A directed evolution method for improving the thermal stability of phospholipase D

Abstract

The invention relates to the field of biotechnology, and discloses a directed evolution method for improving the thermal stability of phospholipase D. In the scheme, PLD (2ZE4) is molecularly docked with substrates PC and L-ser respectively through AutoDock Vina, the binding sites of substrate molecules and enzyme molecules are determined, protein mutation design is performed, several amino groups of protein high stability structures are selected for mutation, primers are designed to construct 14 mutant strains, and the phospholipase D expressed by the mutant strains G381M, A122M, A122L, and R377M has improved activity in synthesizing PS, the thermal stability of the mutant strain R377M is significantly improved, and the enzyme activity can still be maintained at about 80% after being kept warm for 120 minutes.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a directed evolution method for improving the thermal stability of phospholipase D. Background Art

[0002] Phospholipids is a general term for compounds containing phosphate groups. The molecular structure consists of two long hydrophobic carbon chains and a polar head. Phospholipids were first discovered in egg yolks and are the main components of cell membranes of living organisms. They are widely distributed in various cell tissues of animals and plants, and are responsible for signal transduction and promoting metabolism. The main physiological activity of phospholipids is determined by its polar head. According to the type of polar head, phospholipids can be divided into: phosphatidylcholine (PC), phosphatidylerhanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), etc. Although phospholipids are widely distributed in nature and there are many types, the distribution of these phospholipids in nature is very uneven. Among them, PS is an essential active substance present on the cell membrane. It can regulate membrane proteins to be quickly absorbed by the human body, efficiently cross the brain-blood barrier, and safely slow down or even repair damaged cell membranes of nerve cells in the human brain. Thereby improving the patient's cognitive ability, learning ability and memory ability, it has a certain auxiliary effect on the treatment of Alzheimer's disease. It is mainly extracted from soybeans and also exists in brain cells and peanuts. In animals, PS mainly exists in cells of the brain and liver. Since animals are prone to disease, the safety of the product is threatened; in plants, PC has a higher content of phospholipids. Relatively speaking, the content of PS is very small and difficult to extract. Therefore, the synthesis of PS by enzyme conversion came into being, which has the advantages of stable operation and high specificity.

[0003] Phospholipase D (PLD) is a special phospholipid hydrolase that can catalyze the hydrolysis of phospholipids, release the polar head of the original phospholipids, and generate phosphatidic acid (PA). In addition, in the presence of a second substrate (nucleophilic reagents such as serine, ethanolamine, glycerol, etc.), phospholipase D can catalyze the cleavage of the phosphodiester bond of phospholipids and promote the combination of phosphoryl groups with nucleophilic reagents to generate new phospholipids. The phosphatidyl transfer reaction catalyzed by PLD is currently the most widely used and most effective method for artificial synthesis of PS.

[0004] However, the currently known PLD phosphatidyl transfer activity, reaction selectivity and stability need to be improved. Molecular docking technology studies the possibility of substrate-enzyme binding, and molecular dynamics simulation is used to determine the stability of the binding complex. The direction and extent of the reaction are further determined by binding free energy analysis between substrate and enzyme. The rational design of proteins can minimize the experimental workload of site-directed mutagenesis. Considering that each mutation site has 20 mutations (19 substitution mutations and 1 deletion mutation), biomolecular simulation technology can be used to virtually screen mutant libraries, greatly improving the actual mutation efficiency. However, there are almost no reports on the research on virtual mutation to improve the catalytic performance of PLD, resulting in a lack of directionality in the research of PLD site-directed mutagenesis. Summary of the invention

[0005] The present invention aims to provide a directed evolution method for improving the thermal stability of phospholipase D, so as to improve the catalytic performance of PLD.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The invention provides a mutant protein of phospholipase D, wherein a point mutation is performed on an amino acid sequence of phospholipase D, wherein the amino acid sequence of the phospholipase includes a sequence shown in SEQ ID NO.1; and the mutation point is: G381M, A122M, A122L or R377M.

[0008] The present invention also provides a recombinant bacterium expressing the mutant protein.

[0009] Preferably, as an improvement, the basic strain of the recombinant bacteria includes Escherichia coli.

[0010] The present invention also provides a method for constructing the above-mentioned recombinant bacteria, comprising the following steps:

[0011] (1) Cultivating wild-type E. coli TOP10 / pBADKP-PLD2, wherein the vector is plasmid pBADKP and the host cell is E. coli TOP10. The specific construction method is shown in patent CN109136207B;

[0012] (2) Plasmid Mini Kit I was used to extract the plasmid pBADKP containing the gene encoding the amino acid sequence shown in SEQ ID NO. 1 from the strain E. coli TOP10 / pBADKP-PLD2, and the plasmid pBADKP was used as a template for mutation PCR;

[0013] (3) performing PCR amplification on the plasmid using site-directed mutagenesis primers to obtain an amplified product;

[0014] (4) digesting the template DNA of the amplified product without mutation using DpnⅠ enzyme to obtain a digestion product;

[0015] (5) The digestion product is introduced into E. coli top10 competent cells by heat stimulation, and single clones are screened using LB solid medium containing kanamycin resistance to obtain the recombinant bacteria.

[0016] Preferably, as an improvement, the site-directed mutagenesis primers in step (3) include: mutagenesis primers G381M-F and G381M-R for the G381M point mutation, the nucleotide sequence of G381M-F is shown in SEQ ID NO.2, and the nucleotide sequence of G381M-R is shown in SEQ ID NO.3;

[0017] Mutation primers A122M-F and D175L-R for A122M point mutation, the nucleotide sequence of A122M-F is shown in SEQ ID NO.4, and the nucleotide sequence of A122M-R is shown in SEQ ID NO.5;

[0018] Mutation primers A122L-F and A122L-R for A122L point mutation, the nucleotide sequence of A122L-F is shown in SEQ ID NO.6, and the nucleotide sequence of A122L-R is shown in SEQ ID NO.7;

[0019] The mutation primers R377M-F and R377M-R for the R377M point mutation, the nucleotide sequence of R377M-F is shown in SEQ ID NO.8, and the nucleotide sequence of R377M-R is shown in SEQ ID NO.9.

[0020] Preferably, as an improvement, the step (1) of culturing wild-type E. coli TOP10 / pBADKP-PLD2 specifically comprises the following steps: taking frozen bacterial solution of E. coli TOP10 / pBADKP-PLD2 and culturing it at 37° C. and 200 rpm for 12 hours using LB medium.

[0021] Preferably, as an improvement, the LB medium comprises: 10 g / L tryptone, 5 g / L yeast powder and 10 g / L sodium chloride.

[0022] Preferably, as an improvement, the specific steps of step (4) are: adding 2 μL of DpnⅠ to react at 37° C. for 1.5 h to digest the unmutated template DNA to obtain a digestion product.

[0023] Preferably, as an improvement, the PCR amplification procedure in step (3) includes: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 10 s, annealing at 55-65°C for 30 s, extension at 72°C for 2 min, extension at 1 min / kb, 30 cycles; extension at 72°C for 10 min; and holding at 4°C.

[0024] The present invention also provides a method for preparing phospholipase D by using the above-mentioned phospholipase D expressing recombinant bacteria, comprising the following steps: the phospholipase D expressing recombinant bacteria are revived with LB culture medium, transferred to TB culture medium for culture for 5-6 hours, then transferred to TB adjustment culture medium, and an inducer is added to induce the expression of phospholipase D.

[0025] This scheme provides a mutant protein of phospholipase D, and the PLD used is derived from Streptomyces antibioticus, with the PDB number of 2ZE4 ligand-free PLD, and the PLD gene number is Genebank no.MH237968. The software AutoDock Vina is used to perform molecular docking of PLD (2ZE4) with substrates PC and L-ser, respectively, to determine the binding sites of substrate molecules and enzyme molecules, and the FoldX 3b5.1 version of YASARAPlugin is used to calculate the relative folding free energy of phospholipase D, and the stability of PLD is predicted after the amino acids at the binding sites of phospholipase D and substrate molecules lecithin (PC) and L-ser (L-ser) are mutated into 19 other amino acids according to the free energy size, and several amino acid sites of the protein with high stability structure are selected to perform single-point mutation on PLD. Compared with the original strains, the mutant strains G381M, A122M, A122L, and R377M had improved PS conversion rates, and the thermal stability of the mutant R377M was significantly improved, and the enzyme activity could still be maintained at about 80% after 120 minutes of incubation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a diagram showing the binding site results of the docking of PLD (2ZE4) of Example 1 of the present invention with the substrate PC molecule.

[0027] Figure 2 This is a diagram showing the binding site results of the docking of PLD (2ZE4) of Example 1 of the present invention with the substrate L-ser molecule.

[0028] Figure 3 This is a diagram showing the stability results of phospholipase D predicted by FoldX in Example 1 of the present invention.

[0029] Figure 4 This is a graph showing the hydrolase activity and PS yield of phospholipase D of different mutants in Example 3 of the present invention.

[0030] Figure 5 This is a thermal stability curve of different mutant phospholipase D at 60°C in Example 4 of the present invention. DETAILED DESCRIPTION

[0031] The following is further described in detail through specific implementation methods:

[0032] The present invention discloses a phospholipase D mutant, a preparation method and application thereof, a DNA molecule encoding the phospholipase D mutant and a host cell. Personnel in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0033] The present invention uses conventional techniques and methods used in the field of genetic engineering and molecular biology. These general references provide definitions and methods known to those skilled in the art. However, those skilled in the art can adopt other conventional methods, experimental schemes and reagents in the art based on the technical solutions described in the present invention, without being limited to the specific embodiments of the present invention. For example, the present invention can select the following experimental materials and reagents:

[0034] PLD (2ZE4): Derived from Streptomyces antibioticus, the PDB number is 2ZE4 ligand-free PLD, and the PLD gene number is Genebank no.MH237968.

[0035] AutoDockVina: An open source molecular simulation software, mainly used to perform ligand-protein molecular docking.

[0036] FoldX 3b5.1 version of YASARAPlugin: Molecular structure viewing software.

[0037] E.coli TOP10 / pBADKP-PLD2: The wild-type E.coli TOP10 / pBADKP-PLD2 containing the PLD gene from Streptomyces antibioticus (Genebank no. MH237968) was preserved at -80°C by our research group (Lu Yinghua research group, School of Chemistry and Chemical Engineering, Xiamen University). The construction method of this strain has been disclosed in the form of a patent, patent application number: CN201810843255.2.

[0038] LB medium: tryptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L.

[0039] PlasmidMini KitⅠ: plasmid extraction kit.

[0040] Primer X: is an online tool for designing mutation PCR primers.

[0041] TB medium (g·L -1 ): Yeast extract 24, peptone 12, potassium dihydrogen phosphate 2.31, potassium hydrogen phosphate 12.54.

[0042] TB adjusted medium: TB adjusted medium: 10 g L -1 Peptone; 5 g·L -1 Yeast powder; 5g·L -1 Glycerol; 0.1 M potassium phosphate buffer, pH 7.2.

[0043] The present invention will be further described below in conjunction with the embodiments.

[0044] Example 1. A method for constructing a phospholipase D mutant Escherichia coli strain

[0045] The invention considers calculating the relative folding free energy of phospholipase D, and predicting the stability of PLD after the amino acids at the binding sites of phospholipase D and substrate molecules phosphatidylcholine (PC) and L-ser (L-ser) are mutated into 19 other amino acids based on the free energy, thereby determining the directed evolution strategy of phospholipase D.

[0046] 1.1 Determine the binding site of PLD

[0047] The software AutoDockVina was used to perform molecular docking of PLD (2ZE4) with the substrate PC and L-ser, respectively, to determine the binding sites between the substrate molecule and the enzyme molecule. Figure 1 and attached Figure 2 As shown, the binding sites of PC and 2ZE4 are several amino acid sites among 85-95, 120-130, 160-170, 185-190, 340-350, 370-39, and 440-470; when the binding free energy is -5.7780 kcal / mol, the binding sites of L-ser and 2ZE4 are several amino acid sites among 70-90 and 170-205.

[0048] 1.2 Determine the mutation site of PLD

[0049] The effect of mutation on protein is calculated by the free energy difference between the two states of protein. For example, the thermodynamic stability of protein is related to the change of free energy (ΔΔG) between folded and unfolded states. The relative folding free energy of phospholipase D is calculated using FoldX3b5.1 version of YASARAPlugin. The stability of PLD is predicted based on the free energy after the amino acids at the binding sites of phospholipase D and substrate molecules phosphatidylcholine (PC) and L-ser (L-ser) are mutated into 19 other amino acids, thereby determining the directed evolution strategy of phospholipase D.

[0050] Among them, the folding free energy difference before and after PLD mutation is ddG fold As attached Figure 3 As shown in the figure, different colors represent different ranges of ddGfold, where the smaller the ddGfold, the higher the thermal stability of the mutant enzyme. The corresponding relationship between ddGfold (kcal / mol) and thermal stability is: >+1.84, highly unstable; +0.92-+1.84, unstable; +0.46-+0.92, slightly unstable; -0.46-+0.46, neutral; -0.92--0.46, slightly unstable; -1.84--0.92, stable; <-1.84, highly stable. This study selected several amino acid sites with high protein stability structures ( Figure 2 Single point mutation was performed on PLD (in the middle blue area). In this embodiment, the mutation sites selected were S87, A122, A123, Y126, H168, Y191, D202, R377, N459, and G381, and the amino acids at these mutation sites corresponded to Ser, Ala, Ala, Tyr, His, Tyr, Asp, Arg, Asn, and Gly, respectively.

[0051] 1.3 Design of PLD mutation primers

[0052] The wild-type E. coli TOP10 / pBADKP-PLD2 containing the PLD gene (Genebank no.MH237968) from Streptomyces antibioticus was constructed by our research group and stored in a -80℃ refrigerator. The phospholipase D nucleotide sequence expressed by this PLD gene is shown in SEQ ID NO.1. Take an appropriate amount of frozen bacterial liquid and use LB medium (tryptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L) to culture at 37℃, 200rpm for 12h, then use PlasmidMini KitⅠ kit to extract the plasmid pBADKP containing the target gene PLD as a template for mutation PCR. Then use Primer X online software to design mutation primers. The specific mutation sites and primer sequences are shown in Table 1.

[0053] Among them, the upstream and downstream mutation primers of G381M are SEQ ID NO.2 and SEQ ID NO.3, the upstream and downstream mutation primers of A122M are SEQ ID NO.4 and SEQ ID NO.5, the upstream and downstream mutation primers of A122L are SEQ ID NO.6 and SEQ ID NO.7, the upstream and downstream mutation primers of R377M are SEQ ID NO.8 and SEQ ID NO.9; the upstream and downstream mutation primers of Y126M are SEQ ID NO.10 and SEQ ID NO.11; the upstream and downstream mutation primers of Y191M are SEQ ID NO.12 and SEQ ID NO.13; the upstream and downstream mutation primers of A123P are SEQ ID NO.14 and SEQ ID NO.15; the upstream and downstream mutation primers of H168Y are SEQ ID NO.16 and SEQ ID NO.17; the upstream and downstream mutation primers of H168F are SEQ ID NO.18 and SEQ ID NO.19; the upstream and downstream mutation primers of N459P are SEQ ID NO.20 and SEQ ID NO.21; the upstream and downstream mutation primers of S87N are SEQ ID NO.22 and SEQ ID NO.23; the upstream and downstream mutation primers of D202M are SEQ ID NO.24 and SEQ ID NO.25; the upstream and downstream mutation primers of D202V are SEQ ID NO.26 and SEQ ID NO.27; the upstream and downstream mutation primers of D202R are SEQ ID NO.28 and SEQ ID NO.29.

[0054] Table 1 Mutation sites and mutation primers

[0055]

[0056]

[0057] 1.4 PCR amplification

[0058] The plasmid was amplified by PCR using site-directed mutagenesis primers to obtain an amplified product. The specific amplification system and temperature program are shown in Tables 2 and 3, respectively.

[0059] Table 2 Mutation PCR amplification system

[0060]

[0061] Table 3 Mutation PCR amplification procedure

[0062]

[0063] 1.5 Construction of expression vector

[0064] After the PCR amplification was completed, 2 μL of DpnⅠ was added and reacted at 37°C for 1.5 h to digest the unmutated template DNA to obtain the digestion product.

[0065] 1.6 Transformation and screening

[0066] The digested product was introduced into E. coli top10 competent cells by heat stimulation, and single clones were screened by LB solid medium containing kanamycin resistance. After gene sequencing, the target mutant was obtained, mixed with 40% glycerol in a 1:1 ratio, and stored in a -80°C refrigerator for future use.

[0067] Example 2. Expression verification of the 14 mutant strains and the original strain obtained in Example 1

[0068] 1. Extraction of Phospholipase D from Cell Culture Medium

[0069] Take a small amount of bacteria from the glycerol cryopreserved tube of TOP10 / pBADKP-PLD2, inoculate it in a 50mL conical flask containing 10mL LB medium, shake it at 37℃, 200rpm, and culture it overnight (12-16h). According to the 1% inoculation amount, transfer it to a 250mL groove conical flask containing 20mL TB medium, shake it at 37℃, 200rpm, and culture it for 5-6h until the OD600 is 5-6. Centrifuge at 10℃, 5000rpm for 5min to collect the bacteria, add TB adjustment medium according to the same volume before centrifugation, resuspend the cells, and add 0.035%·OD600-1 arabinose and different concentrations of chemicals (such as DTT, ethanol, butanol, antibiotics, etc.), shake it at 18℃, 200rpm, and induce expression for 16h. Take 1mL of bacterial solution and centrifuge at 10000rpm for 5min at 10℃. Resuspend the cells in the precipitate with 1mL of PLD extract (10mM EDTA, 0.05% sodium deoxycholate, 200mM Tris-HCl, pH 7.5). Place at room temperature for 1h, vortex for 5s every 20min. Centrifuge at 4℃, 13000rpm for 10min, and take the supernatant as the intracellular phospholipase D component to measure the enzyme activity.

[0070] Example 3. Determination of the phospholipase D hydrolase activity expressed by the 14 mutant strains and the original strain of Example 2 and determination of the phosphatidylserine (PS) synthesis catalyzed by lecithin

[0071] 1. Determination of phospholipase D hydrolase activity

[0072] The specific steps of the phospholipase D enzyme activity assay method include:

[0073] (1) Substrate solution: 76 mg of phosphatidylcholine (PC, from soybean, >98%, Aladdin Reagent, Shanghai, China), dissolved in 10 mL of 5% Triton X-100 (w / v) solution;

[0074] (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 was mixed with 0.25 mL of reaction solution;

[0075] (3) 0.15 L of substrate solution and the above mixed solution were incubated in a 37°C water bath for 5 min;

[0076] (4) Add 0.1 mL of enzyme sample to the reaction solution and react in a 37°C water bath for 10 min;

[0077] (5) Immediately add 0.2 mL of stop solution (1 M Tris-HCl, pH 8.0, 0.5 M EDTA) and incubate at 100 °C in a metal bath for 15 min; (6) Cool the reaction solution to room temperature, add 0.2 mL of color development 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 at 37 °C in a water bath for 1 h;

[0078] (7) Dilute the reaction solution at a ratio of 1:8 and add 1% Triton X-100. Take 0.2 mL of the sample and measure the absorbance at 500 nm on a microplate reader. Use different concentrations of choline chloride solution instead of the enzyme sample to determine the standard curve. The absorbance value is in the range of 0-0.22, and the choline concentration is in the range of 0-0.25 μmol. The two have a good linear correlation.

[0079] According to the above conditions, the amount of phospholipase D that releases 1 μmol of choline per minute is defined as one enzyme activity unit (U).

[0080] 2. Phospholipase D catalyzes the synthesis of phosphatidylserine (PS) from lecithin

[0081] Take 0.75mL crude enzyme solution and 0.75mL PLD extract solution (10mMEDTA, 0.05% sodium deoxycholate, 200mM Tris-HCl, pH 7.5) containing 1M L-serine and 50mM anhydrous calcium chloride, add 3mL of ether solution with a PC concentration of 10mM, and react at 32°C, 200rpm for two hours. After centrifugation at 4000rpm at room temperature for 3min, take 2mL of the upper ether solution containing PS, blow dry with nitrogen, freeze in a -20°C refrigerator, and then detect the PS concentration by HPLC.

[0082] 3. Detection and analysis of product PS

[0083] The PS product after nitrogen drying was re-dissolved with 2 mL of mobile phase (n-hexane: isopropanol: 1% acetic acid solution = 8:8:1 (V:V:V)) and then the PS content was detected by HPLC. The HPLC conditions were as follows: chromatographic column: ShimNex S-Sil-SB (250 mm × 4.6 mm, 5 μm); column temperature: 30 ° C; flow rate: 1.5 mL / min; injection volume: 10 μL; detector: UV detector; wavelength: 203 nm.

[0084] The yield of PS is calculated as follows:

[0085]

[0086] The hydrolase activity and transacylation activity of PLD expressed by the mutant strains are shown in the attached figure. Figure 4 As shown and Table 4.

[0087] Table 4 Hydrolase activity and transacylation activity of PLD expressed by the original strain and mutant strain

[0088]

[0089]

[0090] After single-point mutation, compared with the original strain (WT), the PS conversion rates of mutants G381M, A122M, A122L, and R377M were improved, while the PS conversion rates of mutants H168Y and H168F were almost zero.

[0091] Example 4. Determination of the thermal stability of enzymes expressed by 11 mutant strains and the original strain

[0092] The crude enzyme solutions of 11 phospholipase D WT, Y126M, Y191M, G381M, A122M, A122L, A123P, R377M, S87N, D202M, and D202V in Example 3, which were substantially unchanged or improved compared with WT, were placed in a 60°C water bath for insulation, and then 0.75 mL of the enzyme solution was taken out every 30 minutes to catalyze the reaction of PC and L-ser to generate PS according to the method in Example 3. Remaining PLD transacyl activity (%) = PS yield after insulation / PS yield before insulation × 100%. The results of the thermal stability test of the phospholipase D expressed by the 11 mutant strains and the original strain are shown in Table 5.

[0093] Table 5 The residual enzyme activity percentage of different mutant enzymes after incubation at 60℃ for different time periods

[0094]

[0095] Compared with WT, the thermal stability of mutants Y126M, G381M, A122M, A122L, A123P, and S87N showed no obvious change, and all were close to inactivation after 90 minutes of incubation. However, the thermal stability of Y191M, D202M, and D202V was significantly reduced, and the enzyme was completely inactivated after 30 minutes of incubation. Surprisingly, the thermal stability of mutant R377M was significantly improved, and the enzyme activity could still be maintained at about 80% after 120 minutes of incubation.

[0096] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical schemes and / or characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the technical scheme of the present invention. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to the specific circumstances. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A mutant protein of phospholipase D, wherein the amino acid sequence of phospholipase D is subjected to a point mutation, wherein the mutation point is: G381M, A122M, A122L or R377M; The amino acid sequence of the phospholipase D is as shown in SEQ ID NO.1; The G381M indicates that the sequence shown in SEQ ID NO.1 is used as the starting sequence, and the amino acid at position 381 is mutated from glycine to methionine; the A122M indicates that the sequence shown in SEQ ID NO.1 is used as the starting sequence, and the amino acid at position 122 is mutated from alanine to methionine; the A122L indicates that the sequence shown in SEQ ID NO.1 is used as the starting sequence, and the amino acid at position 122 is mutated from alanine to leucine; the R377M indicates that the sequence shown in SEQ ID NO.1 is used as the starting sequence, and the amino acid at position 377 is mutated from arginine to methionine.

2. A recombinant bacterium expressing the mutant protein according to claim 1.

3. The recombinant bacterium according to claim 2, characterized in that The basic strain of the recombinant bacteria is Escherichia coli.

4. The method for constructing a recombinant bacterium according to claim 2 or 3, characterized in that: The following steps are involved: (1) Cultivation of wild type E. coli TOP10 / pBADKP-PLD2, where the vector is plasmid pBADKP and the host cell is E. coli TOP10, the specific construction method is shown in patent CN109136207B; (2) Using PlasmidMiniKitⅠ kit from strains E. coli The plasmid pBADKP containing the gene encoding the amino acid sequence shown in SEQ ID NO.1 was extracted from TOP10 / pBADKP-PLD2, and the plasmid pBADKP was used as a template for mutation PCR; (3) Perform PCR amplification on the plasmid using site-directed mutagenesis primers to obtain an amplified product; (4) Using DpnⅠ enzyme to digest the template DNA of the amplified product without mutation to obtain the digestion product; (5) Heat stimulation of digestion products E. coli The top10 competent cells are screened for single clones using LB solid culture medium containing kanamycin resistance to obtain the recombinant bacteria.

5. The construction method according to claim 4, characterized in that: The site-directed mutagenesis primers in step (3) include: mutagenesis primers G381M-F and G381M-R for the G381M point mutation, wherein the nucleotide sequence of G381M-F is shown in SEQ ID NO.2, and the nucleotide sequence of G381M-R is shown in SEQ ID NO.3; Mutation primers A122M-F and D175L-R for A122M point mutation, the nucleotide sequence of A122M-F is shown in SEQ ID NO.4, and the nucleotide sequence of A122M-R is shown in SEQ ID NO.5; Mutation primers A122L-F and A122L-R for A122L point mutation, the nucleotide sequence of A122L-F is shown in SEQ ID NO.6, and the nucleotide sequence of A122L-R is shown in SEQ ID NO.7; The mutation primers R377M-F and R377M-R for the R377M point mutation, the nucleotide sequence of R377M-F is shown in SEQ ID NO.8, and the nucleotide sequence of R377M-R is shown in SEQ ID NO.

9.

6. The construction method according to claim 4, characterized in that: The step (1) cultivates wild type E. coli TOP10 / pBADKP-PLD2 The specific steps include: E. coli The frozen bacterial suspension of TOP10 / pBADKP-PLD2 was cultured in LB medium at 37°C and 200 rpm for 12 h.

7. The construction method according to claim 6, characterized in that: The LB medium contains: 10 g / L tryptone, 5 g / L yeast powder and 10 g / L sodium chloride.

8. The construction method according to claim 4, characterized in that: The specific steps of step (4) are as follows: adding 2 μL of DpnⅠ and reacting at 37°C for 1.5 h to digest the unmutated template DNA to obtain a digestion product.

9. The construction method according to claim 4, characterized in that: The PCR amplification procedure in step (3) includes: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 10 s, annealing at 55-65°C for 30 s, extension at 72°C for 2 min, extension at 1 min / kb, 30 cycles; extension at 72°C for 10 min; and hold at 4°C.

10. A method for preparing phospholipase D using the phospholipase D expressing recombinant bacteria according to claim 4, characterized in that: The phospholipase D expression recombinant was revived in LB medium, transferred to TB medium for culture for 5-6 hours, and then transferred to TB adjustment medium, and an inducer was added to induce the expression of phospholipase D.

Images