A rational directed evolution method to improve the activity of phospholipase D in PS synthesis

Abstract

The invention relates to the field of biotechnology, and discloses a rational directed evolution method for improving the activity of phospholipase D in synthesizing PS. The scheme determines the active center of ZET4 by literature comparison, and then uses AutoDock Vina to perform molecular docking on PLD (2ZE4) with substrates PC and L-ser respectively to determine the binding sites of substrate molecules and enzyme molecules, and performs protein mutation design, and performs single point mutation or two groups of combined mutation on Y135A, Y135I, T181A, T181I, Q387A, Q387I, D392A, D392I and T194I points, wherein the PS yield of the mutant strain constructed by T181I, Q387A and Q387I is improved from 27.46% to 30.1 1%, 36.71%, and 36.90%; the mutant strain T194I phospholipase D had the highest PS yield of 58.32%, which was 112% higher than that of the original strain phospholipase D under the same reaction conditions; the PS yields of the mutants T194I+Q387A and T194I+Q387I reached 71.24% and 73.20%, which were 159% and 167% higher than that of WT, and 22.15% and 25.51% higher than that of T194I (PS yield was 58.32%).

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a rational directed evolution method for improving the activity of phospholipase D in synthesizing PS. 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 phospholipid transfer activity, reaction selectivity and stability of the currently known PLD need to be improved. Summary of the invention

[0005] The present invention aims to provide a rational directed evolution method for improving the activity of phospholipase D in synthesizing PS, so as to improve the activity of phospholipase D in synthesizing PS.

[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 the phospholipase D, wherein the amino acid sequence of the phospholipase D comprises a sequence shown in SEQ ID NO.1; and the mutation point is one or more combinations of Y135A, Y135I, T181A, T181I, Q387A, Q387I, D392A, D392I, and T194I.

[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: Y135A-F and Y135A-R, which are mutagenesis primers for Y135A point mutation, wherein the nucleotide sequence of Y135A-F is shown in SEQ ID NO.2, and the nucleotide sequence of Y135A-R is shown in SEQ ID NO.3;

[0017] Mutation primers Y135I-F and Y135I-R for Y135I point mutation, the nucleotide sequence of Y135I-F is shown in SEQ ID NO.4, and the nucleotide sequence of Y135I-R is shown in SEQ ID NO.5;

[0018] Mutation primers T181A-F and T181A-R for T181A point mutation, the nucleotide sequence of T181A-F is shown in SEQ ID NO.6, and the nucleotide sequence of T181A-R is shown in SEQ ID NO.7;

[0019] Mutation primers T181I-F and T181L-R for T181I point mutation, the nucleotide sequence of T181I-F is shown in SEQ ID NO.8, and the nucleotide sequence of T181I-R is shown in SEQ ID NO.9;

[0020] Mutation primers Q387A-F and Q387A-R for Q387A point mutation, wherein the nucleotide sequence of Q387A-F is shown in SEQ ID NO.10, and the nucleotide sequence of Q387A-R is shown in SEQ ID NO.11;

[0021] Mutation primers Q387I-F and Q387I-R for Q387I point mutation, the nucleotide sequence of Q387I-F is shown in SEQ ID NO.12, and the nucleotide sequence of Q387I-R is shown in SEQ ID NO.13;

[0022] Mutation primers D392A-F and D392A-R for the D392A point mutation, wherein the nucleotide sequence of D392A-F is shown in SEQ ID NO.14, and the nucleotide sequence of D392A-R is shown in SEQ ID NO.15;

[0023] Mutation primers D392I-F and D392I-R for D392I point mutation, the nucleotide sequence of D392I-F is shown in SEQ ID NO.16, and the nucleotide sequence of D392I-R is shown in SEQ ID NO.17;

[0024] The mutation primers D194I-F and D194I-R for the D194I point mutation, the nucleotide sequence of D194I-F is shown in SEQ ID NO.18, and the nucleotide sequence of D194I-R is shown in SEQ ID NO.19.

[0025] 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.

[0026] 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.

[0027] Preferably, as an improvement, the inducer comprises: arabinose, DTT, ethanol, butanol and antibiotics, and the concentration of arabinose is 0.035%·OD 600 -1 After adding the inducer to the TB adjusted medium, the expression was induced for 16 h at 18°C ​​and 200 rpm.

[0028] Preferably, as an improvement, 1 mL of the induced expression bacterial solution is centrifuged at 10°C and 10,000 rpm for 5 min, the precipitated bacteria are extracted with 1 mL of PLD, the cells are resuspended, and placed at room temperature for 1 h, vortexed for 5 s every 20 min; centrifuged at 4°C and 13,000 rpm for 10 min, and the supernatant is taken as the intracellular phospholipase D component.

[0029] Preferably, as an improvement, the PLD extract comprises: 10 mM EDTA, 0.05% sodium deoxycholate and 200 mM Tris-HCl, and the pH value of the PLD extract is 7.5.

[0030] The present invention provides a mutant protein of phospholipase D. The mutant protein uses PLD (2ZE4) to compare the amino acid sequence with PLD reported in the literature and derived from Streptomyces sp.PMF with a PDB number of 1V0S to determine the active center line of the enzyme, and then mutates the amino acids in the active center and the vicinity of the active site of the enzyme into hydrophobic amino acids. The software AutoDock Vina is used to perform molecular docking of PLD (2ZE4) with substrates PC and L-ser, respectively, to determine the binding site of the substrate molecule and the enzyme molecule, and then mutates the amino acids in the binding site on PLD into hydrophobic amino acids, so as to inhibit the hydrolytic enzyme activity of PLD and increase the pocket size of the binding site of PLD and the substrate. Compared with the original strain, the hydrolysis activity of phospholipase D was significantly reduced after the amino acids at the four positions of Y135, T181, Q387, and D392 were mutated to A and I in this scheme, that is, the mutation of the binding site amino acid to a hydrophobic amino acid can inhibit the hydrolase activity of PLD; the PS yield of Y135I was slightly reduced to 24.86%, and there was no significant change in Y135A and T181A. The PS yields of T181I, Q387A, and Q387I increased from 27.46% to 30.11%. , 36.71%, 36.90%; the mutant strain T194I phospholipase D had the highest PS yield of 58.32%, which was 112% higher than that of the original strain phospholipase D under the same reaction conditions; the PS yields of mutants T194I+Q387A and T194I+Q387I reached 71.24% and 73.20%, which were 159% and 167% higher than that of WT, and 22.15% and 25.51% higher than that of T194I (PS yield was 58.32%). BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the structure of the active center of PLD (2ZE4) in Example 1 of the present invention.

[0032] Figure 2 This is a diagram showing the docking results of PLD (2ZE4) of Example 1 of the present invention and the substrate PC molecule.

[0033] Figure 3 This is a diagram showing the docking results of PLD (2ZE4) of Example 1 of the present invention and the substrate L-ser molecule.

[0034] Figure 4 The hydrolase activity and PS yield of the phospholipase D of some mutants designed for MOE molecular docking in Example 3 of the present invention.

[0035] Figure 5 This is the protein electrophoresis diagram of phospholipase D after T194 single point mutation in Example 4 of the present invention.

[0036] Figure 6 The hydrolase activity and PS yield of phospholipase D after single-point mutation of the T194 site in Example 4 of the present invention.

[0037] Figure 7 The hydrolase activity and PS yield after the two-point combination mutation of T194I+Q387A and T194I+Q387I in Example 5 of the present invention. DETAILED DESCRIPTION

[0038] The following is further described in detail through specific implementation methods:

[0039] 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.

[0040] 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:

[0041] PLD (2ZE4): Derived from Streptomyces antibioticus, the PDB number is 2ZE4 ligand-free PLD, and the PLD gene number is Genebank no.MH237968.

[0042] PLD(1V0S): PLD derived from Streptomyces sp.PMF, PDB number 1V0S.

[0043] AutoDockVina: An open source molecular simulation software, mainly used to perform ligand-protein molecular docking.

[0044] 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.

[0045] LB medium: tryptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L.

[0046] PlasmidMini KitⅠ: plasmid extraction kit.

[0047] Primer X: is an online tool for designing mutation PCR primers.

[0048] TB medium (g·L -1 ): Yeast extract 24, peptone 12, potassium dihydrogen phosphate 2.31, potassium hydrogen phosphate 12.54.

[0049] 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.

[0050] The present invention will be further described below in conjunction with the embodiments.

[0051] Example 1. A method for constructing a phospholipase D mutant Escherichia coli strain

[0052] The present invention considers transforming the active site of PLD and the amino acids near the active site into hydrophobic amino acids with less steric hindrance, so as to inhibit the hydrolysis activity of PLD and increase the pocket size of the PLD-substrate binding site, which is beneficial for small molecule substrates to enter the active center and react with PLD, thereby improving the conversion rate of PS.

[0053] 1.1 Determination of the active center and binding site of PLD

[0054] The amino acid sequence of PLD (2ZE4) was compared with that of PLD from Streptomyces sp.PMF reported in the literature and PDB numbered 1V0S, and the amino acid composition of the active center of the enzyme was determined to be D467, N459, H442, K444, H168, N185, K170 and D200. Figure 1 shown.

[0055] 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 2 and attached Figure 3 As shown, when the binding free energy is -5.3260 kcal / mol, the binding sites of PC and 2ZE4 are 31 amino acid sites among 85-95, 120-130, 165-175, 185-195, 340-350, 370-390, and 440-470; when the binding free energy is -5.7780 kcal / mol, the binding sites of L-ser and 2ZE4 are 12 amino acid sites among 70-90, 170-185, and 200-205.

[0056] 1.2 Determine the mutation site of PLD

[0057] After determining the active site of PLD and the binding sites of substrates PC and L-ser with PLD, we first considered transforming the amino acids in the active site of PLD and the attachments of the active site into hydrophobic amino acids with less steric hindrance, so as to inhibit the hydrolytic activity of PLD and increase the size of the pocket of the binding site between PLD and the substrate, which is conducive to the entry of small molecule substrates into the active center to react with PLD, thereby improving the conversion rate of PS. In summary, we first chose to mutate Y135, T181, Q387, D392 and T194 into hydrophobic amino acids with less steric hindrance, alanine (A) and leucine (I), and first mutate Y135A, Y135I, T181A, T181I, Q387A, Q387I, D392A and D392I. Among them, the amino acids corresponding to Y135, T181, Q387, D392 and T194 are Tyr, Thr, Gln, Asp and Thr, respectively.

[0058] 1.3 Design of PLD mutation primers

[0059] 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°C refrigerator. The amino acid sequence expressed by this gene is specifically shown in SEQ ID NO.1 in the sequence list.

[0060] 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°C, 200rpm for 12h, then use Plasmid Mini 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 Y135A, Y135I, T181A, T181I, Q387A, Q387I, D392A and D392I. The specific primer sequences are shown in Table 1. Among them, the upstream and downstream mutation primers of Y135A are SEQ ID NO.2 and SEQ ID NO.3, respectively, the upstream and downstream mutation primers of Y135I are SEQ ID NO.4 and SEQ ID NO.5, respectively, the upstream and downstream mutation primers of T181A are SEQ ID NO.6 and SEQ ID NO.7, respectively, the upstream and downstream mutation primers of T181I are SEQ ID NO.8 and SEQ ID NO.9, respectively, the upstream and downstream mutation primers of Q387A are SEQ ID NO.10 and SEQ ID NO.11, respectively, the upstream and downstream mutation primers of Q387I are SEQ ID NO.12 and SEQ ID NO.13, respectively, the upstream and downstream mutation primers of D392A are SEQ ID NO.14 and SEQ ID NO.15, respectively, and the upstream and downstream mutation primers of D392I are SEQ ID NO.16 and SEQ ID NO.17, respectively.

[0061] Table 1 Mutation primer mutation sites

[0062]

[0063] 1.4 PCR amplification

[0064] 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.

[0065] Table 2 Mutation PCR amplification system

[0066]

[0067] Table 3 Mutation PCR amplification program

[0068]

[0069] 1.5 Construction of expression vector

[0070] 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.

[0071] 1.6 Transformation and screening

[0072] 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.

[0073] Example 2. Expression verification of the 8 mutant strains obtained in Example 1

[0074] 1. Extraction of Phospholipase D from Cell Culture Medium

[0075] 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.

[0076] Example 3. Determination of the phospholipase D hydrolase activity expressed by the 8 mutant strains of Example 2 and the determination of the phosphatidylserine (PS) synthesis catalyzed by lecithin

[0077] 1. Determination of phospholipase D hydrolase activity

[0078] The specific steps of the phospholipase D enzyme activity assay method include:

[0079] (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;

[0080] (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;

[0081] (3) 0.15 L of substrate solution and the above mixed solution were incubated in a 37°C water bath for 5 min;

[0082] (4) Add 0.1 mL of enzyme sample to the reaction solution and react in a 37°C water bath for 10 min;

[0083] (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;

[0084] (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.

[0085] 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).

[0086] 2. Phospholipase D catalyzes the synthesis of phosphatidylserine (PS) from lecithin

[0087] 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.

[0088] 3. Detection and analysis of product PS

[0089] 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.

[0090] The yield of PS is calculated as follows:

[0091]

[0092] The hydrolase activity and transacylase activity of PLD expressed by the mutant strains Y135A, Y135I, T181A, T181I, Q387A, Q387I, D392A, D392I and the original strain WT are shown in the attached figure. Figure 4 As shown and Table 4.

[0093] Table 4 Hydrolase activity and transacylation activity of PLD expressed by the original strain and mutant strain

[0094]

[0095]

[0096] After single-point mutations of other amino acids (Y135, T181, Q387, D392) near the entrance of L-ser to A and I, the hydrolase activities of eight mutants at four sites of Y135, T181, Q387, and D392 on PLD were significantly reduced compared with the original strain (WT), that is, the activity of PLD in hydrolyzing PC to generate PA was significantly inhibited. In terms of PS yield, compared with WT, the PS yield of Y135I was slightly reduced to 24.86%, Y135A and T181A had no significant changes, and the PS yields of T181I, Q387A, and Q387I increased from 27.46% to 30.11%, 36.71%, and 36.90%, respectively.

[0097] Example 4. Single point mutation of T194 site of PLD

[0098] The threonine at position 194 was further mutated to other hydrophobic amino acids, including isoleucine (I), valine (V), phenylalanine (F), cysteine ​​(C), methionine (M), and proline (P). The specific mutation primers are shown in Table 4.

[0099] Table 5 Primers used for single point mutation PCR of T194 The mutant strains, expression verification, hydrolase activity and PS product detection were specifically constructed by referring to the methods of Example 1, Example 2 and Example 3.

[0100] After single point mutation at T194 site, the expression of PLD in the constructed mutant strain is shown in the attached figure. Figure 5 As shown in Table 6. The upstream and downstream mutation primers of T194I are SEQ ID NO.18 and SEQ ID NO.19, respectively; the upstream and downstream mutation primers of T194V are SEQ ID NO.20 and SEQ ID NO.21, respectively; the upstream and downstream mutation primers of T194M are SEQ ID NO.22 and SEQ ID NO.23, respectively; the upstream and downstream mutation primers of T194F are SEQ ID NO.24 and SEQ ID NO.25, respectively; the upstream and downstream mutation primers of T194C are SEQ ID NO.26 and SEQ ID NO.27, respectively; the upstream and downstream mutation primers of T194P are SEQ ID NO.28 and SEQ ID NO.29, respectively.

[0101] Table 6 PLD expression of mutant strains with single point mutation at T194 site

[0102]

[0103]

[0104] From the attached Figure 5 As shown in Table 6, after the W at position 194 was mutated into different hydrophobic amino acids, the hydrolase activity was significantly reduced. Figure 6 It can be seen that the conversion rate of PS of PLD was improved to varying degrees, among which the PS yield of mutant strain T194I phospholipase D was the highest, up to 58.32%, which was 112% higher than that of the original strain phospholipase D under the same reaction conditions.

[0105] The caver software was used to calculate the channel size for substrate molecules to enter the active center of the enzyme before and after mutation. It was found that the acyl entrance radius of 194I was 1.46A, which was larger than the radius of 194W (1.00A). That is, the amino acids at the binding site between the substrate and the enzyme molecule near the active center were mutated into amino acids with less steric hindrance, in order to enlarge the binding pocket and thus facilitate the interaction between the enzyme and the substrate.

[0106] Example 5

[0107] The results of single-point mutations based on Example 3 and Example 4 show that the conversion rate of PS is significantly improved after the T194 amino acid (tryptophan) at the entrance of L-ser into the active center of PLD is mutated to I, and the conversion rate of PS is also improved after Q387 is mutated to A and I. In this example, T194I is combined with Q387A and Q387I respectively to further improve the activity of PLD catalytic synthesis of PS. The mutant strains, expression verification, hydrolase activity and PS product detection are specifically constructed with reference to the methods of Example 1, Example 2 and Example 3. The corresponding PCR primers are shown in Table 1 and Table 5.

[0108] The PLD expression of the strains after T194I combined with Q387A and Q387I mutations was compared with the original strain as shown in the attached figure. Figure 7 As shown, according to the attached Figure 7 It can be seen that the PS yields of mutants T194I+Q387A and T194I+Q387I reached 71.24% and 73.20%, respectively, which were increased by 159% and 167% compared with WT, and increased by 22.15% and 25.51% compared with T194I (PS yield was 58.32%).

[0109] 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, and the amino acid sequence of the phospholipase D is the sequence shown in SEQ ID NO.1; The mutation sites are: single mutation T181I, Q387A, Q387I, T194I, double mutation T194I, Q387A or double mutation T194I, Q387I; The T181I indicates that the sequence shown in SEQ ID NO.1 is used as the starting sequence, and the amino acid at position 181 is mutated from threonine to isoleucine; the Q387A indicates that the sequence shown in SEQ ID NO.1 is used as the starting sequence, and the amino acid at position 387 is mutated from glutamine to alanine; the Q387I indicates that the sequence shown in SEQ ID NO.1 is used as the starting sequence, and the amino acid at position 387 is mutated from glutamine to isoleucine; the T194I indicates that the sequence shown in SEQ ID NO.1 is used as the starting sequence, and the amino acid at position 194 is mutated from threonine to isoleucine.

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 described in step (3) include: Mutation primers T181I-F and T181L-R for T181I point mutation, the nucleotide sequence of T181I-F is shown in SEQ ID NO.8, and the nucleotide sequence of T181I-R is shown in SEQ ID NO.9; Mutation primers Q387A-F and Q387A-R for Q387A point mutation, wherein the nucleotide sequence of Q387A-F is shown in SEQ ID NO.10, and the nucleotide sequence of Q387A-R is shown in SEQ ID NO.11; Mutation primers Q387I-F and Q387I-R for Q387I point mutation, the nucleotide sequence of Q387I-F is shown in SEQ ID NO.12, and the nucleotide sequence of Q387I-R is shown in SEQ ID NO.13; The mutation primers D194I-F and D194I-R for the D194I point mutation, the nucleotide sequence of D194I-F is shown in SEQ ID NO.18, and the nucleotide sequence of D194I-R is shown in SEQ ID NO.

19.

6. 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 holding at 4°C.

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

8. The method according to claim 7, characterized in that: The inducer includes: arabinose, DTT, ethanol, butanol and antibiotics, and the concentration of arabinose is 0.035%·OD 600 -1 After adding the inducer to the TB adjusted medium, the expression was induced for 16 h at 18°C ​​shaker and 200 rpm.

9. The method according to claim 7, characterized in that: Take 1 mL of the induced expression bacterial solution and centrifuge it at 10°C, 10000 rpm for 5 min. Use 1 mL of PLD extraction solution to resuspend the cells in the precipitate and place them at room temperature for 1 h. Vortex and shake for 5 s every 20 min during this period; centrifuge it at 4°C, 13000 rpm for 10 min, and take the supernatant as the intracellular phospholipase D component.

10. The method according to claim 9, characterized in that: The PLD extract comprises: 10 mM EDTA, 0.05% sodium deoxycholate and 200 mM Tris-HCl, and the pH value of the PLD extract is 7.5.

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