Phospholipase D mutant Lip-Gri / NQ, plasmid, recombinant bacterium and application of phospholipase D mutant Lip-Gri / NQ
Patent Information
- Application Number
- CN202511277215.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-09
- Publication Date
- 2025-10-10
- Estimated Expiration
- 2045-09-09
AI Technical Summary
Existing phospholipase D faces problems such as low catalytic activity, hydrolysis side reactions and instability in industrial applications, which limits its application in synthetic biology and industrial catalysis.
By performing site-directed mutagenesis on key amino acid residues in phospholipase D, the phospholipase D mutant Lip-Gri/NQ was constructed. The aspartic acid at position 123 and the glutamate at position 306 were mutated to asparagine and glutamine, respectively, forming a double mutant and improving its catalytic activity.
The catalytic activity of the phospholipase D mutant Lip-Gri/NQ is significantly improved, reaching 11 times that of the wild type. It has better stability and applicability and is suitable for generating functional lipids with specific configurations.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a phospholipase D mutant Lip-Gri / NQ, a plasmid, a recombinant bacterium and applications thereof. Background Art
[0002] Phospholipase D (EC 3.1.4.4) is a class of phosphodiesterases widely found in microorganisms and plants. It catalyzes the hydrolysis of the ester bond between phosphatidic acid and the polar head group in phospholipid molecules and can also undergo phosphatidyl transfer in the presence of alcohols to synthesize novel phospholipids. This enzyme uses calcium ions as a cofactor and is expressed in many actinomycetes and plants. Phospholipase D exhibits excellent regioselectivity and broad substrate adaptability, acting on a variety of phospholipid substrates, such as phosphatidylcholine and phosphatidylethanolamine, to produce the corresponding phosphatidic acid or modified phospholipids. These properties give phospholipase D broad potential for application in food processing, pharmaceutical preparation, liposome construction, and functional lipid synthesis.
[0003] Although phospholipase D-catalyzed synthesis of rare phospholipids has been extensively studied, its industrial application still faces significant challenges, including hydrolytic side reactions, the instability of the free enzyme itself, and low reusability. To further expand the application of phospholipase D in synthetic biology and industrial catalysis, it is necessary to identify the key amino acid residues in the molecule that affect catalytic performance and then construct phospholipase D mutants with enhanced catalytic activity. Summary of the Invention
[0004] In response to the deficiencies in the prior art, the present invention provides a phospholipase D mutant Lip-Gri / NQ, a plasmid, a recombinant bacterium, and applications thereof. By identifying the key amino acid residues in phospholipase D that affect the catalytic performance, the catalytic activity of phospholipase D is improved, laying the foundation for its industrial application.
[0005] The present invention is achieved through the following technical solutions: A phospholipase D mutant Lip-Gri / NQ, the amino acid sequence of the phospholipase D mutant is shown in SEQ ID NO: 1, compared with the wild-type phospholipase D Lip-Gri (GenBank ID: WP_125214065.1), the 123rd and 306th amino acids are subjected to site-directed mutagenesis.
[0006] Furthermore, the wild-type phospholipase D Lip-Gri is derived from Streptomyces griseofuscus .
[0007] The present invention also provides a gene encoding the phospholipase D mutant Lip-Gri / NQ, the nucleotide sequence of which is shown in SEQ ID NO.2.
[0008] The present invention also provides a recombinant plasmid carrying the nucleotide sequence shown in SEQ ID NO. 2, and the expression vector is preferably pET28a.
[0009] The present invention also provides a recombinant engineered strain transformed with the above recombinant plasmid, wherein the expression host is preferably E. coli BL21(DE3).
[0010] The present invention also provides applications of the phospholipase D mutant Lip-Gri / NQ, and the application scope includes but is not limited to using the phospholipase D mutant Lip-Gri / NQ to catalyze phospholipids to generate functional lipids of specific configurations including phosphatidylserine.
[0011] The present invention offers advantages over existing technologies: This invention addresses the low catalytic activity of existing phospholipase D enzymes. Based on the wild-type phospholipase D Lip-Gri, the invention modifies it through site-directed mutagenesis. This results in the first double-mutant Lip-Gri / NQ, in which aspartic acid at position 123 and glutamic acid at position 306 are mutated to asparagine and glutamine, respectively. The resulting mutant exhibits significantly enhanced enzyme activity, reaching 11 times that of the wild-type. The double-mutant Lip-Gri / NQ phospholipase D of the present invention has significant application value in the production of functional lipids with specific configurations, such as phosphatidylserine, from phospholipids. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1 This is a graph showing the effect of temperature on the enzyme activity of the phospholipase D mutant Lip-Gri / NQ; Figure 2 This is a graph showing the effect of pH on the enzyme activity of the phospholipase D mutant Lip-Gri / NQ; Figure 3 The effect of temperature on the stability of phospholipase D mutant Lip-Gri / NQ enzyme; Figure 4 This figure shows the effect of pH on the stability of the phospholipase D mutant Lip-Gri / NQ enzyme. DETAILED DESCRIPTION
[0013] The methods of the present invention are further described below by way of examples with reference to the accompanying drawings. However, the experimental conditions used in the examples can be selected based on existing techniques. For experimental methods where specific conditions are not specified in the examples, conventional conditions or those recommended by the manufacturer can generally be used.
[0014] Example 1. Construction of phospholipase D mutants The wild-type phospholipase D Lip-Gri plasmid was used as a template and PCR amplification was performed using the primer pairs D123NF, D123NR and E306QF, E306QR in Table 1, respectively, to construct the phospholipase D mutants Lip-Gri / 123N and Lip-Gri / 306Q. Compared with the wild-type phospholipase D Lip-Gri, Lip-Gri / 123N mutated the aspartic acid at position 123 to asparagine, and Lip-Gri / 306Q mutated the glutamic acid at position 306 to glutamine. Table 1 Primer sequences .
[0015] Example 2: Detection of hydrolytic activity of phospholipase D single mutants The two single mutants constructed in Example 1 were assayed for enzymatic activity and compared with wild-type phospholipase D Lip-Gri. Phospholipase D activity was determined using an enzyme-linked colorimetric assay as follows: 30 μL of 10 mg / mL phosphatidylcholine substrate solution, 39 μL of 100 mM potassium dihydrogen phosphate buffer (pH 7.0), 20 μL of 20 mM CaCl₂ solution, and 1 μL of 10% v / v Trition X-100 solution were added to the reaction system. The reaction was incubated at 40°C for 20 minutes, and then terminated by boiling in a water bath for 5 minutes. Add 200 μL of colorimetric solution to the cooled reaction mixture, mix thoroughly, and incubate in a 37°C water bath for 60 min for color development. The colorimetric solution consists of 50 U of choline oxidase, 100 U of horseradish peroxidase, 0.10 g of 4-aminoantipyrine, 1.00 g of Triton X-100, and 0.05 g of phenol in 100 mL of Tris-HCl buffer (1 M, pH 8.0). The absorbance of the reaction product at 500 nm is measured using a microplate reader to calculate the enzyme activity. Enzyme activity is defined as the amount of enzyme required to hydrolyze 1 μmol of choline per minute.
[0016] Example 3: Construction, induced expression and purification of the phospholipase D mutant Lip-Gri / NQ The above two sites were combined and mutated, and PCR amplification was performed using the Lip-Gri / 123N plasmid as a template and E306QF and E306QR as primers. Dpn After digestion with I, the cells were transformed into Escherichia coli BL21 (DE3) to construct the phospholipase D mutant Lip-Gri / NQ. Compared with the wild-type phospholipase D Lip-Gri, the aspartic acid at position 123 and the glutamic acid at position 306 of Lip-Gri / NQ were mutated to asparagine and glutamine, respectively.
[0017] The recombinant bacteria containing the phospholipase D mutant Lip-Gri / NQ were inoculated into LB medium and cultured at 37°C for approximately 3 hours. The cells were then incubated with IPTG at a final concentration of 0.1 mM at 25°C for an additional 18 hours. The cells were harvested by centrifugation and disrupted by sonication. The protein was purified using a Ni-NTA affinity column, and the target protein was eluted using NPI-200. The purified phospholipase D mutant Lip-Gri / NQ was then removed by ultrafiltration and concentrated at 4°C to obtain the purified phospholipase D mutant.
[0018] SEQ ID NO:1 amino acid sequence of phospholipase D mutant Lip-Gri / NQ: ; SEQ ID NO: 1 Nucleotide sequence of phospholipase D mutant Lip-Gri / NQ:
[0019] Enzymatic property detection of phospholipase D mutant Lip-Gri / NQ Hydrolytic activity detection of phospholipase D mutant The hydrolytic activity of phospholipase D mutant Lip-Gri / NQ was measured by the activity detection method of phospholipase D in Example 2 above, and was 421.26 U / mg, which was 11.05 times of wild-type phospholipase D Lip-Gri.
[0020] Effect of temperature and pH on the enzymatic activity of phospholipase D mutant Lip-Gri / NQ The obtained phospholipase D mutant Lip-Gri / NQ was diluted with potassium dihydrogen phosphate buffer (100 mM, pH 7.0) to an appropriate concentration, and the enzymatic reaction was carried out at 20-60°C to study the effect of temperature on its enzymatic activity. The obtained phospholipase D mutant Lip-Gri / NQ enzyme solution was diluted with different buffers, and the buffers used for dilution were sodium citrate buffer (50 mM, pH 4.0-6.0), phosphate buffer (50 mM, pH 6.0-8.0) and Tris-HCl buffer (50 mM, pH 8.0-9.0), respectively, and then the reaction was carried out at 40°C to study the effect of pH on its enzymatic activity. The highest value of enzymatic activity was taken as 100%, and the results are shown in Figure 1 and Figure 2 The results show that the optimum temperature of phospholipase D mutant Lip-Gri / NQ is 45°C, and the optimum pH is 7.0.
[0021] (3) Effect of temperature and pH on the stability of phospholipase D mutant Lip-Gri / NQ The obtained phospholipase D mutant Lip-Gri / NQ was diluted with potassium dihydrogen phosphate buffer (100 mM, pH 7.0) to an appropriate concentration, and the residual enzyme activity was tested after incubation at 30°C, 40°C and 50°C for different times to study the effect of temperature on its stability. The obtained phospholipase D mutant Lip-Gri / NQ was incubated in phosphate buffer at different pH (pH 6.0-8.0) for different times, and then the residual enzyme activity was detected to study the effect of pH on its stability. The results are shown in Figure 3 and Figure 4 When Lip-Gri / NQ was stored at 50°C for 8 h, it could retain 50% of its initial enzyme activity. It showed the maximum stability at pH 7.0, and could still retain 60% of the activity after incubation for 20 h.
[0022] The above embodiments are only for illustrating the technical concept and features of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art may make various changes or modifications based on the present invention, and such equivalent forms are also included within the scope defined by the appended claims of this application.
Claims
1. A phospholipase D mutant Lip-Gri / NQ, characterized in that The amino acid sequence of the phospholipase D mutant is shown in SEQ ID NO:
1.
2. The gene encoding the phospholipase D mutant Lip-Gri / NQ according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.
2.
3. A recombinant plasmid, characterized in that: The recombinant plasmid carries the nucleotide sequence shown in SEQ ID NO.2, and the expression vector is pET28a.
4. The recombinant engineered strain transformed with the recombinant plasmid according to claim 3, characterized in that: The expression host is E. coli BL21(DE3).
5. The use of the phospholipase D mutant Lip-Gri / NQ according to claim 1, characterized in that: The application includes but is not limited to using the phospholipase D mutant Lip-Gri / NQ to catalyze phospholipids to generate functional lipids with specific configurations including phosphatidylserine.