organic filter membrane, while the PG product was retained on the filter membrane. The PG product obtained after the reaction was dissolved in the mobile phase, filtered through a 0.22 μm organic filter membrane, and detected by high-performance liquid chromatography (HPLC). The transacylase activity yields of wild-type and mutant G381I phospholipase D are shown in Table 4.

[0072] Table 4. Kinetic parameters and transacylase activity of wild-type and mutant phospholipase D.

[0073] WT 10.85 0.25 0.116 G381I 19.69 0.37 0.206

[0074] The results showed that the G381I mutant K m The value is higher than that of the wild type, indicating that the G381I mutant has a slightly lower substrate affinity for PC, requiring a higher PC concentration to reach the half-maximum rate. Meanwhile, its V... max The yield was 1.48 times that of the wild type, indicating that the G381I mutant has a higher substrate metabolism capacity. Furthermore, the transphosphatidyl acylase activity of the G381I mutant was 1.78 times that of the wild type. Overall, this demonstrates that the transphosphatidyl acylase activity of the G381I mutant is significantly improved in a pure aqueous system, giving it greater industrial application value.

[0075] In summary, according to embodiments of the present invention, a phospholipase D mutant, G381I, was obtained. The yield of phospholipase D mutant catalyzing the production of phosphatidylglycerol was improved; the PG yield of G381I was 1.47 times that of the wild type. This indicates that the phospholipase D mutant obtained by the present invention can effectively improve the yield of phosphatidylglycerol catalyzed by the enzyme under pure aqueous phase conditions, and is more suitable for catalyzing the production of phosphatidylglycerol compared to the wild type. The hydrolytic activity of these phospholipase D mutants is significantly reduced, which is beneficial for reducing hydrolytic side reactions during the synthesis of rare phospholipids (PG). The transphosphatidylase activity of these phospholipase D mutants is significantly increased, and the K+ of the G381I mutant is significantly higher. m The value is higher than that of the wild type, indicating that the substrate affinity of the G381I mutant is slightly decreased. maxThe yield was 1.48 times that of the wild type, indicating that the G381I mutant has a higher substrate metabolism capacity. In addition, the transphosphatidyl acylase activity of the G381I mutant was 1.78 times that of the wild type, indicating that the transphosphatidyl acylase activity of the G381I mutant is significantly improved, and it has higher industrial application value.

[0076] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A phospholipase D mutant, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

02.

2. A nucleic acid molecule, characterized in that: It encodes the phospholipase D mutant as described in claim 1.

3. An expression vector, characterized in that: It is loaded with the nucleic acid molecule as described in claim 2.

4. An expression vector as described in claim 3, characterized in that: It is based on pBADKP.

5. A recombinant cell, characterized in that: Its load has the expression vector as described in claim 3 or 4.

6. The recombinant cell as described in claim 5, characterized in that: It is based on Escherichia coli.

7. A method for preparing phosphatidylglycerol in a pure aqueous phase system, characterized in that: The phospholipase D mutant of claim 1 is used to catalyze the transphosphatidyl reaction of lecithin with glycerol, which is carried out in a buffer system free of organic solvents.

8. The method as described in claim 7, characterized in that: The buffer system uses water as a solvent and contains 10 mM EDTA, 0.05% sodium deoxycholate, 200 mM Tris HCl, 24 mM glycerol and 10 mM CaCl2, with a pH of 7.

5.

9. The application of the phospholipase D mutant according to claim 1 in the preparation of phosphatidylglycerol, characterized in that: It is carried out in a buffer system that does not contain organic solvents.

10. The application as described in claim 9, characterized in that: The buffer system uses water as a solvent and contains 10 mM EDTA, 0.05% sodium deoxycholate, 200 mM Tris HCl, 24 mM glycerol and 10 mM CaCl2, with a pH of 7.5.

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