Highly active phospholipase mutants and uses thereof

Abstract

The application belongs to the technical field of genetic engineering of enzymes, and particularly relates to a phospholipase D mutant and preparation and application thereof. The phospholipase D mutant is obtained by means of molecular biology technology and A379T mutation on the basis of the wild-type phospholipase D amino acid sequence, and the amino acid sequence is shown as SEQ ID NO. 2. Compared with the wild-type phospholipase D, the catalytic activity of the phospholipase D mutant is significantly improved. The phospholipase D mutant obtained by the application provides a certain reference for the rational design of phospholipase D to improve the catalytic performance thereof, and can be applied to the production of various natural rare phospholipids and non-natural phospholipid compounds, and applied to the fields of biology, food, medicine, daily chemicals and the like.

Description

Technical fields:

[0001] This invention belongs to the field of enzyme genetic engineering technology, specifically relating to obtaining phospholipase D mutants with enhanced enzyme activity through site-directed saturation mutagenesis using overlap PCR technology, and their preparation and application. Background technology:

[0002] Phospholipase D (PLD, EC 3.1.4.4) is an enzyme that acts on phosphoryl-oxygen bonds, hydrolyzing the phosphodiester bonds of glycerophospholipids to generate phosphatidic acid and hydroxyl compounds. It can also catalyze the binding of certain hydroxyl-containing compounds to the acyl chains of phospholipids to form new functional phospholipids. Phospholipids are a general term for compounds containing phosphate groups. Their molecular structure consists of two hydrophobic long carbon chains and one polar head. Widely distributed in the animal and plant kingdoms, they are natural biosurfactants and important components of human and animal cell membranes, possessing excellent emulsifying and antioxidant properties. As an important component of cell membranes, they also have numerous physiological functions, including promoting brain and nervous system development, improving memory, lowering blood lipids and cholesterol, anti-aging, and cancer prevention. Therefore, PLD can be used to modify phospholipids to prepare single or rare phospholipids, such as phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidylethanolamine (PE). These enzyme-modified phospholipids have been greatly improved and enhanced in terms of nutritional value and various properties, and are widely used in food, health products, medicine, feed, cosmetics and other fields.

[0003] PLDs are widely found in numerous biological groups, including bacteria, fungi, and higher plants and animals. As early as the 1940s, PLDs were discovered in carrot roots and cabbage leaves. PLDs in plant tissues are mostly extracellular enzymes, making isolation and extraction relatively simple, while PLDs in animal tissues are mostly intracellular enzymes, making isolation and preparation difficult. Subsequently, PLDs were successfully isolated from various bacteria. In comparison, microbially produced PLDs have advantages such as low production cost, short culture cycle, high phosphatidyl transfer reaction activity, and good substrate specificity, thus attracting considerable attention. Currently reported PLD-producing microorganisms mainly include *Streptomyces*, *Corynebacterium*, *Escherichia*, *Pseudomonas*, *Bacillus*, and *Salmonella*. Compared to other microbial PLDs, *Streptomyces*-derived PLDs have higher commercial value due to their high catalytic activity and broad substrate specificity, meeting the needs of industrial production.

[0004] Although phospholipids can be extracted from egg yolks and soybeans, this requires large amounts of organic solvents, leading to high purification costs and limiting their application. PLD-catalyzed phosphatidyl transfer reactions are the primary process for preparing rare phospholipids. Compared to traditional physical extraction and chemical synthesis methods, PLD biocatalysis offers advantages such as high selectivity, environmental friendliness, ease of large-scale production, and mild reaction conditions for preparing rare and difficult-to-isolate single phospholipids and their derivatives. Therefore, using PLD enzymatic synthesis of rare phospholipids can address the urgent need for high-quality phospholipids in functional food development and promote the development of my country's food industry. However, wild-type PLDs have low phosphatidyl transfer activity, resulting in low product conversion efficiency, which is insufficient for industrial applications. Furthermore, the PLD-catalyzed reaction occurs in a two-phase system, which significantly reduces the activity of the PLD. To address these practical application problems, strategies such as directed evolution, rational design, and chemical modification can be used to improve the function and characteristics of natural enzymes, effectively enhancing catalytic activity, expanding the substrate range, and improving enzyme stability, thereby meeting the needs of industrial applications.

[0005] Directed evolution of proteins is a crucial method for improving protein function and activity. By increasing gene mutation rates and designing specialized screening and selection methods, enzymes with specific properties can be rapidly obtained. Directed evolution of enzymes typically involves three steps: ① constructing a gene mutant library through random mutation, site-directed mutagenesis, or recombination of protein-coding sequences; ② targeted screening and selection to obtain mutants with improved phenotypes; ③ using these mutants as the starting point for the next round of gene diversification, iterating through directed evolution until the mutant with optimal performance is obtained. Directed evolution technology has greatly promoted the development of many fields such as enzyme engineering, metabolic engineering, and medicine. It has achieved significant results in enhancing protein stability and substrate specificity, and altering or enhancing protein activity. Site-directed mutagenesis has successfully modified a large number of enzyme molecules, resulting in industrial enzymes with higher activity and better stability than natural enzymes. Overlap extension PCR technology uses primers with complementary ends to form overlapping strands in the PCR products. In subsequent amplification reactions, the overlapping strands extend, splicing together amplified fragments from different sources. Overlap extension PCR has a wide range of unique applications in site-directed mutagenesis, construction of fusion genes, synthesis of long gene fragments, gene knockout, and amplification of target genes.

[0006] Therefore, in this invention, the PLD gene derived from Streptomyces antibioticus is molecularly modified by overlapping PCR, and high-throughput screening is performed using Bacillus subtilis and Bacillus amyloliquefaciens expression systems to obtain a PLD mutant gene with enhanced enzyme activity. Summary of the Invention:

[0007] Based on the problems existing in the current technology, in order to further promote the application of PLD in the industrial field, it is necessary to further improve its existing properties. The purpose of this invention is to provide a mutant of a high-activity PLD.

[0008] The technical approach to achieving the objective of this invention is summarized as follows:

[0009] Wild-type PLD gene from *Streptomyces antibioticus* was obtained using basic molecular biology techniques. Overlap PCR was used to saturate the wild-type PLD gene, and the PLD mutant A379T and its encoding gene pldmA379T were screened using a *Bacillus subtilis* expression system. A recombinant vector was reconstructed, and its efficient expression in *Bacillus subtilis* and *Bacillus amyloliquefaciens* was achieved. PLD mutants with enhanced enzyme activity were obtained through fermentation and extraction techniques.

[0010] One of the technical solutions provided by the present invention is a PLD mutant, the amino acid sequence of which is shown in SEQ ID NO.2, and is obtained by A379T amino acid mutation in the wild-type PLD shown in SEQ ID NO.1;

[0011] Furthermore, the present invention also provides the coding gene of the PLD mutant, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0012] The second technical solution provided by the present invention is a recombinant vector or recombinant strain containing the coding gene of the above-mentioned PLD mutant;

[0013] Furthermore, the expression vector used in the recombinant vector is the shuttle vector pBSA43;

[0014] Furthermore, the host used for the recombinant strain is Bacillus subtilis WB600 or Bacillus amyloliquefaciens CGMCC No.11218.

[0015] The third technical solution provided by this invention is the application of the above-mentioned recombinant vector or recombinant strain, especially its application in the production of the PLD mutant shown in SEQ ID NO.2.

[0016] The fourth technical solution provided by the present invention is the application of the PLD mutant shown in SEQ ID NO.2, especially in the preparation of phospholipids and their derivatives, and more particularly in the preparation of non-natural phospholipid derivatives.

[0017] Furthermore, the application of the PLD mutant shown in SEQ ID NO.2 in the catalytic synthesis of phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylglucose, phosphatidylglucosamine, phosphatidylfructose, etc.;

[0018] Furthermore, the application forms of the PLD mutant include, but are not limited to, enzyme solutions, enzyme powders, emulsions, gels, and immobilized enzymes.

[0019] Beneficial effects:

[0020] 1. This invention utilizes overlap PCR technology to perform site-directed saturation mutagenesis on wild-type PLD, obtaining a mutant A379T with enhanced enzyme activity. The specific activity of WT is 0.33 U / mg, while the specific activity of A379T is 1.91 U / mg, which is 5.8 times that of WT. Furthermore, the PS yield of A379T is 4.05 times that of WT.

[0021] 2. This invention uses Bacillus subtilis expression system and Bacillus amyloliquefaciens expression system to achieve efficient expression of PLD mutants with enhanced enzyme activity in different ways. Attached image description:

[0022] Figure 1 Electrophoresis diagram of PCR amplification of wild-type PLD gene in this invention

[0023] Where: M is DNA Marker, and 1 is PLD gene;

[0024] Figure 2 The image shows the enzyme digestion verification of the recombinant plasmid pBSA43-pldmA379T of this invention. In the image, M is the DNA Marker, 1 is the electrophoresis image of the recombinant plasmid pBSA43-pldmA379T from Bacillus subtilis after double digestion with BamHI and NotI, and 2 is the electrophoresis image of the recombinant plasmid pBSA43-pldmA379T from Bacillus amyloliquefaciens after double digestion with BamHI and NotI.

[0025] Figure 3 This is a thin-layer chromatography image of the synthesis of phosphatidylserine catalyzed by wild-type PLD and mutant A379T in this invention.

[0026] Figure 4 SDS-PAGE image of the purified sample of mutant A379T of this invention.

[0027] Where: M is the protein marker, and 1 is the A379T purified sample;

[0028] Figure 5 The optimal temperature curves for wild-type PLD and mutant PLD in this invention are shown.

[0029] Wherein: WT is wild-type PLD, and A379T is mutant A379T;

[0030] Figure 6 The optimal pH curves for wild-type PLD and mutant PLD in this invention.

[0031] Wherein: WT is wild-type PLD, and A379T is mutant A379T;

[0032] Figure 7 This is a temperature stability diagram of wild-type PLD and mutant PLD of the present invention.

[0033] Wherein: WT is wild-type PLD, and A379T is mutant A379T;

[0034] Figure 8 This is a pH stability diagram of wild-type PLD and mutant PLD of the present invention.

[0035] Wherein: WT is wild-type PLD, and A379T is mutant A379T. Detailed implementation method:

[0036] The technical content of the present invention will be further described below with reference to the embodiments. However, the present invention is not limited to these embodiments, and the scope of protection of the present invention cannot be limited by the following embodiments.

[0037] The culture media and solutions used in the embodiments of this invention are as follows:

[0038] Wash Buffer (mM): Weigh 29.25g NaCl, 2.42g Tris, and 3.5g imidazole, dissolve them in ultrapure water, and bring the volume to 1L. After removing impurities by filtration through a 0.22μm microporous membrane, store in a refrigerator at 4℃ protected from light.

[0039] Elution Buffer (mM): Weigh 29.25g NaCl, 2.42g Tris, and 13.6g imidazole, dissolve them in ultrapure water, and bring the volume to 1L. After removing impurities by filtration through a 0.22μm microporous membrane, store in a refrigerator at 4℃ protected from light.

[0040] Lysis Buffer (mM): Weigh 29.25g NaCl, 2.42g Tris, and 1.4g imidazole and dissolve them completely in ultrapure water. Make up to 1L, filter through a 0.22μm microporous membrane to remove impurities, and store in a refrigerator at 4℃ protected from light.

[0041] LB medium (g / L): Yeast extract 5.0, tryptone 10.0, NaCl 10.0, add 800mL of water to dissolve, and after complete dissolution, continue to add water to make up to 1L.

[0042] SP salt solution (g / L): K2HPO4 18.34, KH2PO4 6.0, (NH4)2SO4 2.0, sodium citrate 1.0, MgSO4·7H2O 0.2. Dissolve in 800mL of water, and after complete dissolution, continue to add water to make up to 1L.

[0043] SPI medium (200mL): 195.2mL SP salt solution, 0.8mL 5% casein hydrolysate, 2mL 10% yeast extract, 2mL 5% glucose solution. Mix well and dispense 5mL into sterilized empty test tubes. Store at 4℃.

[0044] SPII medium: 292.8 mL SP salt solution, 1.2 mL 5% casein hydrolysate, 3 mL 10% yeast extract, 3 mL 5% glucose solution, 1.5 mL 100 mM calcium chloride, 1.5 mL 50 mM magnesium chloride. Mix well and dispense 2 mL into sterilized empty test tubes. Store at 4°C.

[0045] LBS medium (g / L): Sorbitol 91.085, NaCl 10, yeast extract 5, tryptone 10, add 800mL of water to dissolve, and after complete dissolution, continue to add water to make up to 1L.

[0046] Fermentation medium (g / L): 64g corn flour, 40g soybean meal, 4g disodium hydrogen phosphate, 0.3g potassium dihydrogen phosphate, 0.7g high-temperature amylase, add 800mL water to dissolve, and after complete dissolution, continue to add water to make up to 1L.

[0047] All of the above-mentioned solid culture media were supplemented with 2% agar.

[0048] In this invention, the following definitions are used:

[0049] 1. Nomenclature of amino acids and DNA nucleic acid sequences

[0050] The IUPAC nomenclature, a recognized system for naming amino acid residues, is used, employing either three-letter codes or single-letter forms. DNA nucleic acid sequences are named using the IUPAC nomenclature.

[0051] 2. Identification of PLD mutants

[0052] The term "amino acid replaced at the original amino acid position" is used to represent the mutated amino acid in the PLD mutant. For example, Ala379Thr indicates that the amino acid at position 379 is replaced by Thr from the wild-type PLD, and the position number corresponds to the amino acid sequence number of the wild-type PLD in SEQ ID NO. 1.

[0053] In this invention, lowercase italic pld represents the coding gene of wild-type PLD, and lowercase italic pldmA379T represents the coding gene of mutant A379T, as shown in the table below.

[0054] wild type Ala379 — 1 3 A379T Ala379Thr GCA→ACG 2 4

[0055] In this invention, the amino acid sequence of wild-type PLD is as shown in SEQ ID NO.1: ADTPPTPHLDAIERSLRDTSPGLEGSVWQRTDGNRLDAPDGDPAGWLLQTPGCWGDAGCKDRAGTRRLLDKMTRNIADARHTVDISSLAPFPNGGFEDAVVDGLKASVAAGHSPRVRILVGAAPIYHLNVVPSRYRDELIGKLGAAAGKVTLNVASMTTSKTSLSWNHSKLLVVDGKTAITGGINGWKDDYLDTAHPVSDVDMALSGPAARSAGKYLDTLWDWTCRNASDPAKVWLATSNGASCMPSMEQD EAGSAPAEPTGDVPVIAVGGLGVGIKESDPSSGYHPDLPTAPDTKCTVGLHDNTNADRDYDTVNPEENALRSLIASARSHVEISQQDLNATCPPLPRYDIRTYDTLAGKLAAGVKVRIVVSDPANRGAV GSGGYSQIKSLDEISDTLRTRLVALTGDNEKASRALCGNLQLASFRSSDAAKWADGKPYALHHKLVSVDDSAFYIGSKNLYPAWLQDFGYIVESPAAAQQLKTELLDPEWKYSQQAAATPAGCPARQAG

[0056] In this invention, the amino acid sequence of the PLD mutant A379T is shown in SEQ ID NO.2: ADTPPTPHLDAIERSLRDTSPGLEGSVWQRTDGNRLDAPDGDPAGWLLQTPGCWGDAGCKDRAGTRRLLDKMTRNIADARHTVDISSLAPFPNGGFEDAVVDGLKASVAAGHSPRVRILVGAAPIYHLNVVPSRYRDELIGKLGAAAGKVTLNVASMTTSKTSLSWNHSKLLVVDGKTAITGGINGWKDDYLDTAHPVSDVDMALSGPAARSAGKYLDTLWDWTCRNASDPAKVWLATSNGASCMPSMEQD EAGSAPAEPTGDVPVIAVGGLGVGIKESDPSSGYHPDLPTAPDTKCTVGLHDNTNADRDYDTVNPEENALRSLIASARSHVEISQQDLNATCPPLPRYDIRTYDTLAGKLAAGVKVRIVVSDPANRGTV GSGGYSQIKSLDEISDTLRTRLVALTGDNEKASRALCGNLQLASFRSSDAAKWADGKPYALHHKLVSVDDSAFYIGSKNLYPAWLQDFGYIVESPAAAQQLKTELLDPEWKYSQQAAATPAGCPARQAG

[0057] The present invention will be further explained and illustrated below through specific embodiments.

[0058] Example 1: Obtaining the wild-type PLD gene

[0059] 1. Genomic DNA was extracted from Streptomyces antibioticus TCCC 21059 stored in the applicant's laboratory using the OMEGA Bacterial DNA Kit. The extraction steps are as follows:

[0060] (1) Activation of strain: Dip a glycerol tube into the Streptomyces spore solution with an inoculation loop, streak in three zones, and incubate at 28℃ for 4-5 days;

[0061] (2) Pick a single colony from the culture plate and inoculate it into 5 mL of liquid LB medium. Incubate at 220 r / min and 37 ℃ for 12 h.

[0062] (3) Take an appropriate amount of culture solution and dispense it into 2mL EP tubes. Centrifuge at 12000r / min for 2min and discard the supernatant.

[0063] (4) Add 250 μL of ddH2O to resuspend the bacterial cells, and add 50 μL of lysozyme. Incubate at 37°C for 20 min.

[0064] (5) Add 100 μL BTL Buffer and 20 μL proteinase K, and vortex.

[0065] (6) In a 55℃ water bath for 40-50 minutes, shake to mix every 20-30 minutes;

[0066] (7) Add 5 μL of RNase, invert and mix several times, and let stand at room temperature for 5 min;

[0067] (8) Centrifuge at 12000r / min for 2min to remove undigested portion, transfer the supernatant to a new EP tube, add 220μL BDL buffer, and incubate at 65℃ for 15min.

[0068] (9) Add 220 μL of anhydrous ethanol and mix by blowing and sucking.

[0069] (10) Transfer the liquid in the EP tube into the recovery column and let it stand for 1 min. Centrifuge at 12000 r / min for 1 min. Pour the filtrate back into the recovery column and repeat twice. Discard the waste liquid.

[0070] (11) Add 500 μL HBC buffer, centrifuge at 12000 r / min for 1 min, and discard the filtrate.

[0071] (12) Add 700 μL DNA wash buffer, let stand for 1 min, centrifuge at 12000 r / min for 1 min, and discard the filtrate.

[0072] (13) Add 500 μL DNA wash buffer, let stand for 1 min, centrifuge at 12000 r / min for 1 min, and discard the filtrate.

[0073] (14) 12000r / min for 2min, discard the waste liquid tube, and put the recovery column into a new EP tube.

[0074] (15) Place in a 55℃ metal bath to dry for 10 minutes.

[0075] (16) Add 50 μL of sterile water at 55℃, let stand at room temperature for 5 min, centrifuge at 12000 r / min for 2 min, discard the recovery column, and the liquid in the EP tube is the genome.

[0076] 2. Amplification of wild-type PLD gene

[0077] Using the extracted genome of *Streptomyces violaceum* as a template, a pair of primers was designed upstream and downstream of the ORF frame to introduce restriction enzyme sites BamHI and NotI, respectively. The primers used in this invention are as follows:

[0078] Upstream primer P1:

[0079] 5'-CGCGGATCCGCAGATACGCCGCC-3'

[0080] Downstream primer P2:

[0081] 5'-AAGGAAAAAAGCGGCCCGCTTAGTGGTGGTGGTGGTGGTGACCAGCTTG GCGAGC-3'

[0082] Using P1 and P2 as upstream and downstream primers, amplification was performed using the genome of Streptomyces antibiotics as a template.

[0083] The PCR amplification reaction system was 50 μL, and its composition was as follows:

[0084]

[0085]

[0086] Note: The reagents required above are from Takara Bio Inc.

[0087] The amplification program was as follows: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 53℃ annealing for 20 s, 72℃ extension for 8 s, for 30 cycles; 72℃ extension for 10 min. The PCR amplification products were subjected to 0.8% agarose gel electrophoresis, yielding a band of approximately 1500 bp (see...). Figure 1 The PCR product was recovered using a small-volume DNA recovery kit to obtain the wild-type PLD original gene pld (SEQ ID NO. 3) of the present invention. pld and pBSA43 plasmid were double-digested with restriction endonucleases BamHI and NotI, respectively. The pld recovered from the gel was ligated with the pBSA43 vector to obtain the recombinant plasmid pBSA43-pld, which was then transformed into Bacillus subtilis WB600 to obtain the recombinant strain WB600 / pBSA43-pld.

[0088] Example 2: Obtaining the PLD mutant A379T

[0089] 1. Based on overlap PCR technology, a saturation mutation was performed on amino acid position 379 of wild-type PLD to construct a novel PLD mutant library with the mutation occurring at position A379. The mutation primers are designed as follows:

[0090] Mutated upstream primer 379-F:

[0091] 5'-AATAGAGGCNNKGTTGGAAGC-3'

[0092] Mutated downstream primer 379-R:

[0093] 5'-GCTTCCAACMNNGCCTCTATT-3'

[0094] In the first step of the overlap PCR reaction system, P1 and 379-R were used as upstream and downstream primers, respectively, and P2 and 379-F were used as upstream and downstream primers, respectively. The plasmid pBSA43-pld was used as a template to perform PCR1 reaction, and upstream and downstream fragments were obtained respectively.

[0095] The reaction system for upstream fragment amplification is as follows:

[0096] 379-R 2μL pBSA43-pld 2μL Primer Star Max Enzyme 25μL <![CDATA[ddH2O]]> 19μL

[0097] The reaction system for downstream fragment amplification is as follows:

[0098] 379-F 2μL pBSA43-pld 2μL Primer Star Max Enzyme 25μL <![CDATA[ddH2O]]> 19μL

[0099] The amplification program was as follows: 98℃ pre-denaturation for 30 min; 98℃ denaturation for 10 s, 53℃ annealing for 20 s, 72℃ extension for 7 s, for 30 cycles; 72℃ extension for 10 min.

[0100] 2. After gel extraction and recovery of upstream and downstream fragments, PCR 2 was performed. The reaction system was as follows:

[0101] Downstream segments 2.0μL Primer Star Max Enzyme 25μL <![CDATA[ddH2O]]> 21μL

[0102] The amplification program was as follows: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 53℃ annealing for 20s, 72℃ extension for 8s, for 5 cycles; 72℃ extension for 10min.

[0103] 3. After PCR 2, add 2 μL each of primers P1 and P2 to the system. The PCR 3 amplification program is as follows: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 54℃ annealing for 20 s, 72℃ extension for 10 s, for 30 cycles; 72℃ extension for 10 min. The PCR amplification products are subjected to 0.8% agarose gel electrophoresis and recovered using a small-volume DNA recovery kit. The PCR products and vector plasmid are double-digested with BamHI and NotI, purified, and recovered. The PCR products are ligated with the BamHI and NotI-digested vector plasmid pBSA43, transformed into Bacillus subtilis WB600, plated on LB solid medium containing kanamycin resistance, and incubated statically at 37℃ for 12 h to obtain transformants. A site-directed mutant gene library of phospholipase D at amino acid position 379 is obtained.

[0104] 4. Initial screening of high-activity mutants: Under aseptic conditions, liquid LB medium containing Kan resistance was aliquoted into sterile 48-well plates, 1 ml per well. Then, a single clone of each transformant was picked up with a sterile toothpick and transferred to the 48-well plate, ensuring that only a small amount of bacteria was picked up each time. The 48-well plates were transferred to a shaker and incubated at 220 rpm and 37°C for 48 h. Then, the plates were centrifuged at 5000 rpm for 10 min at 4°C. 500 μL of the fermentation supernatant was added to 1.5 mL of 4.4 mM PC (dissolved in ether) and 1 mL of 52.8 mM L-serine (dissolved in 0.2 M pH 5.5 acetate-sodium acetate buffer). The mixture was incubated at 40°C and 200 rpm for 20 min in a water bath. Extraction was performed overnight in the dark using chloroform:methanol (2:1, v / v). The lower layer was collected for TLC analysis. Mutants with high activity were detected (see...). Figure 3 ).

[0105] Thin-layer chromatography was used to initially screen transformants containing mutants. The specific steps are as follows:

[0106] (1) Preparation of developing solvent: n-butanol / glacial acetic acid / 95% ethanol / water / 0.1% ninhydrin solution (4:1:1:2:2, v / v / v / v / v);

[0107] (2) Pour the developing solvent into the developing tank and let it stand for 20 minutes;

[0108] (3) Draw a line 1.5cm from the bottom of the silicone plate;

[0109] (4) Spotting: Spot the test sample and the standard sample onto the silicone plate using a capillary tube. The spotting radius should not exceed 10 mm, and the samples should be spaced at least 1 cm apart. Each sample should be spotted 3 times.

[0110] (5) After the sample has been spotted and evaporated on the silicone plate, tilt it into the display tank. The silicone plate should not contact the inner wall of the display tank.

[0111] (6) When the developing solvent reaches about 1 cm from the top, remove the silica gel plate, dry it at 105℃ for 5-10 min, and place it in an iodine bath for 30 s. Serine will develop purple-red spots, PS will develop purple spots, and PC will develop yellow spots. The mutant with the highest activity enhancement was obtained (see...). Figure 3 ), extract plasmids, and verify via enzyme digestion (e.g. Figure 2 The sample was sent for testing in lane 1 (as shown in the image), and a high-activity mutant A379T was obtained. The corresponding coding gene is pldmA379T. The plasmid containing this gene was named pBSA43-pldmA379T, and the recombinant strain was named Bacillus subtilis recombinant strain WB600 / pBSA43-pldmA379T.

[0112] 5. Secondary screening of high-activity mutants

[0113] (1) Fermentation preparation of crude enzyme solution: The wild-type strain WB600 / pBSA43-pld and the mutant strain WB600 / pBSA43-pldm A379T with the highest activity obtained from the initial screening were activated in solid plate medium, and single colonies were picked and inoculated into 5 mL of liquid LB medium (Kan resistant), and cultured at 37℃ and 220 rpm for 8 h with shaking. The inoculum was transferred to 50 mL of liquid LB medium (Kan resistant) at a 2% inoculation rate, and cultured at 37℃ and 220 rpm for 48 h with shaking. The supernatant was centrifuged and the crude enzyme solution of wild type and A379T was obtained.

[0114] (2) Preparation of pure enzyme solution: Wash the Ni in the chromatography column with ddH2O 2+ The resin was rinsed to remove residual ethanol, and then two column volumes of Lysis Buffer were added to balance the pH of the resin.

[0115] The wild-type and A379T crude enzyme solutions obtained in step (1) were respectively mixed with the pretreated Ni 2+ Mix the resin thoroughly (100 rpm) for 60 min, then pour the mixture into the chromatography column to allow Ni to bind. 2+ The resin deposits inside the column. After the filtrate has drained, add 10 mL of Wash Buffer to wash away the weakly binding proteins.

[0116] After the Wash Buffer has drained, add 10 mL of Elution Buffer to elute the target protein bound to the resin, and collect the filtrate containing the target protein that flows out at this time.

[0117] The collected filtrate contained a high concentration of imidazole, which affected subsequent experiments. Therefore, the imidazole was replaced with Tris-HCl (pH 7.0, 50 mM), and the purified enzyme solution was collected and analyzed by SDS-PAGE. The results are as follows: Figure 4 As shown, a single strip with a size of 54kDa is obtained.

[0118] (3) Enzyme activity assay: After purification, the pure enzyme solution was used for catalysis. The reaction was carried out in a two-phase system, mainly including 3 mL of 0.022 M PC (dissolved in ether), 2 mL of 0.264 M L-serine (dissolved in 0.2 mol / L pH 5.5 acetate-sodium acetate buffer) and 1 mL of pure enzyme solution. The reaction was carried out in a 40℃ water bath for 20 min.

[0119] After the reaction was complete, chloroform / methanol (2:1, v / v) solution was added for extraction, followed by determination by high-performance liquid chromatography (HPLC). HPLC detection conditions:

[0120] The chromatographic column was a Venusil XBP Silica (5 μm, 2.1 × 150 mm), the mobile phase was acetonitrile / methanol / 85% phosphoric acid (95:5:0.8, v / v / v), the UV detection wavelength was 205 nm, the flow rate was set to 0.3 mL / min, the column temperature was maintained at 25 °C, and the injection volume was 10 μL.

[0121] Enzyme activity definition: Under the above catalytic conditions (unless otherwise specified, pH is 5.5 and temperature is 40℃), the amount of enzyme required to produce 1 μmol PS per minute using PC and L-serine as substrates is defined as one unit of enzyme activity, denoted as U / mL.

[0122] (4) Optimal temperature: The enzyme activity of pure enzyme solutions of wild type (WT) and mutant (A379T) was measured at 30, 40, 50, 60, 70 and 80 °C, respectively. The highest activity was taken as 100%, and the relative activity at each temperature was calculated. Figure 5 As shown, the optimal temperature for both the wild type and the mutant is 60℃.

[0123] Optimal pH: Wild-type (WT) and mutant (A379T) purified enzyme solutions were placed in acetate-sodium acetate buffer solutions at pH 4.0, 5.0, 6.0, 7.0, and 8.0, and enzyme activity was measured at 60°C. The highest activity was taken as 100%, and the relative activity at each pH was calculated. Figure 6 As shown, the optimal pH for both the wild type and the mutant is 6.0.

[0124] Temperature stability: Wild-type (WT) and mutant (A379T) pure enzyme solutions were incubated at 40℃, 50℃, and 60℃ for 2 hours, respectively. After incubation, residual enzyme activity was measured under optimal conditions. The activity of the untreated enzyme was set as 100%, which was used as a reference for calculating residual enzyme activity. Figure 7 As shown, after incubation at 40℃ for 2 hours, the residual enzyme activity of A379T showed no significant change compared to the initial enzyme activity, while the residual enzyme activity of WT was 74%. After incubation at 50℃ for 2 hours, the residual enzyme activity of A379T was 68%, while the residual enzyme activity of WT was only 31% of the initial enzyme activity. After incubation at 60℃ for 2 hours, the residual enzyme activity of A379T was 47%, while the residual enzyme activity of WT was only about 9%. These results indicate that both WT and A379T maintain good stability at 40℃, and the thermostability of the mutant A379T is higher than that of WT at 50℃-60℃.

[0125] pH stability: Pure enzyme solutions of wild-type (WT) and mutant (A379T) were stored at 4°C for 5 days in buffer solutions with pH values ​​of 6.0, 7.0, and 8.0, respectively. Residual enzyme activity was measured under optimal conditions, with the activity of the untreated enzyme set as 100%, and this was used as a reference for calculating residual enzyme activity. Figure 8 As shown, after 5 days of low-temperature storage at pH 6.0-8.0, the residual activity of A379T remained almost unchanged, and the residual enzyme activity of WT was also above 90% after 5 days of low-temperature storage at pH 6.0-8.0. These results indicate that both WT and A379T maintain good stability within the pH range of 6.0-8.0.

[0126] Specific enzyme activity assay: Under the optimal reaction conditions of 60℃ and 6.0, the enzyme activity of pure enzyme solutions of wild type (WT) and mutant was measured and the specific enzyme activity was calculated. The results showed that the specific activity of mutant (A379T) was 1.91 U / mg and that of WT was 0.33 U / mg.

[0127] Example 3: Construction of a PLD-expressing recombinant strain with enhanced Bacillus amyloliquefaciens enzyme activity

[0128] 1. Constructing a recombinant strain of Bacillus amyloliquefaciens with enhanced PLD expression activity.

[0129] (1) Preparation of competent cells of Bacillus amyloliquefaciens CGMCC No.11218

[0130] ① Bacillus amyloliquefaciens was streaked in three zones on antibiotic-free LB solid medium and incubated at 37°C for 24 hours;

[0131] ② Pick a single colony and inoculate it into 5 mL of LBS medium. Incubate at 37°C and 220 rpm for 12 h.

[0132] ③ Inoculate the seed culture into 100 mL of LBS medium at an inoculum rate of 2%, and incubate at 37°C and 220 rpm for 2-3 hours until OD (Organic Depth). 600 =0.4-0.6;

[0133] ④ Centrifuge at 6000 rpm for 10 minutes using a low-temperature centrifuge (4℃), and discard the supernatant;

[0134] ⑤ Resuspend the bacterial cells in 30 mL of washing buffer (0.5 M sorbitol, 0.5 M mannitol, 10% glycerol), centrifuge at 6000 rpm for 10 min using a low-temperature centrifuge (4℃), and discard the supernatant.

[0135] ⑥ Repeat step ⑤ twice;

[0136] ⑦ Add 10 mL of resuspension buffer (0.5 M sorbitol, 0.5 M mannitol, 10% glycerol, 14% PEG6000), mix by pipetting and aspiration to resuspend the bacteria.

[0137] ⑧ After fully suspending the bacterial cells, dispense them into sterilized, pre-cooled 1.5 mL EP tubes, 100 μL per tube, and store at -80°C.

[0138] (2) Electroporation of Bacillus amyloliquefaciens

[0139] ① First clean the electric rotary cup with 75% alcohol, then rinse it with water.

[0140] ② Mix 10 ng of recombinant plasmid pBSA43-pldmA379T and 100 μL of competent cells, then transfer to an electroporation vessel and incubate on ice for 2 min;

[0141] ③ After 2500V electric shock for 4-6ms, immediately add 1mL of resuscitation solution (LB + 0.5M sorbitol + 0.38M mannitol) and resuscitate for 3h. The resuscitation conditions are 37℃ and 220r / min.

[0142] ④ After resuscitation, centrifuge at 4000 r / min for 5 min to collect the bacterial cells, discard the supernatant and leave 100 μL. Mix the remaining 100 μL of supernatant with the bacterial cells by pipetting and aspiration. After mixing, spread it evenly on a Kan resistance selection plate and finally invert it and incubate it in a 37℃ incubator.

[0143] ⑤ Select transformants, extract plasmids, and verify via enzyme digestion (e.g.) Figure 2 (As shown in lane 2 of the middle swimming pool), the recombinant strain of Bacillus amyloliquefaciens CGMCC No.11218 / pBSA43-pldmA379T was obtained.

[0144] Wild-type PLD recombinant strain CGMCC No.11218 / pBSA43-pld was prepared using the same method described above.

[0145] Example 4: Expression and preparation of PLD mutant A379T in Bacillus amyloliquefaciens

[0146] 1. Recombinant strains activated by streak plating in three zones: CGMCC No.11218 / pBSA43-pldmA379T and CGMCC No.11218 / pBSA43-pld;

[0147] 2. Pick a single colony and inoculate it into 5 mL of LB medium containing kanamycin resistance, and incubate at 37°C and 220 rpm for 12 h with shaking.

[0148] 3. Transfer the inoculum to a kanamycin-resistant fermentation medium at a rate of 2% and ferment at 37°C and 220 rpm for 48 h.

[0149] 4. Centrifuge at 12000 rpm for 10 min, collect the fermentation supernatant, and obtain the crude enzyme solution of A379T mutant and the crude enzyme solution of wild-type PLD, respectively.

[0150] 5. Enzyme activity assay

[0151] The transesterification activity of PLD was determined by measuring the amount of PS generated under the action of PLD using PC and L-serine as substrates. The reaction was carried out in a biphasic system, mainly consisting of 3 mL of 0.022 M PC (dissolved in ether), 2 mL of 0.264 M L-serine (dissolved in 0.2 mol / L pH 6.0 acetate-sodium acetate buffer), and 1 mL of crude enzyme solution, reacted in a 60 °C water bath for 20 min. After the reaction was completed, chloroform / methanol (2:1, v / v) solution was added for extraction.

[0152] Enzyme activity assays showed that, in the Bacillus amyloliquefaciens expression system, the wild-type enzyme activity was 3.5 U / mL, and the A379T enzyme activity was 21.6 U / mL.

[0153] Example 6: Preparation of functional phospholipids PS

[0154] Take 3 mL of 0.022 M PC (dissolved in ether), 2 mL of 0.264 M L-serine (dissolved in 0.2 mol / L pH 6.0 acetate-sodium acetate buffer), and 1 mL of crude enzyme solution (prepared in Example 5). React at 40 °C for 3 h with a magnetic stirrer. Then, extract with 9 mL of chloroform:methanol (2:1) to obtain PS. HPLC was then used for liquid phase detection. The PS content was calculated based on the standard curve. PS yield (mol%) = PS yield / initial PC amount × 100%. The PS yield of WT was 11.26%, and the PS yield of A379T was 45.6%.

[0155] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that those skilled in the art can make various modifications, combinations, and improvements to the above embodiments without departing from the concept of this patent, and these modifications and combinations all fall within the scope of protection of this patent. Therefore, the scope of protection of this patent should be determined by the claims.

Claims

1. A phospholipase D mutant, characterized in that, The phospholipase D mutant was obtained by mutating the amino acid sequence of wild-type phospholipase D by A379T, and the amino acid sequence of the mutant is shown in SEQ ID No.

2.

2. The encoding gene of the phospholipase D mutant according to claim 1.

3. The encoding gene as described in claim 2, characterized in that, The nucleotide sequence is shown in SEQ ID No.

4.

4. A recombinant vector or recombinant strain containing the encoding gene of claim 2.

5. The recombinant vector as described in claim 4, characterized in that, The expression vector used was pBSA43 plasmid.

6. The recombinant strain according to claim 4, characterized in that, The host bacteria used were Bacillus subtilis WB600 or Bacillus amyloliquefaciens CGMCC No.11218.

7. The use of the recombinant vector or recombinant strain of claim 4 in the production of the phospholipase D mutant of claim 1.

8. The application of the phospholipase D mutant according to claim 1, characterized in that, It is used in the preparation of phosphatidylserine.

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