Phospholipase D mutant with improved thermal stability and preparation method thereof

Abstract

The invention discloses a phospholipase D mutant with improved thermal stability and a preparation method thereof. The amino acid sequence of the phospholipase D mutant is as shown in SEQ ID NO.4; according to the phospholipase D mutant A426P with improved thermal stability, the mutant is incubated for 30 min at 55 DEG C and 60 DEG C respectively, the residual enzyme activity is 85% and 58% respectively, the activity of wild-type enzyme is only 56% and 7% under the same treatment condition, the thermal stability of the mutant enzyme is remarkably improved, and the phospholipase D mutant A426P can be used as a phospholipase D mutant. The problem that phospholipase D is not ideal in thermal stability in biological catalytic reaction is solved.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and enzymes, and in particular relates to a phospholipase D mutant with improved thermal stability and a preparation method thereof. Background Art

[0002] Phospholipase D (PLD) can catalyze two reactions: (1) acyl hydrolysis reaction; (2) acyl transfer reaction. Phospholipase D is widely present in plants (such as cabbage and peanuts), mammals (such as humans and mice), and microorganisms (such as Streptomyces). The transesterification activity of Streptomyces-derived phospholipase D is significantly higher than that of other species, and it can catalyze the reaction under mild conditions. It can be widely used in the modification of glycerol phospholipids and the production of rare phospholipid products, and has important application value in the fields of food and medicine. For example, in the process of food processing and production, phospholipase D can be used as a dispersant and emulsifier. In addition, phospholipase D can catalyze the synthesis of phosphatidylserine from phosphatidylcholine and serine. Phosphatidylserine is an important component of cell membranes and plays an important role in improving memory and alleviating Alzheimer's disease.

[0003] In 2000, Leiros et al. determined the three-dimensional structure of a phospholipase D protein from Streptomyces, the first protein in the phospholipase D superfamily to have its crystal structure determined. The protein is composed of 35 secondary structures, including two β-sheets flanked by 18 α-helices. Since the publication of the first crystal structure, a growing number of phospholipase D crystal structures have been characterized, laying the foundation for the rational computational design of phospholipase D. However, secretory expression of phospholipase D in wild Streptomyces requires long fermentation cycles, low yields, complex purification processes, and high purity, resulting in high costs. Therefore, efficient heterologous expression of phospholipase D has important application value.

[0004] Currently, recombinant expression of Streptomyces-derived phospholipase D has been achieved in Escherichia coli, Pichia pastoris, Bacillus subtilis, and . However, due to E. coli's weak ability to secrete and express foreign proteins, and the high toxicity of phospholipase D when expressed in E. coli, expression of phospholipase D in E. coli cells inhibits cell growth, with the recombinant protein primarily present as inclusion bodies. The yield of active recombinant phospholipase D is very low. Pichia pastoris possesses a strong ability to secrete and express foreign proteins, making high-density fermentation amenable. However, recombinant expression of foreign proteins in Pichia pastoris can be subject to overglycosylation. Huang Lin et al. used Pichia pastoris to express phospholipase D from Streptomyces septatus, resulting in an activity of only 2.4 U / ml. Bacillus subtilis has a strong ability to secrete and express foreign proteins. By optimizing expression elements such as protein secretion signal peptides, promoters, and ribosome binding sites (RBS), the shake flask expression level of recombinant phospholipase D can reach 24 U / ml. However, compared with Pichia pastoris, Bacillus subtilis secretory expression has a higher content of impurities, accounting for more than 50% of the total secretory expression protein. Therefore, the development of new phospholipase D foreign protein expression systems is of great significance. In addition, in the process of enzymatic synthesis of phosphatidylserine, phospholipase D needs to have high thermal stability. However, the thermal stability of wild-type phospholipase D is relatively poor, and it is necessary to improve its thermal stability by molecular modification of phospholipase D. Summary of the Invention

[0005] The present invention aims to provide a phospholipase D mutant with improved thermal stability and a preparation method thereof, which can obtain the phospholipase D mutant with improved thermal stability and a genetically engineered bacterium R. rhodochrous-pDD103-PLD expressing phospholipase D.

[0006] The present invention adopts the following technical solution: a phospholipase D mutant with improved thermal stability, whose amino acid sequence is as SEQ ID NO.4.

[0007] A method for preparing a phospholipase D mutant with improved thermal stability, comprising:

[0008] Step 1: Clone the gene shown in SEQ ID No. 2 into the pDD103 expression vector to obtain the recombinant plasmid pDD103-PLD; transform the obtained recombinant plasmid pDD103-PLD into Rhodococcus rhodochrous competent cells to obtain recombinant Rhodococcus rhodochrous-pDD103-PLD;

[0009] Step 2: inoculating the recombinant Rhodococcus cell monoclone into liquid LB medium and culturing at 30°C overnight for 12-16 hours to obtain a seed solution; inoculating the overnight cultured seed solution into liquid TB medium at a 1% inoculum volume and culturing at 30°C for 36-48 hours;

[0010] Step 3: The culture solution is centrifuged to remove the bacterial cells to obtain a crude phospholipase D enzyme solution, and the crude enzyme solution collected by centrifugation is subjected to nickel column affinity chromatography and molecular sieve filtration desalting to obtain a phospholipase D mutant.

[0011] The beneficial effects of the present invention are:

[0012] The present invention utilizes computer-assisted semi-rational design to molecularly modify phospholipase D from Streptomyces antibioticus, screens and obtains phospholipase D mutants with improved thermal stability, and recombinantly expresses and purifies the recombinant phospholipase D in Rhodococcus rhodochrous.

[0013] The present invention provides a phospholipase D mutant A426P with improved thermal stability. When incubated at 55°C and 60°C for 30 minutes, the residual enzyme activity of the mutant is 85% and 58%, respectively, while the wild-type enzyme has only 56% and 7% residual activity under the same treatment conditions. The mutant enzyme has significantly improved thermal stability, solving the problem of unsatisfactory thermal stability of phospholipase D in biocatalytic reactions.

[0014] The present invention constructs a recombinant expression vector pDD103-PLD and realizes efficient secretory expression in Rhodococcus rhodochrous cells, thereby solving the problems of low expression and excessive glycosylation of phospholipase D in recombinant expression in Pichia pastoris, and also solves the problem of excessively high impurity protein content in the fermentation broth in recombinant expression of phospholipase D in Bacillus subtilis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The phospholipase D recombinant expression vector pDD103-PLD constructed by the present invention; Kanamycin represents the kanamycin resistance gene, pAL5000 represents the Rhodococcus replicon, pMB1 represents the Escherichia coli replicon, Promoter represents the constitutive promoter in the pDD103 plasmid, and Phospholipase D represents the phospholipase D gene sequence;

[0016] Figure 2 The nucleic acid electrophoresis diagram of the wild-type phospholipase D gene of the present invention; M: Marker; 1: wild-type phospholipase D gene;

[0017] Figure 3 This is the SDS-PAGE electrophoresis diagram of the fermentation supernatant of the wild-type phospholipase D recombinantly expressed by Rhodococcus in the present invention; M: Marker; 1: Phospholipase D

[0018] Figure 4This is the minimum free energy heat map after FoldX predicts the virtual saturation mutation of amino acid residues in the present invention;

[0019] Figure 5 is the fermentation supernatant activity of wild-type (WT) phospholipase D and mutant enzymes in the present invention;

[0020] Figure 6 The residual enzyme activity of the wild-type (WT) phospholipase D and mutant enzyme fermentation broth incubated at 55°C for 30 min;

[0021] Figure 7 The figure is an SDS-PAGE electrophoresis diagram of the purified protein of the recombinant phospholipase D mutant A426P of the present invention; M: Marker; 1: purified phospholipase D mutant A426P;

[0022] Figure 8 2 is a graph comparing the thermal stability of the recombinant phospholipase D mutant A426P and the wild-type enzyme in the present invention. DETAILED DESCRIPTION

[0023] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0024] The present invention discloses a phospholipase D mutant with improved thermal stability, the amino acid sequence of which is shown as SEQ ID NO.4.

[0025] SEQ ID NO.4:

[0026] MTSDQRPARLPTHKGKLLAPHRLHRLIPVSVALTTVCAALPSSTAYAADTPPTPHLDAIERSLRDTSPGLEGSVWQRTDGNRLDAPDGDPAGWLLQTPGCWGDAGCKDRAGTRRLLDKMTRNIADARHTVDISSLAPFPNGGFEDAVVDGLKAVVAAGHSPRVRILVGAAPIYHLNVVPSRYRDELIGKLGAAAGKVTLNVASMTTSKTSLSWNHSKLLVVDGKTAITGGINGWKDDYLDTAHPVSDVDMALSGPAAASAGKYLDTLWDWTCRNASDPAKVWLATSNGASCMPSMEQDEAGSAPAEPTGDVPVIAVGGLGVGIKESDPSSGYHPDLPTAPDTKCTVGLHDNTNADRDYDTVNPEENALRSLIASARSHVEISQQDLNATCPPLPRYDIRTYDTLAGKLAAGVKVRIVVSDPANRGPVGSGGYSQIKSLDEISDTLRTRLVALTGDNEKASRALCGNLQLASFRSSDAAKWADGKPYALHHKLVSVDDSAFYIGSKNLYPAWLQDFGYIVESPAAAQQLKTELLDPEWKYSQQAAATPAGCPARQAG

[0027] The phospholipase D mutant of the present invention has a nucleotide sequence as shown in SEQ ID NO.3.

[0028] SEQ ID NO.3:

[0029]

[0030] The phospholipase D mutant of the present invention is obtained by mutation of the parent amino acid sequence, and the parent amino acid sequence is SEQ ID NO.1.

[0031] SEQ ID NO.1:

[0032] MTSDQRPARLPTHKGKLLAPHRLHRLIPVSVALTTVCAALPSSTAYAADTPPTPHLDAIERSLRDTSPGLEGSVWQRTDGNRLDAPDGDPAGWLLQTPGCWGDAGCKDRAGTRRLLDKMTRNIADARHTVDISSLAPFP NGGFEDAVVDGLKAVVAAGHSPRVRILVGAAPIYHLNVVPSRYRDELIGKLGAAAGKVTLNVASMTTSKTSLSWNHSKLLVVDGKTAITGGINGWKDDYLDTAHPVSDVDMALSGPAAASAGKYLDTLWDWTCRNASDP AKVWLATSNGASCMPSMEQDEAGSAPAEPTGDVPVIAVGGLGVGIKESDPSSGYHPDLPTAPDTKCTVGLHDNTNADRDYDTVNPEENALRSLIASARSHVEISQQDLNATCPPLPRYDIRTYDTLAGKLAAGVKVRIV VSDPANRGAVGSGGYSQIKSLDEISDTLRTRLVALTGDNEKASRALCGNLQLASFRSSDAAKWADGKPYALHHKLVSVDDSAFYIGSKNLYPAWLQDFGYIVESPAAAQQLKTELLDPEWKYSQQAAATPAGCPARQAG

[0033] The phospholipase D mutant of the present invention is obtained by mutation of the original nucleotide, and the sequence of the original nucleotide is SEQ ID NO.2.

[0034] SEQ ID NO.2:

[0035]

[0036] Example

[0037] Expression and activity determination of phospholipase D

[0038] The reagent materials required for this example are as follows:

[0039] Escherichia coli Top10 was maintained in our laboratory. Rhodococcus rhodochrous was purchased from the China General Microbiological Culture Collection (CGMCC 4.1147). The Rhodococcus expression plasmid pDD103 was purchased from Addgene (Plasmid No. 119887). The DNA sequence of the vector can be downloaded from the Addgene website (https: / / www.addgene.org / ).

[0040] LB medium recipe: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride. TB medium recipe: 12 g / L peptone, 24 g / L yeast extract, 17 mM potassium phosphate (KH2PO4), 72 mM potassium phosphate (K2HPO4), 4 ml / L glycerol. MB medium recipe: 5 g / L yeast extract, 15 g / L tryptone, 5 g / L soy peptone, 5 g / L sodium chloride, 2.0% glycine, and 1.8% sucrose, pH 7.2.

[0041] The steps for expressing wild-type phospholipase D are as follows:

[0042] (1) The protein sequence of phospholipase D from Streptomyces antibioticus (PDB ID: 2ZE4) was obtained from the database (https: / / www.rcsb.org). The amino acid sequence is shown in SEQ ID NO.1. The phospholipase D gene sequence was artificially synthesized based on the codon preference of Bacillus subtilis (nucleotide sequence is shown in SEQ ID NO.2). The phospholipase D gene was connected to the expression vector pDD103 by seamless cloning to construct the Rhodococcus expression vector pDD103-PLD. The expression vector map is shown in the attached figure. Figure 1 As shown, the nucleic acid electrophoresis of phospholipase D gene is as follows Figure 2 The constructed recombinant plasmid was then transformed into competent E. coli Top10 cells and cultured on plates containing 50 μg / mL kanamycin for 12-16 hours until a single colony appeared. A single E. coli Top10 colony containing the pDD103-PLD recombinant plasmid was inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C for 12-16 hours. The plasmid was then extracted and the plasmid DNA concentration was determined.

[0043] (2) The above plasmid was electroporated into Rhodococcus rhodochrous competent cells, and the cells were spread on LB solid plates containing 34 μg / mL kanamycin and cultured at 30°C for 48-72 hours.

[0044] The preparation method of Rhodococcus competent cells is as follows:

[0045] 1. Pick a single colony of Rhodococcus rhodochrous from an LB solid plate and incubate in 20 mL of LB medium at 30°C, 200 rpm / min, for 36 h.

[0046] 2. Take 3-4 mL of culture medium, centrifuge at 4000 rpm for 3 minutes, wash with 1.0 mL of sterile water, centrifuge again, and resuspend in 1.0 mL of sterile water. Pipette the suspension into 100 mL of MB medium (5 g / L yeast extract, 15 g / L tryptone, 5 g / L soy peptone, 5 g / L NaCl, 2.0% glycine, and 1.8% sucrose, pH 7.2). Incubate at 30°C, 200 rpm until OD reaches 0. 600 The value reaches between 0.8-1.0.

[0047] 3. Transfer the culture solution into a sterilized 50ml centrifuge tube and centrifuge at 4000rpm for 5 minutes to collect the bacteria.

[0048] 4. Add 15 mL of pre-cooled sterile distilled water, wash gently, and centrifuge at 4000 rpm for 5 minutes to collect the bacteria.

[0049] 5. Add 15 mL of pre-cooled 5% sterile glycerol, wash gently, and centrifuge at 4000 rpm for 5 minutes to collect the bacteria.

[0050] 6. Add 10 mL of pre-cooled 10% sterile glycerol, wash gently, centrifuge at 4000 rpm for 5 minutes to collect the cells, and repeat the same steps once more.

[0051] 7. Resuspend the cells in 5 mL of pre-cooled 10% sterile glycerol and dispense 100 μL aliquots into sterile centrifuge tubes for storage at -80°C.

[0052] The electroporation method for Rhodococcus competent cells is as follows:

[0053] 1. Add 1 μL of plasmid DNA (100 ng) to 100 μL of Rhodococcus competent cells, mix gently, and place on ice for 30 minutes.

[0054] 2. Transfer the competent cell-plasmid mixture into a pre-chilled 0.2 cm electroporation cuvette. Set the electroporation parameters to 2.5 kV and 5.0 ms pulses.

[0055] 3. Immediately after electroporation, add 1 mL of LB medium and culture at 30°C with shaking for 2 h.

[0056] 4. Pipette 100uL of the transformation solution and spread it on a solid LB plate (34μg / mL kanamycin) and culture at 30℃ for 2-3 days until a single colony appears.

[0057] (3) Pick a single clone from the plate and transfer it to 20 mL LB medium. Culture it at 30°C and 200 rpm for 36 hours to obtain the first-level seed solution. Inoculate the first-level seed solution into a shake flask containing 200 mL TB medium at a ratio of 2%, and culture it at 30°C and 200 rpm for 24-36 hours. Since the phospholipase D gene itself has a secretion signal peptide, the recombinantly expressed phospholipase D protein will be secreted into the culture medium. Then, centrifuge it at 10,000 rpm for 10 minutes to collect the culture supernatant. Determine the activity of phospholipase D in the supernatant, and use SDS-PAGE electrophoresis to analyze the expression of recombinant phospholipase D in the fermentation supernatant. The electrophoresis of the fermentation supernatant of Rhodococcus expressing wild-type phospholipase D is shown in the attached figure. Figure 3 As shown, it was shown that phospholipase D was successfully secreted and expressed in Rhodococcus.

[0058] Phospholipase D activity is measured using an enzyme-linked colorimetric assay. Phospholipase D catalyzes the conversion of soybean lecithin, a substrate, to choline. Choline oxidase then reacts with choline to produce hydrogen peroxide (H2O2). In the presence of 4-aminoantipyrine and horseradish peroxidase, H2O2 generates a red quinoneimine compound, the absorbance of which is measured at 505 nm using a microplate reader. Phospholipase D activity can be quantitatively determined by measuring choline content. Enzyme activity is defined as the amount of enzyme solution required to catalyze the conversion of 1 μmol of choline per minute at 37°C (1 unit / mL).

[0059] 1. Preparation of buffer and reagents for phospholipase D activity assay

[0060] A: Substrate buffer: Tris-HCl (40 mM, pH = 7.5), dissolved with 0.1% (v / v) Triton X-100, 15 mM CaCl2.

[0061] B: Substrate lecithin solution: 44 mg soybean lecithin was dissolved in 5 mL of solution A, sonicated on ice until dissolved, and stored at -20°C.

[0062] C: Reaction termination solution: 100 mM EDTA.

[0063] D: 4-Aminoantipyrine solution: 7.6 mM, stored at 4°C in the dark.

[0064] E: Phenol solution: 66 mM, stored at 4°C in the dark.

[0065] F: Choline oxidase solution: 12.5 U / mL, store at -20°C in the dark.

[0066] G: Peroxidase solution: 12.5 U / mL, store at -20°C in the dark.

[0067] Note: Solution C, Solution D, Solution E, Solution F, and Solution G are all prepared using 40 mM Tris-HCl (pH = 7.5).

[0068] 2. Phospholipase D Activity Assay Steps

[0069] 1) Thoroughly mix 60 μL of the prepared substrate lecithin solution and 40 μL of the phospholipase D enzyme solution, and shake on a constant temperature shaker at 37°C for 20 min.

[0070] 2) Add 50 μL of reaction stop solution to terminate the reaction, incubate in a boiling water bath for 5 min, and then cool on ice for 5 min.

[0071] 3) Centrifuge at 6000 rpm for 5 min, aspirate all the supernatant (about 150 μL), add 60 μL of choline oxidase, 200 μL of phenol solution, 200 μL of 4-aminoantipyrine solution and 40 μL of horseradish peroxidase, and react in a 37°C constant temperature metal bath for 20 min.

[0072] 4) After the reaction is completed, 505 The absorbance of the reaction solution was measured at 37 nm, and the enzyme activity was calculated according to the standard curve.

[0073] The method for preparing the choline chloride standard curve is as follows:

[0074] 1 mg / mL Choline Chloride Standard Solution: Dissolve 100 mg of choline chloride in a small amount of distilled water and dilute to 100 mL in a volumetric flask. Replace 40 μL of crude phospholipase D enzyme solution in the enzyme activity assay with the 1 mg / mL choline chloride standard solution. Adjust the volume of the choline chloride solution to 0, 4, 8, 12, 16, 20, 24, 28, and 32 μL, respectively, and then fill up to 40 μL with water. Perform the assay according to the above method. After the reaction is complete, measure the absorbance of the reaction solution. Based on the measured absorbance, create a choline standard curve.

[0075] PLD enzyme activity calculation formula:

[0076]

[0077] Where,

[0078] C—Choline chloride concentration calculated according to the standard curve (μmol / mL);

[0079] V1—volume of fermentation broth taken (mL);

[0080] V2—total volume of reaction system (mL);

[0081] T—reaction time (min).

[0082] Construction of phospholipase D mutant library

[0083] The enzyme's 3D structure was first constructed using Swiss-model, using PDB ID: 2ZE4. Following homology modeling, molecular dynamics (MD) simulations were performed to assess the stability of the phospholipase D model in a dynamic environment. MD simulations were performed using Gromacs 2019.6, employing the amber99sb force field and the TIP3P water model, under constant temperature and pressure (NVT and NPT) conditions, with periodic boundary conditions. The optimized crystal structure was initially placed in the center of a cubic box, ensuring a minimum distance of 1 nm between the protein surface and the box edge. The system was then structurally optimized. The final structure served as the initial structure for subsequent MD simulations. Next, position-restrained equilibrium was performed using 100 ps of NVT and NTP equilibrium, followed by 100 ns of molecular dynamics simulations at 300 K. All bond lengths were constrained using the LINCS algorithm, with a time step of 2 fs. Electrostatic interactions were calculated using the particle-mesh Ewald (PME) method.

[0084] Phospholipase D was subjected to full-sequence virtual saturation mutation using FoldX software, and the free energy change ΔΔG was calculated. The overall structural stability of the protein (ΔG) can be evaluated by free energy, and the lower the value, the better the thermal stability. The change in thermal stability caused by the mutation can be calculated by the ΔG change (i.e., ΔΔG) between the wild type and the mutant. If ΔΔG < -1 kcal / mol, it means that the mutation has improved protein stability. The appropriate mutation site is selected based on the size of the free energy change. The minimum free energy heat map after virtual saturation mutation of the candidate amino acid site is shown in the attached figure. Figure 4 The virtual screening results of mutation sites are as follows: A157R, A279R, E299L, A300L, G331W, N351F, A426P, S429W, G430A.

[0085] Primers were designed to amplify the nine mutants using the wild-type phospholipase D gene plasmid pDD103-PLD as a template. The PCR protocol was as follows: initial denaturation at 98°C for 2 minutes; 30 cycles of denaturation at 98°C for 15 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 2 minutes; and termination extension at 72°C for 5 minutes. The PCR products were digested with Dpn I at 37°C for 4 hours. The digested products were transformed into competent E. coli Top10 cells and cultured on LB plates at 37°C for 12-16 hours until single colonies emerged. Single colonies were then selected for sequencing to obtain the different phospholipase D mutants. The recombinant plasmids for the nine mutants were then isolated and used for future reference. The primer sequences for PCR amplification of the phospholipase D mutants are shown in Table 1.

[0086] Table 1 Primers used for site-directed mutagenesis

[0087]

[0088]

[0089] Expression and screening of phospholipase D mutants

[0090] The obtained phospholipase D mutant plasmid was electroporated into Rhodococcus competent cells for expression. The specific steps are as follows:

[0091] (1) The phospholipase D mutant plasmid was electroporated into Rhodococcus competent cells and cultured on LB plates containing 34 μg / mL kanamycin at 30°C for 48-72 h until a single colony was grown;

[0092] (2) Single clones were picked from the plate and transferred to 20 mL of LB medium. The culture was incubated at 30°C and 200 rpm for 36 h to obtain a primary seed solution. The primary seed solution was inoculated into a shake flask containing 200 mL of TB medium at a ratio of 2%, and the culture was incubated at 30°C and 200 rpm for 24-36 h. The culture supernatant was then collected by centrifugation at 10,000 rpm for 10 min.

[0093] (3) The activities of wild-type phospholipase D and mutant fermentation supernatants were measured respectively. The results are shown in the attached table. Figure 5 All nine mutants were secreted and expressed in Rhodococcus. Three mutants, N351F, S429W, and G430A, showed significantly increased activity compared to the wild-type enzyme. The S429W mutant showed 2.5 times the activity of the wild-type enzyme. The remaining six mutants showed no significant difference in activity compared to the wild-type enzyme.

[0094] Next, the thermal stability of the different mutant enzymes was determined. The activity of the wild-type enzyme and each mutant enzyme that had not been heat-treated was set to 100%. The fermentation supernatant of the wild-type and mutant enzymes was treated at 55°C for 30 minutes, and then the residual enzyme activity was measured. The ratio of the residual activity after heat treatment to the enzyme activity before heat treatment was calculated, which was the residual enzyme activity rate after heat treatment. The results are shown in the attached figure. Figure 6 Compared with wild-type phospholipase D, the G331W and A426P mutant enzymes showed significantly improved thermostability. After treatment at 55°C for 30 minutes, the residual enzyme activity of wild-type phospholipase D was 56%, while the residual enzyme activity of phospholipase D mutant A426P was 85% after treatment at 55°C for 30 minutes. However, the thermostability of the S429W mutant enzyme was significantly reduced.

[0095] Characterization of Phospholipase D Mutant A426P and Its Catalytic Synthesis of Phosphatidylserine

[0096] (1) Thermal stability analysis of purified phospholipase D mutant A426P

[0097] The fermentation supernatant of mutant enzyme A426P was collected by centrifugation and filtered using a 0.22 μm filter membrane. The supernatant was then applied to a nickel ion affinity chromatography column and eluted with an equilibrium buffer (PBS containing 30 mM imidazole). Finally, the target protein was eluted with an elution buffer containing 300 mM imidazole. The eluted protein solution was then desalted with a molecular sieve and the purified recombinant enzyme was collected. The purified recombinant phospholipase D was detected by SDS-PAGE, as shown in the attached figure. Figure 7 As shown, according to the grayscale analysis results, the purity exceeded 95%.

[0098] The purified wild-type phospholipase D and mutant enzyme A426P were then incubated at different temperatures for 30 minutes, and the enzyme activity was measured. The residual enzyme activity after treatment at different temperatures was calculated. The activity of the untreated wild-type phospholipase D and mutant A426P was taken as 100%, and the residual enzyme activity after treatment at different temperatures was calculated. The results are shown in Figure 2. Figure 8 Compared with the wild-type phospholipase D, the mutant enzyme A426P has significantly improved thermal stability. After incubation at 55°C and 60°C for 30 minutes, the residual enzyme activity of the mutant enzyme was 85% and 58%, respectively, while the wild-type enzyme only had 56% and 7% residual activity under the same treatment conditions.

[0099] (2) Phospholipase D mutant A426P catalyzes the synthesis of phosphatidylserine

[0100] Phosphatidylserine was synthesized using wild-type phospholipase D and mutant enzyme A426P. The reaction system for phosphatidylserine synthesis consisted of 10 ml of 0.1 M acetic acid-sodium acetate buffer, 10 mmol / L phosphatidylcholine, 50 mmol / L L-serine, 10 mmol / L calcium chloride, and 10 U / ml recombinant phospholipase D. The reaction was shaken at 45°C for 8 hours, and the phosphatidylserine yield was determined.

[0101] The phosphatidylserine content in the reaction solution was determined using high-performance liquid chromatography (HPLC). Chromatographic conditions: a Vensil XBP Silica column (5 μm, 250 mm × 4.6 mm), a mobile phase of V (acetonitrile): V (methanol): V (phosphoric acid) = 100:5:1, a flow rate of 0.50 ml / min, a sample load of 10 μl, a UV detector at a wavelength of 205 nm, and a column oven temperature of 25°C.

[0102] The results showed that the conversion rate of phosphatidylserine synthesized by wild-type phospholipase D was 26%, while the conversion rate of phosphatidylserine synthesized by phospholipase D mutant A426P was 37%. Compared with the wild-type enzyme, the significant increase in the yield of phosphatidylserine synthesized by mutant enzyme A426P may be due to the significant improvement in enzyme thermal stability. Figure 8 As shown, the mutant enzyme A426P has very good stability under 45 degrees, while the wild-type enzyme has only 83% residual enzyme activity after being treated at 45 degrees for 30 minutes. It is possible that as the catalytic reaction time increases, the activity gradually decreases, which in turn affects the enzyme catalytic efficiency.

[0103] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

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

4.

2. A phospholipase D mutant with improved thermostability according to claim 1, characterized in that Its nucleotide sequence is shown as SEQ ID NO.

3.

3. The phospholipase D mutant with improved thermostability according to claim 1, characterized in that The phospholipase D mutant is obtained by mutation of the parent amino acid sequence, and the parent amino acid sequence is SEQ ID NO.

1.

4. The phospholipase D mutant with improved thermostability according to claim 1, characterized in that The phospholipase D mutant is obtained by mutation of the original nucleotide, and the sequence of the original nucleotide is SEQ ID NO.

2.

5. The phospholipase D mutant with improved thermostability according to claim 1, characterized in that The phospholipase D mutant was incubated at 55°C and 60°C for 30 min, and the residual enzyme activities were 85% and 58%, respectively.

6. A method for preparing a phospholipase D mutant with improved thermal stability, characterized in that: include: Step 1: Clone the gene shown in SEQ ID No. 2 into the pDD103 expression vector to obtain the recombinant plasmid pDD103-PLD; transform the obtained recombinant plasmid pDD103-PLD into Rhodococcus rhodochrous competent cells to obtain recombinant Rhodococcus rhodochrous-pDD103-PLD; Step 2: inoculating the recombinant Rhodococcus cell monoclone into liquid LB medium and culturing at 30°C overnight for 12-16 hours to obtain a seed solution; inoculating the overnight cultured seed solution into liquid TB medium at a 1% inoculum volume and culturing at 30°C for 36-48 hours; Step 3: The culture solution is centrifuged to remove the bacterial cells to obtain a crude phospholipase D enzyme solution, and the crude enzyme solution collected by centrifugation is subjected to nickel column affinity chromatography and molecular sieve filtration desalting to obtain a phospholipase D mutant.

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