CN116769750A

CN116769750A - High-activity phospholipase mutant and application thereof

Info

Publication number: CN116769750A
Application number: CN202310694637.4A
Authority: CN
Inventors: 刘逸寒; 张淑静; 路福平
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2023-06-12
Filing date: 2023-06-12
Publication date: 2023-09-19

Abstract

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

Description

High-activity phospholipase mutant and application thereof

Technical field:

the invention belongs to the technical field of enzyme genetic engineering, and in particular relates to a phospholipase D mutant with improved enzyme activity obtained by performing site-directed saturation mutation through an overlap PCR technology, and preparation and application thereof.

The background technology is as follows:

phospholipase D (PLD, EC 3.1.4.4) is an enzyme that acts on the phosphoryl oxygen bond, hydrolyses the phosphodiester bond of glycerophospholipids to phosphatidic acid and hydroxy compounds, and catalyzes the binding of certain hydroxy-containing compounds to the acyl chain of phospholipids to form new functional phospholipids. The phospholipid is a generic name of a compound containing phosphate groups, the molecular structure of the phospholipid is composed of 2 hydrophobic long carbon chains and 1 polar head, the phospholipid is widely distributed in animal and plant kingdoms, is a natural biosurfactant, is also an important component of cell membranes of human beings, animals and plants, and has good emulsifying property and oxidation resistance; at the same time, it also has many physiological functions of promoting the development of brain nervous system, improving memory, reducing blood fat, reducing cholesterol, anti-aging and preventing cancer, etc. Thus, the PLD can be used to modify phospholipids to produce single or rare phospholipids such as Phosphatidic Acid (PA), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylethanolamine (PE), and the like. The enzyme modified phospholipids have improved nutritive value and various properties, and are widely used in the fields of foods, health products, medicines, feeds, cosmetics, etc.

PLD is widely found in numerous biological groups such as bacteria, fungi and higher animals and plants, and has been found from carrot roots and cabbage leaves as early as 40 s of the last century. Wherein, PLD in plant tissues is mostly extracellular enzyme, the separation and extraction work is relatively simple, and PLD in animal tissues is mostly intracellular enzyme, and the separation and the preparation are difficult. Later, PLD was successfully isolated in a variety of bacteria. In contrast, PLD produced by microorganisms has the advantages of low production cost, short culture period, high phosphatidyl transfer reaction activity, good substrate specificity and the like, and is therefore attracting attention. The PLD-producing microorganisms which have been reported to date mainly include Streptomyces (Streptomyces), corynebacterium (Corynebacterium), escherichia (Escherichia), pseudomonas (Pseudomonas), bacillus (Bacillus), salmonella (Salmonella) and the like. Compared with other microbial PLDs, PLDs derived from Streptomyces have higher commercial value due to their high catalytic activity and broad substrate specificity, which are in line with the demands of industrial production.

Although phospholipids can be extracted from egg yolk and soybean, a large amount of organic solvent is required to be consumed, resulting in high purification cost and limited application fields. PLD-catalyzed phosphatidyl transfer is the most prominent process for the preparation of rare phospholipids. Compared with the traditional physical extraction method and chemical synthesis method, PLD biocatalysis is utilized to prepare single phospholipid and phospholipid derivatives which are rare in nature and difficult to separate and purify, and the PLD biological catalytic preparation method has the advantages of good selectivity, environmental protection, simple mass production, mild reaction and the like. Therefore, the utilization of PLD enzymatic synthesis of rare phospholipids can solve the urgent need of functional food development on high-quality phospholipids, and promote the development of the food industry in China. However, the wild PLD has low transphosphatidylation activity, which results in low product conversion efficiency and difficulty in meeting the requirements of industrial application. In addition, PLD-catalyzed reactions are carried out in two-phase systems, which also greatly reduce the activity of PLD. In order to solve the practical application problems, the functions and characteristics of the natural enzyme, such as directed evolution, rational design, chemical modification and the like, are improved through a protein engineering strategy, so that the catalytic activity of the enzyme is effectively improved, the substrate range is enlarged, the enzyme stability is improved, and the requirements of industrial application are further met.

Protein directed evolution is one of the important means for improving the functions and activities of proteins, and enzymes with specific properties are rapidly obtained by increasing the mutation rate of genes and designing a specific screening and selecting method. Directed evolution of enzymes generally involves three steps: (1) constructing a gene mutant library by carrying out random mutation, site-directed mutation or recombination on a protein coding sequence; (2) directional screening, selection to obtain mutants with improved phenotype; (3) and taking the mutant as a starting point of the next round of gene diversification, and performing directed evolution iteration until the mutant with the optimal performance is obtained. The directed evolution technology greatly promotes the development of a plurality of fields such as enzyme engineering, metabolic engineering, medicine and the like, achieves great results in the aspects of enhancing the stability and substrate specificity of the protein, changing or enhancing the activity of the protein and the like, successfully reforms a large number of enzyme molecules through the site-directed mutagenesis technology, and obtains the industrial enzyme with higher activity and better stability than the natural enzyme. Overlapping extension PCR techniques employ primers with complementary ends to allow the PCR products to form overlapping strands, such that amplified fragments of different origins are spliced together in overlapping fashion by extension of the overlapping strands in subsequent amplification reactions. Overlap extension PCR has wide and unique application in site-directed mutation of genes, construction of fusion genes, synthesis of long fragment genes, gene knockout, amplification of target genes and the like.

Therefore, in the invention, the PLD gene derived from the antibiotic streptomycete is subjected to molecular modification by overlapping PCR, and high-throughput screening is performed by using a bacillus subtilis and bacillus amyloliquefaciens expression system, so that the PLD mutant gene with improved enzyme activity is obtained.

The invention comprises the following steps:

based on the problems existing in the prior art, in order to further promote the application of PLD in the industrial field, the existing properties of PLD need to be further improved, and the invention aims to provide a mutant of PLD with high activity.

The technical route for achieving the purpose of the invention is summarized as follows:

the method comprises the steps of obtaining a wild PLD gene from streptomyces avicola (streptomyces anitiiotics) through a basic molecular biology technical means, carrying out saturation mutation on the wild PLD gene by utilizing an overlap PCR technology, screening by utilizing a bacillus subtilis expression system to obtain a PLD mutant A379T and a coding gene pldMA379T thereof, reconstructing a recombinant vector, realizing high-efficiency expression of the PLD mutant in bacillus subtilis and bacillus amyloliquefaciens, and obtaining the PLD mutant with improved enzyme activity through fermentation, extraction and other technologies.

One of the technical schemes provided by the invention is a PLD mutant, the amino acid sequence of the mutant is shown as SEQ ID NO.2, and the mutant is obtained by carrying out A379T amino acid mutation on wild PLD shown as SEQ ID NO. 1;

furthermore, the invention also provides a coding gene of the PLD mutant, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 4.

The second technical scheme provided by the invention is a recombinant vector or recombinant strain containing the encoding gene of the PLD mutant;

further, the expression vector used by the recombinant vector is a shuttle vector pBSA43;

further, the host used by the recombinant strain is bacillus subtilis WB600 or bacillus amyloliquefaciens CGMCC No.11218.

The third technical scheme provided by the invention is the application of the recombinant vector or recombinant strain, in particular to the application in producing PLD mutant shown in SEQ ID NO. 2.

The fourth technical scheme provided by the invention is the application of PLD mutant shown in SEQ ID NO.2, especially the application in preparing phospholipid and derivatives thereof, more especially the application in preparing non-natural phospholipid derivatives;

further, the PLD mutant shown in SEQ ID NO.2 is applied to the catalytic synthesis of Phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylglucose, phosphatidylamino sugar, phosphatidylfructose and the like;

further, the PLD mutant can be used in forms including, but not limited to, enzyme solutions, enzyme powders, emulsions, gels, immobilized enzymes, and the like.

The beneficial effects are that:

1. the invention uses overlap PCR technology to make site-directed saturation mutation on wild PLD to obtain mutant A379T with improved enzyme activity. The specific activity of WT was 0.33U/mg, the specific activity of A379T was 1.91U/mg, 5.8 times that of WT, and the PS yield of A379T was 4.05 times that of WT.

2. The invention respectively uses a bacillus subtilis expression system and a bacillus amyloliquefaciens expression system to realize the high-efficiency expression of PLD mutants with improved enzyme activity in different modes.

Description of the drawings:

FIG. 1 PCR amplification electrophoretogram of wild-type PLD gene of the invention

Wherein: m is DNA Marker,1 is PLD gene;

FIG. 2 is a diagram showing the cleavage assay of the recombinant plasmid pBSA43-pldmA379T of the present invention, wherein: m is a DNA Marker,1 is a BamHI and NotI double-restriction electrophoresis chart of a recombinant plasmid pBSA43-pldma379T in bacillus subtilis, and 2 is a BamHI and NotI double-restriction electrophoresis chart of a recombinant plasmid pBSA43-pldma379T in bacillus amyloliquefaciens.

FIG. 3 is a thin layer chromatography of wild-type PLD of the invention catalyzed by mutant A379T to phosphatidylserine;

FIG. 4 is a SDS-PAGE of purified mutant A379T samples according to the invention

Wherein: m is a protein Marker,1 is an A379T purified sample;

FIG. 5 shows the optimum temperature profile of wild-type PLD and mutant PLD of the invention

Wherein: WT is wild-type PLD, a379T is mutant a379T;

FIG. 6 shows the pH optimum curve of wild-type PLD and mutant PLD of the present invention

Wherein: WT is wild-type PLD, a379T is mutant a379T;

FIG. 7 is a graph showing the temperature stability of wild-type PLD and mutant PLD of the present invention

Wherein: WT is wild-type PLD, a379T is mutant a379T;

FIG. 8 is a graph showing the pH stability of wild-type PLD and mutant PLD of the present invention

Wherein: WT is wild-type PLD, a379T is mutant a379T.

The specific embodiment is as follows:

the technical contents of the present invention will be further described with reference to examples, but the present invention is not limited to these examples, and the scope of the present invention is not limited to the following examples.

The culture medium and the solution used in the embodiment of the invention are as follows:

wash Buffer (mM): weighing NaCl 29.25g,Tris 2.42g, dissolving 3.5g of imidazole in ultrapure water to a volume of 1L, removing impurities by using a 0.22 mu m microporous membrane, and then placing in a refrigerator at the temperature of 4 ℃ for light-shielding storage.

ElutionBuffer (mM): weighing NaCl 29.25g,Tris 2.42g, dissolving 13.6g of imidazole in ultrapure water to a volume of 1L, removing impurities by using a 0.22 mu m microporous membrane, and then placing in a refrigerator at the temperature of 4 ℃ for light-shielding storage.

Lysis Buffer (mM): weighing NaCl 29.25g,Tris 2.42g, dissolving imidazole 1.4g in ultrapure water, fixing the volume to 1L, removing impurities by using a 0.22 mu m microporous membrane, and then placing in a refrigerator at 4 ℃ for light-shielding storage.

LB medium (g/L): yeast extract 5.0, tryptone 10.0, naCl 10.0, and 800mL of water are added for dissolution, and water is added to fix volume to 1L after complete dissolution.

SP salt solution (g/L): k (K) ₂ HPO ₄ 18.34，KH ₂ PO ₄ 6.0，(NH ₄ ) ₂ SO ₄ 2.0, sodium citrate 1.0, mgSO ₄ ·7H ₂ O0.2, adding 800mL of water for dissolution, and continuously adding water to fix the volume to 1L after complete dissolution.

SPI medium (200 mL): 195.2mL of SP salt solution, 0.8mL of 5% casein hydrolysate, 2mL of 10% yeast juice and 2mL of 5% glucose solution, respectively taking 5mL of mixed solution, subpackaging into sterilized empty test tubes, and preserving at 4 ℃.

SPII medium: 292.8mL of SP salt solution, 1.2mL of 5% casein hydrolysate, 3mL of 10% yeast juice, 3mL of 5% glucose solution, 1.5mL of 100mM calcium chloride and 1.5mL of 50mM magnesium chloride, respectively taking 2mL of mixed solution, packaging into sterilized empty test tubes, and preserving at 4 ℃.

LBS medium (g/L): sorbitol 91.085, naCl 10, yeast extract 5, tryptone 10 and 800mL of water are added for dissolution, and water is added for volume fixation to 1L after complete dissolution.

Fermentation medium (g/L): 64g of corn flour, 40g of bean cake powder, 4g of disodium hydrogen phosphate, 0.3g of potassium dihydrogen phosphate, 0.7g of high-temperature amylase, and 800mL of water to dissolve, and continuously adding water to fix the volume to 1L after complete dissolution.

The solid media of the above media were each supplemented with 2% agar.

The following definitions are employed in the present invention:

1. nomenclature of amino acids and DNA nucleic acid sequences

Using the accepted IUPAC nomenclature for amino acid residues, three letter codes or single letter forms are used. The DNA nucleic acid sequence uses accepted IUPAC nomenclature.

Identification of PLD mutants

"amino acid substituted at the original amino acid position" is used to denote the mutated amino acid in the PLD mutant. As with Ala379Thr, it is indicated that the amino acid at position 379 is replaced by Thr from Ala of the wild-type PLD, the numbering of the position corresponding to the amino acid sequence numbering of the wild-type PLD in SEQ ID NO. 1.

In the present invention, lower case italics PLD indicates the gene encoding the wild-type PLD, lower case italics pldmA379T indicates the gene encoding the mutant a379T, and the information is as follows.

PLD	Amino acid mutation site	Gene mutation site	Amino acid SEQ ID No.	Nucleotide SEQ ID No.
					Wild type	Ala379	—	1	3
A379T	Ala379Thr	GCA→ACG	2	4

In the invention, the amino acid sequence of the wild PLD is shown as SEQ ID NO. 1: ADTPPTPHLDAIERSLRDTSPGLEGSVWQRTDGNRLDAPDGDPAGWLLQTPGCWGDAGCKDRAGTRRLLDKMTRNIADARHTVDISSLAPFPNGGFEDAVVDGLKASVAAGHSPRVRILVGAAPIYHLNVVPSRYRDELIGKLGAAAGKVTLNVASMTTSKTSLSWNHSKLLVVDGKTAITGGINGWKDDYLDTAHPVSDVDMALSGPAARSAGKYLDTLWDWTCRNASDPAKVWLATSNGASCMPSMEQDEAGSAPAEPTGDVPVIAVGGLGVGIKESDPSSGYHPDLPTAPDTKCTVGLHDNTNADRDYDTVNPEENALRSLIASARSHVEISQQDLNATCPPLPRYDIRTYDTLAGKLAAGVKVRIVVSDPANRGAVGSGGYSQIKSLDEISDTLRTRLVALTGDNEKASRALCGNLQLASFRSSDAAKWADGKPYALHHKLVSVDDSAFYIGSKNLYPAWLQDFGYIVESPAAAQQLKTELLDPEWKYSQQAAATPAGCPARQAG

In the invention, the amino acid sequence of PLD mutant A379T is shown in SEQ ID NO. 2: ADTPPTPHLDAIERSLRDTSPGLEGSVWQRTDGNRLDAPDGDPAGWLLQTPGCWGDAGCKDRAGTRRLLDKMTRNIADARHTVDISSLAPFPNGGFEDAVVDGLKASVAAGHSPRVRILVGAAPIYHLNVVPSRYRDELIGKLGAAAGKVTLNVASMTTSKTSLSWNHSKLLVVDGKTAITGGINGWKDDYLDTAHPVSDVDMALSGPAARSAGKYLDTLWDWTCRNASDPAKVWLATSNGASCMPSMEQDEAGSAPAEPTGDVPVIAVGGLGVGIKESDPSSGYHPDLPTAPDTKCTVGLHDNTNADRDYDTVNPEENALRSLIASARSHVEISQQDLNATCPPLPRYDIRTYDTLAGKLAAGVKVRIVVSDPANRGTVGSGGYSQIKSLDEISDTLRTRLVALTGDNEKASRALCGNLQLASFRSSDAAKWADGKPYALHHKLVSVDDSAFYIGSKNLYPAWLQDFGYIVESPAAAQQLKTELLDPEWKYSQQAAATPAGCPARQAG

The invention will be further illustrated by the following examples.

Example 1: obtaining of wild-type PLD Gene

1. Genomic DNA of the applicant laboratory-stored antibiotic Streptomyces (Streptomyces antibioticus) TCCC 21059 was extracted using a kit (OMEGA: bacterial DNA Kit) as follows:

(1) Strain activation: dipping streptomycete spore liquid from an glycerol pipe by using an inoculating loop, and culturing for 4-5 days at the constant temperature of 28 ℃ by using three dividing lines;

(2) Picking single bacterial colony from a flat plate for culturing thalli, inoculating the single bacterial colony into 5mL of liquid LB culture medium, and culturing for 12h at 220r/min and 37 ℃;

(3) Taking a proper amount of culture bacterial liquid, subpackaging the culture bacterial liquid into a 2mL EP tube, centrifuging for 2min at 12000r/min, and discarding the supernatant;

(4) Add 250. Mu.L ddH ₂ O, re-suspending the thalli, adding 50 mu L of lysozyme, and preserving the temperature for 20min at 37 ℃;

(5) 100. Mu.L of BTL Buffer and 20. Mu.L of proteinase K were added and vortexed;

(6) Carrying out water bath at 55 ℃ for 40-50min, and oscillating and uniformly mixing every 20-30 min;

(7) Adding 5 mu L of RNase, reversing and mixing for several times, and standing at room temperature for 5min;

(8) Centrifuge 12000r/min for 2min, remove undigested fractions, transfer supernatant to fresh EP tube, add 220. Mu.L BDL buffer, water bath at 65℃for 15min.

(9) 220 mu L absolute ethyl alcohol is added, and the mixture is blown and sucked uniformly.

(10) Transferring the liquid in the EP pipe into a recovery column, standing for 1min, centrifuging for 1min at 12000r/min, pouring the filtrate into the recovery column again, repeating for two times, and pouring out the waste liquid.

(11) Add 500. Mu.L HBC buffer, centrifuge for 1min at 12000r/min, discard the filtrate.

(12) 700 mu L DNA wash buffer was added, left stand for 1min, centrifuged at 12000r/min for 1min, and the filtrate was discarded.

(13) 500 mu L DNA wash buffer was added, left stand for 1min, centrifuged at 12000r/min for 1min, and the filtrate was discarded.

(14) 12000r/min was allowed to air-space for 2min, the waste tube was discarded, and the recovery column was placed in a new EP tube.

(15) And (5) placing in a metal bath at 55 ℃ for drying for 10min.

(16) 50. Mu.L of 55℃sterile water was added, left to stand at room temperature for 5min, centrifuged at 12000r/min for 2min, and the recovery column was discarded, and the liquid in the EP tube was the genome.

2. Amplification of wild-type PLD Gene

The genome of the extracted antibiotic streptomycete is taken as a template, a pair of primers are designed at the upstream and downstream of the ORF frame, and restriction enzyme cutting sites BamHI and NotI are respectively introduced, and the primers used in the invention are as follows:

the upstream primer P1:

5’-CGCGGATCCGCAGATACGCCGCC-3’

downstream primer P2:

5’-AAGGAAAAAAGCGGCCGCTTAGTGGTGGTGGTGGTGGTGACCAGCTTG GCGAGC-3’

p1 and P2 are used as the upstream and downstream primers, and the genome of the antibiotic streptomycete is used as the template for amplification.

The reaction system for PCR amplification was 50. Mu.L, and the composition thereof was:

note that: the above reagents were obtained from Takara, takara Bio Inc.

The amplification procedure was: pre-denaturation at 98 ℃ for 30s; denaturation at 98℃for 10s, annealing at 53℃for 20s, extension at 72℃for 8s, 30 cycles; extending at 72℃for 10min. The PCR amplified product was subjected to 0.8% agarose gel electrophoresis to obtain a band of about 1500bp (see FIG. 1), the PCR product was recovered with a small amount of DNA recovery kit to obtain the wild-type PLD original gene PLD (SEQ ID NO. 3) of the present invention, PLD and pBSA43 plasmids were digested with restriction enzymes BamHI and NotI, respectively, and the gel-cut recovered PLD was ligated with pBSA43 vector to obtain recombinant plasmid pBSA43-PLD, which was transformed into Bacillus subtilis WB600 to obtain recombinant strain WB600/pBSA43-PLD.

Example 2: acquisition of PLD mutant A379T

1. Based on overlapping PCR technology, carrying out saturation mutation on 379th amino acid of wild PLD, constructing a novel PLD mutation library with mutation at A379th, and designing mutation primers as follows:

mutation of the upstream primer 379-F:

5’-AATAGAGGCNNKGTTGGAAGC-3’

mutation of the downstream primer 379-R:

5’-GCTTCCAACMNNGCCTCTATT-3’

in the first step of overlapping PCR reaction system, P1 and 379-R are used as upstream and downstream primers, P2 and 379-F are used as upstream and downstream primers, and plasmid pBSA43-pld is used as template to perform PCR1 reaction to obtain upstream fragment and downstream fragment.

The reaction system for amplifying the upstream fragment is as follows:

P1	2μL
		379-R	2μL
pBSA43-pld	2μL
		PrimerStar Max enzyme	25μL
ddH ₂ O	19μL

The reaction system for amplifying the downstream fragment is as follows:

P2	2μL
		379-F	2μL
pBSA43-pld	2μL
		PrimerStar Max enzyme	25μL
ddH ₂ O	19μL

The amplification procedure was: pre-denaturation at 98 ℃ for 30min; denaturation at 98℃for 10s, annealing at 53℃for 20s, extension at 72℃for 7s for 30 cycles; extending at 72℃for 10min.

2. And (3) cutting the gel, recovering the upstream fragment and the downstream fragment, and then carrying out PCR 2, wherein the reaction system is as follows:

upstream fragment	2.0μL
		Downstream fragment	2.0μL
PrimerStar Max enzyme	25μL
		ddH ₂ O	21μL

The amplification procedure was: pre-denaturation at 98 ℃ for 30s; denaturation at 98℃for 10s, annealing at 53℃for 20s, extension at 72℃for 8s, 5 cycles; extending at 72℃for 10min.

3. After the end of PCR 2, 2. Mu.L of each of the primers P1 and P2 was added to the system, and the PCR 3 amplification procedure was performed as follows: pre-denaturation at 98 ℃ for 30s; denaturation at 98℃for 10s, annealing at 54℃for 20s, extension at 72℃for 10s, 30 cycles; extending at 72℃for 10min. The PCR amplified product is subjected to 0.8% agarose gel electrophoresis, a small amount of DNA recovery kit is used for recovering the PCR product, bamHI and NotI double digestion is carried out on the PCR product and a carrier plasmid, the PCR product is purified and recovered, the PCR product is connected with a carrier plasmid pBSA43 subjected to BamHI and NotI double digestion, bacillus subtilis WB600 is transformed, the PCR product is coated on LB solid medium containing kanamycin resistance, and the PCR product is subjected to stationary culture in a37 ℃ incubator for 12 hours, so that a transformant is obtained. A library of site-directed mutant genes for amino acid 379 of phospholipase D was obtained.

4. Primary screening of high-activity mutants: under aseptic conditions, liquid LB medium containing Kan resistance was dispensed into aseptic 48-well plates with 1ml each, and then, each transformant was picked up with sterilized toothpicks and single-cloned into 48-well plates, so that a small amount of bacteria was just stained each time during the picking process. The 48-well plate was transferred to shaking culture at 220rpm at 37℃for 48h. Then, the mixture was centrifuged at 5000rpm for 10min by a low-temperature centrifuge (4 ℃ C.), 500. Mu.L of the fermentation supernatant was added to 1.5mL of 4.4mM PC (diethyl ether-dissolved), 1mL of 52.8mM L-serine (0.2M pH5.5 acetic acid-sodium acetate buffer-dissolved), and the mixture was catalyzed at 200rpm in a water bath at 40℃for 20min with chloroform: methanol (2:1, v/v) was extracted overnight in the dark, and the lower layer was subjected to TLC detection to detect mutants with high activity (see FIG. 3).

The transformant containing the mutant is subjected to primary screening by using a thin layer chromatography technology, and the specific steps are as follows:

(1) Preparing a developing agent: n-butanol/glacial acetic acid/95% ethanol/water/0.1% ninhydrin solution (4:1:1:2:2, v/v/v/v/v);

(2) Pouring the developing agent into a developing cylinder, and standing for 20min;

(3) Scribing at a position 1.5cm away from the lower end of the silica gel plate;

(4) Spotting: sample application is carried out on the sample to be tested and the standard sample by using a capillary on a silica gel plate, the sample application radius is not more than 10mm, at least 1cm is arranged between the samples, and each sample point is 3 times;

(5) After the sample after sample application volatilizes on a silica gel plate, the sample is obliquely placed into a spreading cylinder, and the silica gel plate cannot be contacted with the inner wall of the spreading cylinder;

(6) Taking out the silica gel plate when the developing agent moves up to about 1cm from the top end, drying at 105 ℃ for 5-10min, putting into an iodine jar for 30s, developing serine into purple spots, developing PS into purple spots, and developing PC into yellow spots. Obtaining the mutant with highest activity (see figure 3), extracting plasmid, enzyme cutting and verifying (shown as lane 1 in figure 2), and obtaining the high-activity mutant A379T, wherein the corresponding coding gene is pldma379T, the plasmid containing the gene is named pBSA43-pldma379T, and the recombinant strain is named bacillus subtilis recombinant strain WB600/pBSA43-pldm A379T.

5. Rescreening of high-activity mutants

(1) Preparing crude enzyme liquid by fermentation: the wild type strain WB600/pBSA43-pld and the highest activity mutant strain WB600/pBSA43-pldm A379T obtained by preliminary screening are respectively activated in a solid flat-plate culture medium, single colonies are picked up and inoculated in 5mL of liquid LB culture medium (Kan resistance), shake-cultured at 37 ℃ and 220rpm for 8 hours, transferred into 50mL of liquid LB culture medium (Kan resistance) with an inoculum size of 2 percent, shake-cultured at 37 ℃ and 220rpm for 48 hours, and the supernatant is centrifugally obtained, thus obtaining wild type and A379T crude enzyme liquid.

(2) Preparing pure enzyme solution: with ddH ₂ O cleaning Ni in chromatographic column ²⁺ Resin, rinse off residual ethanol, then add two column volumes of Lysis Buffer to balance the pH of the resin;

respectively mixing the wild type crude enzyme solution and the A379T crude enzyme solution prepared in the step (1) with pretreated Ni ²⁺ Mixing resin, combining (100 r/min) for 60min, and pouring into chromatographic column to obtain Ni ²⁺ Depositing resin in column, filtering to remove filtrate, addingAdding 10mL Wash Buffer to Wash out the foreign protein with weak binding force;

after Wash Buffer is drained, 10mL Elution Buffer is added to elute the target protein bound on the resin, and the filtrate containing the target protein flowing out at the moment is collected;

the collected filtrate contained imidazole at a higher concentration, which had an influence on the subsequent experiments, so that imidazole was replaced with Tris-HCl (pH 7.0, 50 mM), and the purified enzyme solution was collected and analyzed by SDS-PAGE, and as a result, a single band of 54kDa was obtained as shown in FIG. 4.

(3) The enzyme activity determination method comprises the following steps: the purified pure enzyme solution is catalyzed, the reaction is carried out in a biphasic system and mainly comprises 3mL of 0.022M PC (dissolved by diethyl ether), 2mL of 0.264M L-serine (dissolved by 0.2mol/L of pH5.5 acetic acid-sodium acetate buffer solution) and 1mL of pure enzyme solution, and the reaction is carried out in a water bath at 40 ℃ for 20min.

After the completion of the reaction, a chloroform/methanol (2:1, v/v) solution was added to extract, followed by measurement by high performance liquid chromatography. High performance liquid chromatography detection conditions:

the chromatographic column is Venusil XBP Silica (5 μm, 2.1X106 mm), the mobile phase is acetonitrile/methanol/85% phosphoric acid (95:5:0.8, v/v/v), the ultraviolet detection wavelength is 205nm, the flow rate is set to 0.3mL/min, the column temperature is maintained at 25 ℃, and the sample injection amount is 10 mu L.

Definition of enzyme activity: under the above-mentioned catalytic conditions (pH 5.5, temperature 40 ℃ C.) the amount of enzyme required to produce 1. Mu. Mol of PS per minute using PC and L-serine as substrates was defined as one enzyme activity unit, and was designated U/mL.

(4) Optimum temperature: the relative activities of the wild-type (WT) and mutant (A379T) were calculated by performing enzyme activity assays at 30, 40, 50, 60, 70 and 80℃respectively, taking the respective highest activities as 100%. As shown in FIG. 5, the optimal temperatures for both the wild type and mutant were 60 ℃.

Optimum pH: the enzyme activities of the wild-type (WT) and mutant (A379T) were measured by placing them in acetic acid-sodium acetate buffers at pH 4.0, pH 5.0, pH6.0, pH7.0 and pH8.0 at 60℃to calculate the relative activities at the respective pHs using the respective highest activities as 100%. As shown in FIG. 6, the optimal pH was 6.0 for both the wild type and mutant.

Temperature stability: pure enzyme solutions of the Wild Type (WT) and the mutant (A379T) were incubated at 40 ℃,50 ℃ and 60 ℃ for 2 hours, respectively, and residual enzyme activities were measured under the optimum conditions after the incubation was completed, and the activity of the enzyme which was not subjected to the incubation treatment was set to 100%, which was the reference calculation of residual enzyme activities. As shown in fig. 7, after incubation at 40 ℃ for 2 hours, the residual enzyme activity of a379T was not significantly changed from the initial enzyme activity, whereas the residual enzyme activity of WT was 74%. After incubation for 2h at 50 ℃, the residual enzyme activity of a379T was 68% and the residual enzyme activity of WT was only 31% of the initial enzyme activity. After incubation at 60℃for 2h, the residual enzyme activity of A379T was 47% and the residual enzyme activity of WT was only about 9%. The results show that both WT and A379T can maintain good stability at 40℃and that the thermal stability of mutant A379T is higher than that of WT at 50℃to 60 ℃.

pH stability: the residual enzyme activities were calculated by storing the pure enzyme solutions of the Wild Type (WT) and the mutant (A379T) in buffers of pH (6.0, 7.0 and 8.0) at a low temperature of 4℃for 5 days, and measuring the residual enzyme activities under the optimum conditions, and setting the enzyme activities without incubation to 100%. As shown in FIG. 8, the residual activity of A379T was hardly changed after 5d of low temperature storage at pH 6.0-8.0, and the residual enzyme activity was also 90% or more after 5d of low temperature storage at pH 6.0-8.0. The above results demonstrate that both WT and A379T maintain good stability in the pH range of 6.0-8.0.

Specific enzyme activity determination: the enzyme activities of the pure enzyme solutions of the Wild Type (WT) and the mutant are respectively measured under the reaction conditions of the optimal temperature of 60 ℃ and the optimal pH of 6.0, and the specific enzyme activities are calculated, and the result shows that the specific activity of the mutant (A379T) is 1.91U/mg and the specific activity of the WT is 0.33U/mg.

Example 3: construction of PLD expression recombinant Strain with enhanced Bacillus amyloliquefaciens enzyme Activity

1. Construction of PLD high-expression recombinant Strain with enhanced Bacillus amyloliquefaciens enzyme Activity

(1) Preparation of Bacillus amyloliquefaciens CGMCC No.11218 competent

(1) Carrying out three-area lineation on bacillus amyloliquefaciens on an antibiotic-free LB solid culture medium, and culturing at 37 ℃ for 24 hours;

(2) single colony is selected and inoculated in 5mL LBS culture medium, and shake culture is carried out for 12h at 37 ℃ and 220 rpm;

(3) inoculating the seed solution into 100mL LBS medium at 37deg.C and 220rpm, and culturing for 2-3h to OD ₆₀₀ ＝0.4-0.6；

(4) Centrifuging at 6000rpm for 10min with a low temperature centrifuge (4deg.C), and discarding supernatant;

(5) the cells were resuspended in 30mL of wash buffer (0.5M sorbitol, 0.5M mannitol, 10% glycerol), centrifuged at 6000rpm for 10min with a low temperature centrifuge (4 ℃), and the supernatant discarded;

(6) repeating two steps (5);

(7) adding 10mL of resuspension buffer (0.5M sorbitol, 0.5M mannitol, 10% glycerol, 14% PEG 6000), blowing and mixing to suspend the bacteria;

(8) after the bacterial cells were sufficiently suspended, they were packed in sterile pre-chilled 1.5mL EP tubes, each containing 100. Mu.L of the cells, and stored at-80 ℃.

(2) Bacillus amyloliquefaciens

(1) Firstly, washing an electric rotating cup with 75% alcohol, and then washing the electric rotating cup with molecular water;

(2) 10ng of recombinant plasmid pBSA43-pldma379T and 100 mu L of competent are evenly mixed and transferred into an electric rotating cup, and the mixture is subjected to ice bath for 2min;

(3) immediately adding 1mL of resuscitation fluid (LB+0.5M sorbitol+0.38M mannitol) after 4-6ms of electric shock at 2500V for 3h at 37deg.C and 220r/min;

(4) after resuscitating, centrifugally collecting thalli at 4000r/min and 5min, pouring the supernatant to leave 100 mu L, blowing and sucking the remaining 100 mu L of supernatant and thalli, uniformly mixing, uniformly coating the mixture on a Kan-resistance-containing screening plate, and finally, inversely placing the mixture in a37 ℃ incubator for culturing.

(5) Picking up the transformant, extracting the plasmid, and performing enzyme digestion verification (shown as lane 2 in FIG. 2) to obtain the bacillus amyloliquefaciens recombinant strain CGMCC No.11218/pBSA43-pldma379T.

The wild PLD recombinant strain CGMCC No.11218/pBSA43-PLD was prepared by the same method as described above.

Example 4: expression and preparation of PLD mutant A379T in bacillus amyloliquefaciens

1. Activating recombinant strain CGMCC No.11218/pBSA43-pldMA379T, CGMCC No.11218/pBSA43-pld by a plate three-zone line;

2. picking single colony, inoculating in 5mL LB culture medium containing kanamycin resistance, and shake culturing at 37deg.C and 220r/min for 12 hr;

3. the cells were inoculated in a 2% inoculum size into a fermentation medium containing kanamycin resistance, and cultured at 37℃for 48 hours at 220 r/min.

4.12000rpm, centrifuging for 10min, and collecting fermentation supernatant to obtain crude enzyme solution of A379T mutant and wild PLD crude enzyme solution.

5. Enzyme activity assay

The transesterification activity of PLD was determined by the amount of PS produced under the action of PLD using PC and L-serine as substrates. The reaction was carried out in a biphasic system, essentially comprising 3mL of 0.022M PC (dissolved in diethyl ether), 2mL of 0.264M L-serine (0.2 mol/L pH6.0 acetic acid-sodium acetate buffer solution) and 1mL of crude enzyme solution, and was reacted in a water bath at 60℃for 20min. After the completion of the reaction, a chloroform/methanol (2:1, v/v) solution was added to extract.

The enzyme activity measurement result shows that in the bacillus amyloliquefaciens expression system, the wild type enzyme activity is as follows: 3.5U/mL, A379T enzyme activity was 21.6U/mL.

Example 6: preparation of functional Phospholipids PS

3mL of 0.022M PC (dissolved in diethyl ether), 2mL of 0.264M L-serine (0.2 mol/L pH6.0 acetic acid-sodium acetate buffer solution) and 1mL of crude enzyme solution (prepared in example 5) were taken, the reaction temperature was 40℃and reacted for 3 hours under stirring by a magnetic stirrer, followed by 9mL of chloroform: PS was obtained by methanol (2:1) extraction, then liquid phase detection was performed by HPLC, PS content was calculated from standard curve, PS yield (mol%) =ps yield/initial PC amount×100%, PS yield of WT was 11.26% and PS yield of a379T was 45.6%.

The above examples merely represent a few embodiments of the present invention, which are described in more detail and are not to be construed as limiting the scope of the patent. It should be noted that, for a person skilled in the art, the above embodiments may also make several variations, combinations and improvements, without departing from the scope of the present patent. Therefore, the protection scope of the patent is subject to the claims.

Claims

1. A phospholipase D mutant is characterized in that the phospholipase D mutant is obtained by carrying out A379T mutation on the basis of the amino acid sequence of wild type phospholipase D, and the amino acid sequence of the mutant is shown as SEQ ID No. 2.

2. The phospholipase D mutant of claim 1, wherein the amino acid sequence of the mutant has greater than 75% homology with the sequence of SEQ ID No.2 of claim 1.

3. A gene encoding the phospholipase D mutant of claim 1.

4. The coding gene of claim 3, wherein the nucleotide sequence is set forth in SEQ ID No. 4.

5. A recombinant vector or recombinant strain comprising the coding gene of claim 3.

6. The recombinant vector according to claim 5, wherein the expression vector used is a pBSA43 plasmid.

7. The recombinant strain of claim 5, wherein the host bacteria are bacillus subtilis WB600 and bacillus amyloliquefaciens CGMCC No.11218.

8. Use of the recombinant vector or recombinant strain of claim 5 for the production of the phospholipase D mutant of claim 1.

9. Use of a phospholipase D mutant according to claim 1.

10. The use according to claim 9, in the preparation of glycerophospholipids and derivatives thereof.