Phospholipase d mutant lip-gri / nq, plasmid, recombinant bacteria and application thereof

Abstract

The application relates to a phospholipase D mutant Lip-Gri / NQ, a plasmid, a recombinant bacterium and application thereof, belongs to the field of enzyme engineering, the amino acid sequence of the phospholipase D mutant is shown as SEQ ID NO: 1, the gene for encoding the phospholipase D mutant Lip-Gri / NQ is shown as SEQ ID NO. 2. The application also provides a recombinant plasmid containing the recombinant plasmid phospholipase D mutant Lip-Gri / NQ gene and an engineering bacterium, and application of the phospholipase D mutant Lip-Gri / NQ in catalyzing phospholipid to generate specific configuration functional lipids including phosphatidylserine. The application is obtained by site-directed mutagenesis on wild-type phospholipase D Lip-Gri, the enzyme activity of the mutant is significantly improved, and is 11 times that of the wild type. Streptomyces griseofuscus The enzyme activity of the mutant is significantly improved, and is 11 times that of the wild type.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a phospholipase D mutant Lip-Gri / NQ, a plasmid, a recombinant bacterium, and their applications. Background Technology

[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 perform transphosphatidyl groups in the presence of alcohol to synthesize novel phospholipids. This enzyme uses calcium ions as a cofactor and is expressed in many actinomycetes and plants. Phospholipase D exhibits good regioselectivity and broad substrate adaptability, acting on a variety of phospholipid substrates, such as phosphatidylcholine and phosphatidylethanolamine, to generate the corresponding phosphatidic acid or modify phospholipids. These characteristics give phospholipase D broad application potential in food processing, pharmaceutical preparation, liposome construction, and functional lipid synthesis.

[0003] Although the synthesis of rare phospholipids catalyzed by phospholipase D has been extensively studied, significant challenges remain in its industrial application, including hydrolytic side reactions, the instability of the free enzyme, and low reusability. To further expand the application of phospholipase D in synthetic biology and industrial catalysis, it is necessary to identify key amino acid residues in its molecule that affect catalytic performance, and then construct phospholipase D mutants with enhanced catalytic activity. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a phospholipase D mutant Lip-Gri / NQ, a plasmid, a recombinant bacterial strain, and their applications. By identifying key amino acid residues in phospholipase D that affect its catalytic performance, the catalytic activity of phospholipase D is improved, laying the foundation for its industrial application.

[0005] This invention is achieved through the following technical solution:

[0006] A phospholipase D mutant, Lip-Gri / NQ, has the amino acid sequence shown in SEQ ID NO:1, which is a site-directed mutation at amino acids 123 and 306 compared to wild-type phospholipase D Lip-Gri (GenBank ID: WP_125214065.1).

[0007] Furthermore, the wild-type phospholipase D Lip-Gri is derived from... Streptomyces griseofuscus .

[0008] 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.

[0009] The present invention also provides a recombinant plasmid carrying the nucleotides shown in SEQ ID NO.2, and the expression vector is preferably pET28a.

[0010] This invention also provides a recombinant engineered strain obtained by transformation containing the above-mentioned recombinant plasmid, wherein the preferred expression host is... E. coli BL21(DE3).

[0011] The present invention also provides applications of the phospholipase D mutant Lip-Gri / NQ, the applications of which include, but are not limited to, using the phospholipase D mutant Lip-Gri / NQ to catalyze the generation of phospholipids into specific conformational functional lipids, including phosphatidylserine.

[0012] The beneficial effects of this invention compared to existing technologies are as follows: Addressing the problem of low catalytic activity in existing phospholipase D enzymes, this invention, based on wild-type phospholipase D Lip-Gri, modifies it through site-directed mutagenesis, resulting in the first double-mutant Lip-Gri / NQ enzyme where aspartic acid at position 123 and glutamic acid at position 306 are mutated to asparagine and glutamine, respectively. The resulting mutant exhibits significantly increased enzyme activity, 11 times that of the wild-type. This double-mutant phospholipase D Lip-Gri / NQ enzyme has significant application value in the generation of specific conformational functional lipids such as phosphatidylserine from phospholipids. Attached Figure Description

[0013] Figure 1 Figure 1 shows the effect of temperature on the activity of the phospholipase D mutant Lip-Gri / NQ.

[0014] Figure 2 The graph shows the effect of pH on the activity of the phospholipase D mutant Lip-Gri / NQ.

[0015] Figure 3 Figure 1 shows the effect of temperature on the stability of the phospholipase D mutant Lip-Gri / NQ enzyme.

[0016] Figure 4 The figure shows the effect of pH on the stability of the phospholipase D mutant Lip-Gri / NQ enzyme. Detailed Implementation

[0017] The method of the present invention will be further described below with reference to the accompanying drawings and embodiments. However, the experimental conditions used in the embodiments can be selected based on existing technologies. For experimental methods where specific conditions are not specified in the embodiments, they can generally be operated under conventional conditions or according to the conditions recommended by the manufacturer.

[0018] Example 1: Construction of phospholipase D mutant

[0019] Using the plasmid of wild-type phospholipase D Lip-Gri as a template, PCR amplification was performed using primer pairs D123NF, D123NR and E306QF, E306QR in Table 1, respectively, to construct phospholipase D mutants Lip-Gri / 123N and Lip-Gri / 306Q. Compared with wild-type phospholipase D Lip-Gri, Lip-Gri / 123N mutated aspartic acid at position 123 to asparagine, and Lip-Gri / 306Q mutated glutamic acid at position 306 to glutamine.

[0020] Table 1 Primer Sequences

[0021] .

[0022] Example 2: Detection of hydrolytic activity of phospholipase D single mutant

[0023] The enzyme activity of the two single mutants constructed in Example 1 above was measured and compared with that of wild-type phospholipase D Lip-Gri. The activity of phospholipase D was determined using an enzyme-linked colorimetric assay, specifically as follows: 30 μL of phosphatidylcholine substrate solution (10 mg / mL), 39 μL of potassium dihydrogen phosphate buffer (100 mM, pH 7.0), 20 μL of CaCl2 solution (20 mM), and 1 μL of Tritium X-100 (10%, v / v) solution were added to the reaction system. 10 μL of enzyme solution was added to the reaction system, and the reaction was incubated at 40°C for 20 min, followed by a boiling water bath for 5 min to terminate the reaction. Add 200 μL of colorimetric solution to the cooled reaction solution, mix well, and incubate at 37°C for 60 min for color development. The colorimetric solution is 100 mL Tris-HCl buffer (1 M, pH 8.0) containing 50 U choline oxidase, 100 U horseradish peroxidase, 0.10 g 4-aminoantipyrine, 1.00 g Triton X-100, and 0.05 g phenol. Measure the absorbance of the reaction product at 500 nm using a microplate reader and calculate the enzyme activity. Enzyme activity is defined as the amount of enzyme required to hydrolyze and produce 1 μmol of choline per minute; one enzyme activity unit (U) is defined as this.

[0024] Example 3: Construction, induction of expression and purification of phospholipase D mutant Lip-Gri / NQ

[0025] The two sites were combined and mutated. Using the Lip-Gri / 123N plasmid as a template, PCR amplification was performed with E306QF and E306QR primer pairs. The product was then subjected to... DpnAfter digestion, the enzyme was transformed into E. coli BL21(DE3) to construct the phospholipase D mutant Lip-Gri / NQ. Compared with the wild-type phospholipase D Lip-Gri, Lip-Gri / NQ mutated aspartic acid at position 123 and glutamic acid at position 306 to asparagine and glutamine, respectively.

[0026] The recombinant bacteria containing the phospholipase D mutant Lip-Gri / NQ were inoculated into LB medium and cultured at 37°C for approximately 3 h. Then, they were cultured for another 18 h at 25°C with IPTG to a final concentration of 0.1 mM. The bacterial cells were collected by centrifugation, sonicated, and the protein was purified using a Ni-NTA affinity column. The target protein was eluted with an NPI-200, followed by ultrafiltration at 4°C for desalting and concentration to obtain the purified phospholipase D mutant Lip-Gri / NQ.

[0027] The amino acid sequence of SEQ ID NO:1 phospholipase D mutant Lip-Gri / NQ:

[0028] ;

[0029] The nucleotide sequence of SEQ ID NO:1 phospholipase D mutant Lip-Gri / NQ:

[0030]

[0031] Example 4: Enzymatic Properties of Phospholipase D Mutant Lip-Gri / NQ

[0032] Hydrolytic activity assay of phospholipase D mutant

[0033] Using the phospholipase D activity detection method described in Example 2 above, the hydrolytic activity of the phospholipase D mutant Lip-Gri / NQ was measured to be 421.26 U / mg, which is 11.05 times that of the wild-type phospholipase D Lip-Gri.

[0034] Effects of temperature and pH on the activity of the phospholipase D mutant Lip-Gri / NQ

[0035] The obtained phospholipase D mutant Lip-Gri / NQ was diluted to an appropriate concentration with potassium dihydrogen phosphate buffer (100 mM, pH 7.0), and enzymatic reactions were carried out at 20–60 °C to study the effect of temperature on enzyme activity. The obtained phospholipase D mutant Lip-Gri / NQ enzyme solution was diluted with different buffers: 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), and then the reaction was carried out at 40 °C to study the effect of pH on enzyme activity. The highest enzyme activity was taken as 100%, and the results are as follows: Figure 1 and Figure 2 As shown in the figure. The results show that the optimal temperature for the phospholipase D mutant Lip-Gri / NQ is 45℃, and the optimal pH is 7.0.

[0036] (3) Effects of temperature and pH on the stability of phospholipase D mutant Lip-Gri / NQ enzyme

[0037] The obtained phospholipase D mutant Lip-Gri / NQ was diluted to an appropriate concentration with potassium dihydrogen phosphate buffer (100 mM, pH 7.0) and incubated at 30℃, 40℃, and 50℃ for different times, followed by testing its residual enzyme activity to investigate the effect of temperature on its stability. The obtained phospholipase D mutant Lip-Gri / NQ was also incubated in phosphate buffers at different pH values ​​(pH 6.0-8.0) for different times, and then the residual enzyme activity was measured to investigate the effect of pH on its stability. The results are as follows: Figure 3 and Figure 4 As shown, Lip-Gri / NQ retains 50% of its initial enzyme activity when stored at 50°C for 8 h. It exhibits maximum stability at pH 7.0, retaining 60% of its activity even after 20 h of incubation.

[0038] The above embodiments are only for illustrating the technical concept and features of the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make various modifications or alterations to the present invention, and these equivalent forms are also covered within the scope defined by the appended claims.

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 as described in 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 obtained by transformation with the recombinant plasmid according to claim 3, characterized in that, The host is E. coli BL21(DE3).

5. The application of the phospholipase D mutant Lip-Gri / NQ according to claim 1, characterized in that, The application described is to utilize the phospholipase D mutant Lip-Gri / NQ to catalyze the production of phospholipids from phosphatidylserine.

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