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CN110129300B - a phospholipase D - Google Patents

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CN110129300B
CN110129300B CN201910528141.3A CN201910528141A CN110129300B CN 110129300 B CN110129300 B CN 110129300B CN 201910528141 A CN201910528141 A CN 201910528141A CN 110129300 B CN110129300 B CN 110129300B
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刘逸寒
路福平
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Tianjin University of Science and Technology
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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 technical scheme includes that site-directed mutagenesis is carried out on a wild phospholipase D gene by means of a recombinant DNA technology to obtain a phospholipase D gene with high activity, then the high-activity phospholipase D gene is expressed in a bacillus subtilis expression system and a pichia pastoris expression system (including a pichia pastoris free expression system and a pichia pastoris cell surface display system), a recombinant strain for producing the high-activity phospholipase D is obtained, after expression, the specific enzyme activity of the high-activity phospholipase D is detected to be improved by 38-140% compared with that of the wild phospholipase D, and the highest values of the fermentation enzyme activities of the high-activity phospholipase D in the bacillus subtilis expression system, the pichia pastoris expression system and the pichia pastoris cell surface display system are respectively 36.8U/ml, 48.1U/ml and 140.2U/(g.cell dry weight).

Description

Phospholipase D
The application is a divisional application of an invention patent application 201610402557.7, the application date of 201610402557.7 is 2016, 6 and 2, the application number is 201610402557.7, and the invention name is a novel phospholipase D and a method for preparing phosphatidic acid and phosphatidylserine by the phospholipase D.
The technical field is as follows:
the invention belongs to the technical field of enzyme genetic engineering, and particularly relates to a phospholipase D mutant with improved specific enzyme activity obtained by in vitro directed evolution of an overlapped PCR (polymerase chain reaction) technology, and provides a method for preparing phosphatidic acid and phosphatidylserine under catalysis of high-activity phospholipase D.
Background art:
phospholipase D (PLD) is widely distributed and is found in various animals, plants and microorganisms. PLD acts on phosphoryl oxygen bond P-O in phospholipid molecule, and its biocatalysis is mainly embodied in two reactions: (1) hydrolyzing the terminal phosphate ester bond on the phospholipid to produce phosphatidic acid and a hydroxyl compound; (2) when another hydroxyl-containing compound is present, it catalyzes the association of a phosphatidyl group with it to form a new phospholipid, i.e., a transphosphatidylation reaction.
Phosphatidic Acid (PA) is a simple and common phospholipid, widely exists in animal and plant cells, and is a basic component of animal and plant cell biological membranes. PA consists of a glycerol backbone, a fatty acid group at position 1 or 2, a phosphate group at position 3. PA is a direct product of PLD action, and PA can drive Ca2+Various enzymes in cells are activated or synergistically activated, and meanwhile, PA can also promote mitosis of cells, promote formation of superoxide in cells, cause contraction of muscles, promote secretion of hormones, induce aggregation of platelets and the like. Based on the above effects, PA can be applied to the food industry, the pharmaceutical industry, the cosmetics industry and the like.
Phosphatidylserine (PS) is a ubiquitous phospholipid, is usually located in the inner layer of a cell membrane, is an important component of the cell membrane, is one of important components of the brain cell membrane due to participating in a series of membrane functional reactions, and is particularly in the nervous system of a human body, and has a remarkable effect on improving memory, relieving pressure, repairing brain injury, treating hyperkinetic syndrome and depression of children, preventing senile dementia and the like. But the phosphatidylserine with certain purity has rare content in nature, so that the preparation of the phosphatidylserine product with high purity and good quality has important significance.
The preparation of phosphatidic acid or phosphatidylserine by an enzymatic method refers to the reaction of PLD catalytic substrate (such as PC) to generate phosphatidic acid or phosphatidylserine under a certain condition. The production of phosphatidic acid or phosphatidylserine is different in the type of PLD-catalyzed reaction, the former being a hydrolysis reaction, and the latter being a modification of the phospholipid head group by transphosphatidylation (transphosphatidylation) reaction using PLD.
The in vitro directed evolution of enzyme molecules belongs to the irrational design of proteins and belongs to the category of protein engineering. The general approach is to create molecular diversity at the molecular level using molecular biology approaches, and to combine sensitive, high-throughput screening techniques to obtain ideal mutants in a short time. Unlike the method of artificially creating special evolution conditions to simulate natural evolution mechanism and to modify enzyme gene in vitro to obtain modified enzyme with some expected characteristics, it needs to know the spatial structure, active site, catalytic mechanism and other factors of protein in advance and modify its DNA with site-directed mutation.
Site-directed mutagenesis, also known as rational design, is the insertion, deletion or substitution of a nucleotide sequence of a certain length into a known DNA sequence, and is a very useful means in gene research work due to its rapid and efficient improvement of the properties and characterization of a target protein expressed by DNA. The overlapping PCR technology used by the invention is one of site-directed mutagenesis technologies, and the technology can simply and quickly carry out in-vitro gene splicing on two or more gene segments through terminal complementation and overlapping extension. The overlapping PCR technology can obtain products which are difficult to obtain by means of restriction enzyme digestion, is convenient and quick, and has unique advantages in site-directed mutagenesis of large-fragment genes, gene fragment deletion and chimeric connection of a plurality of coding sequences.
Bacillus subtilis belongs to gram-positive bacteria. The use of a plurality of bacillus subtilis in the fermentation industry has a long history, is free from pathogenicity, does not produce any endotoxin, belongs to human intestinal bacteria for promoting the growth of beneficial anaerobic bacteria, produces organic acids such as lactic acid and the like, reduces the pH value of intestinal tracts, indirectly inhibits the growth of other pathogenic bacteria, can efficiently secrete various proteins, and has the advantages of being clear in the background research of bacillus in the field of microbial genetics, unobvious in codon preference, simple in fermentation, rapid in growth, free from special requirements on culture media and the like.
Pichia pastoris belongs to unicellular lower eukaryote, and is an ideal tool for expressing exogenous genes. It has the characteristics of easy culture, fast propagation, convenient gene engineering operation, high density fermentation, etc. of prokaryotes, and can strictly regulate the expression of exogenous genes by using methanol because of containing a special strong AOX (alcohol oxidase gene) promoter. In addition, the culture cost is low, and the product is easy to separate. The used fermentation medium is very cheap, the common carbon sources are glycerol or glucose and methanol, and the rest is inorganic salt. Can stably inherit exogenous protein genes, and as a eukaryotic expression system, pichia pastoris has a subcellular structure of eukaryote and has post-translational modification processing functions of glycosylation, fat acylation, protein phosphorylation and the like. Compared with the traditional eukaryotic expression systems such as saccharomyces cerevisiae, the pichia pastoris expression system becomes the most important tool and model for modern molecular biology research. In addition, the pichia pastoris cell surface display system has the advantages of post-translational processing capacity of exogenous genes, folding processing and proper glycosylation of proteins and the like, and the whole cell catalyst obtained by the system can be recycled, so that the production cost is reduced.
In the invention, the high-activity phospholipase D gene and the mutant gene thereof are expressed in a bacillus subtilis expression system and a pichia pastoris expression system respectively to obtain the high-activity phospholipase D, and the high-activity phospholipase D is purified and then reacts with a substrate to prepare phosphatidic acid and phosphatidylserine in a catalytic manner.
The invention content is as follows:
the invention aims to provide high-activity phospholipase D and a method for preparing phosphatidic acid and phosphatidylserine by using the same.
In order to achieve the above purpose, one of the technical solutions provided by the present invention is: a phospholipase D mutant which is based on the phospholipase D amino acid sequence shown in SEQ ID No.2, wherein at least one of the amino acids at positions 139, 209, 256, 388 and 519 is replaced by the following amino acid: 139 th position: asp139 Ile; position 209: asn209 Ile; 256th bit: asp256 Thr; position 388: gln388 Cys; 519 th position: asp519 Val;
the mutant gene is obtained by cloning a phospholipase D wild-type gene plD (shown as SEQ ID NO: 1) by using a Streptomyces heuchei TCCC 21102 genome as a template, constructing a recombinant vector through enzyme digestion, connection and the like, and then performing site-specific mutagenesis on the wild-type phospholipase D gene by an overlapping PCR technology (see Table 1).
In order to achieve the above purpose, the second technical solution provided by the present invention is: reconstructing a recombinant vector from the mutant gene, performing high-efficiency expression in bacillus subtilis WB600 and pichia pastoris GS115 to obtain a recombinant strain for producing high-activity phospholipase D, and performing fermentation, extraction and other technologies to obtain the high-activity phospholipase D.
In order to achieve the above purpose, the third technical solution provided by the present invention is: the high-activity phospholipase D prepared by the method is used for catalyzing phosphatidylcholine to prepare phosphatidic acid and catalyzing phosphatidylcholine and serine to prepare phosphatidylserine respectively.
The following definitions are used in the present invention:
1. nomenclature for amino acid and DNA nucleic acid sequences
The accepted IUPAC nomenclature for amino acid residues is used, in the form of a three letter code. DNA nucleic acid sequences employ the accepted IUPAC nomenclature.
2. Identification of phospholipase D mutants
"amino acid substituted by original amino acid position" is used to indicate a mutated amino acid in the phospholipase D mutant. Such as Asn209Ile, the amino acid at position 209 is replaced by Asn of the wild-type phospholipase D to Ile. The numbering of positions corresponds to SEQ ID NO: 2. nucleotide changes are also indicated by "nucleotides substituted at the original nucleotide position", the position numbering corresponding to SEQ ID NO:1, nucleotide sequence number of wild-type phospholipase D.
In the present invention, plD represents the amino acid sequence of wild-type phospholipase D, i.e., the original sequence (shown in SEQ ID NO: 2). Each mutant is represented by plDm plus a number x, x is 139, 209, 256, 388, 519 respectively, wherein 139 represents the substitution of 139 th amino acid by Asp to Ile, 209 represents the substitution of 209 th amino acid by Asn to Ile, 256 represents the substitution of 256th amino acid by Asp to Thr, 388 represents the substitution of 388 th amino acid by Gln to Cys, and 519 represents the substitution of 519 th amino acid by Asp to Val; x may also be xm-…-xnIn form (1) and represents a combined mutant of a plurality of positions, such as a mutant that plDm139-209 represents replacement of amino acid 139 by Asp to Ile and replacement of amino acid 209 by Asn to Ile. The coding gene of each mutant is shown in italics with its amino acid representation, for example, the coding gene of mutant plDm139 is plDm 139.
In the present invention, the combinatorial mutation of amino acids comprises the following:
plDm139-209、plDm139-256、plDm139-388、plDm139-519、plDm209-256、plDm209-388、plDm209-519、plDm256-388、plDm256-519、plDm388-519、plDm139-209-256、plDm139-209-388、plDm139-209-519、plDm139-256-388、plDm139-256-519、plDm139-388-519、plDm209-256-388、plDm209-256-519、plDm209-388-519、plDm256-388-519、plDm139-209-256-388、plDm139-209-256-519、plDm139-209-388-519、plDm139-256-388-519、plDm209-256-388-519、plDm139-209-256-388-519；
table 1: sequence comparison table
Figure GDA0003012420260000031
Figure GDA0003012420260000041
Figure GDA0003012420260000051
The expression host of the phospholipase D and the mutant thereof is Bacillus subtilis WB600, and the expression vector is pBSA 43;
the expression host of the phospholipase D and the mutant thereof is Pichia pastoris GS115, and the expression vector is pPIC 9K;
the host cell of the phospholipase D mutant is Pichia pastoris GS115, and the display vector is pPIC 9K-Flo.
The experimental steps of the invention are as follows:
1. the process of constructing high activity phosphatidase D mutant encoding gene includes the following steps:
(1) connecting a wild phospholipase D gene from Streptomyces halstedii TCCC 21102 with a vector pUC-T to construct a recombinant plasmid pUC-T-plD, and carrying out site-specific mutagenesis on the wild phospholipase D gene by overlapping PCR to obtain a high-activity phospholipase D mutant encoding gene;
(2) pUC-T-plDmx containing a gene encoding the high-activity phospholipase D mutant was stored.
2. A bacillus subtilis recombinant strain containing high-activity phospholipase D genes and a process for preparing the high-activity phospholipase D by using the bacillus subtilis recombinant strain comprise the following steps:
(1) carrying out enzyme digestion on the stored pUC-T-plDmx containing the coding gene of the high-activity phospholipase D mutant, and connecting the obtained coding gene of the high-activity phospholipase D mutant with a vector escherichia coli-bacillus subtilis shuttle plasmid pBSA43 to obtain a new recombinant vector;
(2) and (3) transforming the recombinant vector into the bacillus subtilis WB600 to obtain a recombinant strain, and then fermenting the recombinant strain to obtain the high-activity phospholipase D.
3. A pichia pastoris recombinant strain containing a high-activity phospholipase D gene and a process for preparing the high-activity phospholipase D by using the pichia pastoris recombinant strain comprise the following steps:
(1) carrying out enzyme digestion on the stored pUC-T-plDmx containing the high-activity phospholipase D mutant coding gene, and connecting the obtained high-activity phospholipase D mutant coding gene with an expression vector pPIC9K to obtain a new recombinant vector;
(2) transforming the recombinant vector into pichia pastoris GS115, and screening the obtained recombinant strain by using geneticin and measuring the enzyme activity of phospholipase D to obtain a high-yield strain of high-activity phospholipase D;
(3) then fermenting to prepare the high-activity phospholipase D.
4. The pichia pastoris cell surface display recombinant strain containing high-activity phospholipase D and the process for preparing the high-activity phospholipase D whole-cell catalyst by using the pichia pastoris cell surface display recombinant strain comprise the following steps:
(1) carrying out enzyme digestion on the stored pUC-T-plDmx containing the high-activity phospholipase D mutant coding gene, and connecting the obtained high-activity phospholipase D mutant coding gene with a pichia pastoris display vector pPIC9K-Flo to obtain a new recombinant vector;
(2) and transforming the recombinant vector into a host strain pichia GS115 to obtain a pichia cell surface display phospholipase D recombinant strain.
(3) Fermenting the recombinant strain to prepare the yeast cell surface display high-activity phospholipase D whole-cell catalyst.
5. The method for producing phosphatidic acid and phosphatidylserine by using the phospholipase D disclosed by the invention.
Has the advantages that:
1. the invention utilizes the overlap PCR technology to carry out site-directed mutagenesis on wild-type phospholipase D (plDm139, plDm209, plDm256, plDm388, plDm519, plDm139-209, plDm139-256, plDm139-388, plDm139-519, plDm209-256, plDm209-388, plDm209-519, plDm256-388, plDm256-519, plDm388-519, plDm139-209-, plDm139-209-256-388-519) to obtain high-activity phospholipase D, wherein the enzyme activity of the high-activity phospholipase D is at least 38.2% higher than that of the wild-type phospholipase D at 37 ℃.
2. According to the invention, a bacillus subtilis expression system, a pichia pastoris expression system and a pichia pastoris surface display system are respectively used, and the highest values of the fermentation enzyme activity of the high-activity phospholipase D in each expression system are respectively 36.8U/ml, 48.1U/ml and 140.2U/(g.cell dry weight), which are improved by about 140% compared with the wild type.
3. The conversion rate of phosphatidic acid and phosphatidylserine produced by the high-activity phospholipase D is 72.4% and 78.5% respectively, particularly, the phospholipase D is displayed on the surface of yeast by the yeast display system, yeast cells are used as an immobilized carrier while producing the phospholipase, and the cost is greatly reduced.
Description of the drawings:
FIG. 1 is a PCR amplification electrophoretogram of wild-type phospholipase D gene of the present invention
Wherein: m is DNA Marker, 1 is phospholipase D gene;
FIG. 2 is the restriction enzyme digestion verification map of recombinant plasmid pBSA43-plDmx of the present invention
Wherein: m is DNA Marker, 1 is pBSA43-plDmx and is subjected to double enzyme digestion by BamHI and HindIII;
FIG. 3 is the restriction enzyme digestion verification map of recombinant plasmid pPIC9K-plDmx of the present invention
Wherein: m is DNA Marker, 1 is pPIC9K-plDmx and is subjected to EcoRI and NotI double enzyme digestion;
FIG. 4 shows the restriction enzyme digestion verification of the recombinant plasmid pPIC9K-Flo-plDmx of the present invention
Wherein: m is DNA Marker, 1 is pPIC9K-Flo through double enzyme digestion of SnaBI and EcoRI, 2 is pPIC9K-Flo through single enzyme digestion of SnaBI, and 3 is pPIC9K-Flo-plDmx through double enzyme digestion of SnaBI and EcoRI.
The specific implementation mode is as follows:
the technical content of the present invention is further illustrated by the following examples, but the present invention is not limited to these examples, and the following examples should not be construed as limiting the scope of the present invention.
Example 1: acquisition of wild-type phospholipase D Gene
1. The wild-type phospholipase D gene is derived from Streptomyces heuchensis (Streptomyces halstedii) TCCC 21102, and the genomic DNA thereof is extracted.
The extraction method of the genomic DNA of the streptomyces heucheiensis comprises the following steps:
(1) a loopful of the bacterium was picked from the plate on which the bacterium was cultured, inoculated in 50mL of an appropriate medium, and cultured at 26 ℃ and 150r/min for 2 to 3 days.
(2) Then 1mL of the culture medium was centrifuged at 8000r/min for 20min in a 1.5mL EP tube, the supernatant was decanted, and resuspended in 200. mu.L of solution I or sterile water.
(3) Adding 20-50 μ L of 50mg/mL lysozyme, and digesting at 37 deg.C for 0.5-1 h.
(4) 100 mu L of 2% SDS solution is added, and the reaction is carried out fully until the bacterial suspension is viscous.
(5) Equal volumes of Tris-equilibrated phenol were added: chloroform-1: 1, mix well, centrifuge at 12000rpm for 5min, transfer the supernatant to another EP tube.
(6) The extraction was repeated twice until no protein layer appeared, and finally, the extraction was performed once with chloroform of the same volume.
(7) Adding equal volume of isopropanol to precipitate DNA, centrifuging at 12000r/min for 5min, discarding supernatant, washing with 500 μ L75% ethanol for 2 times, and centrifuging at 12000r/min for 5min after each blow-beating.
(8) Placing the EP tube in filter paper or metal bath at 55 deg.C, air drying until no alcohol smell, dissolving with TE buffer solution or sterilized water, and storing at-20 deg.C.
2. Designing amplification primers of a wild phospholipase D gene from the phospholipase D gene, wherein the sequences are as follows:
upstream P1(SEQ ID NO: 5): ATGATCAAGGTTGGTGGTGTTGCTG
Downstream P2(SEQ ID NO: 6): TTAACCCTGACACAAACCTCTAGCGTAATCGT
The reaction system for PCR amplification is 50 μ L, and comprises the following components:
2×LA buffer 25μL
dNTPs(2.5mmol/L) 2μL
upstream primer P1 (20. mu. mol/L) 5μL
Downstream primer P2 (20. mu. mol/L) 5μL
Genomic DNA of Streptomyces heuchei 2μL
LA Taq DNA polymerase 0.5μL
ddH2O 10.5μL
Total volume 50μL
The setting of the amplification program is as follows:
a. pre-denaturation at 95 deg.C for 5 min;
b. denaturation: 30s at 95 ℃;
c. annealing: 45 ℃ at 70 ℃;
d. extension: 90s at 72 ℃;
e.b-d for 30 cycles;
f. extension at 72 ℃ for 10 min.
The PCR product was subjected to agarose gel electrophoresis to find a band of 1683bp (see FIG. 1) of the wild-type phospholipase D gene, and then the PCR product was recovered by a small amount of DNA recovery kit to obtain plD, which is the wild-type phospholipase D gene.
Example 2: the high-activity phospholipase D gene is obtained, and the final amino acid sequence is shown in the table 1 by taking an Asn209Ile single amino acid mutant as an example.
1. The wild-type phospholipase D gene is connected with a T vector.
The purified plD was ligated with pUC-T vector, and the recombinant plasmid was transformed into E.coli DH 5. alpha. by EcoRI and MluI double digestion, it was successfully verified that the wild-type phospholipase D gene was cloned into the T vector.
2. Site-directed mutagenesis of a Single site by overlapping PCR techniques
Carrying out site-directed mutagenesis based on an overlapping PCR technology to obtain high-activity phospholipase D, and designing primers as follows:
upstream P1(SEQ ID NO. 5): ATGATCAAGGTTGGTGGTGTTGCTG
Downstream P2(SEQ ID NO. 6): TTAACCCTGACACAAACCTCTAGCGTAATCGT
Overlapping primer P3(SEQ ID NO. 7): ATGGAAGCAACGATCAAAGTGATGTTAGCAGCAGCA
Overlapping primer P4(SEQ ID NO. 8): TGCTGCTGCTAACATCACTTTGATCGTTGCTTCCAT
The overlapping primers P3 and P4 contained a mutation at amino acid residue 209.
Carrying out PCR amplification on a recombinant plasmid pUC-T-plD, namely a recombinant vector formed by connecting a wild phospholipase D gene with a pUC-T vector, serving as a template;
PCR1, reaction system 50 μ L, consisting of:
2×LA buffer 25μL
dNTPs(2.5mmol/L) 2μL
upstream primer P1 (20. mu. mol/L) 5μL
Downstream primer P4 (20. mu. mol/L) 5μL
Recombinant plasmid pUC-T-plD 2μL
LA TaqDNA polymerase 0.5μL
ddH2O 10.5μL
Total volume 50μL
PCR2, reaction system 50 μ L, consisting of:
2×LA buffer 25μL
dNTPs(2.5mmol/L) 2μL
upstream primer P3 (20. mu. mol/L) 5μL
Downstream primer P2 (20. mu. mol/L) 5μL
Recombinant plasmidParticle pUC-T-plD 2μL
LA TaqDNA polymerase 0.5μL
ddH2O 10.5μL
Total volume 50μL
The PCR1 and PCR2 amplification programs were set up as:
a. pre-denaturation at 95 deg.C for 5 min;
b. denaturation: 30s at 95 ℃;
c. annealing: 30s at 68 ℃;
d. extension: 45s at 72 ℃;
e.b-d for 10 cycles;
f. extension at 72 ℃ for 10 min.
PCR3, the reaction system is as follows:
2×buffer 25μL
dNTPs(2.5mmol/L each) 2μL
PCR1 product 1μL
PCR2 product 1μL
LA TaqDNA polymerase 0.5μL
ddH2O 20.5μL
Total volume 50μL
The PCR3 amplification program was set up as:
a. pre-denaturation at 95 deg.C for 5 min;
b. denaturation: 30s at 95 ℃;
c. annealing: 45s at 70 ℃;
d. extension: 90s at 72 ℃;
e.b-d for 10 cycles;
f. extension at 72 ℃ for 10 min.
PCR4, the reaction system is as follows:
2×buffer 25μL
dNTPs(2.5mmol/L each) 2μL
upstream primer P1 (20. mu. mol/L) 5μL
Downstream primer P2 (20. mu. mol/L) 5μL
PCR3 product 2μL
LA TaqDNA polymerase 0.5μL
ddH2O 10.5μL
Total volume 50μL
The PCR4 amplification program was set up as:
a. pre-denaturation at 95 deg.C for 5 min;
b. denaturation: 30s at 95 ℃;
c. annealing: 45s at 70 ℃;
d. extension: 90s at 72 ℃;
e.b-d for 30 cycles;
f. extension at 72 ℃ for 10 min.
Sequencing is carried out on the finally obtained PCR product (Beijing Hua great bioengineering company), and the result shows that the phospholipase D gene segment plDm209 of Asn209Ile is obtained by amplification at the moment.
Example 3: obtaining a phospholipase D variant with a plurality of amino acid mutations on the basis of single amino acid mutation, taking the overlapping PCR technology for carrying out Asp139Ile, Asp256Thr, Gln388Cys and Asp519Val mutation on the basis of the Asn209Ile mutant as an example, the final amino acid sequence is shown as SEQ ID NO: 4, respectively.
The specific strategy is as follows: double mutations are first effected on the basis of a single mutation, followed by mutations of the third, fourth and fifth amino acids.
The mutation of Asp519Val is firstly realized on the basis of Asn209Ile, the steps are consistent with the steps in the example 2, and overlapping primers are designed as follows:
upstream P1(SEQ ID NO. 5): ATGATCAAGGTTGGTGGTGTTGCTG
Downstream P2(SEQ ID NO. 6): TTAACCCTGACACAAACCTCTAGCGTAATCGT
Overlapping primer P5(SEQ ID NO. 9): CAACAACGTAACCGAAGACTTGCAACCAGGATGGG
Overlapping primer P6(SEQ ID NO. 10): CCCATCCTGGTTGCAAGTCTTCGGTTACGTTGTTG
Overlapping primers P5 and P6 contained mutations to amino acid residue 519.
Performing PCR amplification on a recombinant plasmid pUC-T-plDm209, namely a recombinant vector in which a gene encoding the mutant plDm209 is linked to a pUC-T vector, as a template;
PCR1, reaction system 50 μ L, consisting of:
2×LA buffer 25μL
dNTPs(2.5mmol/L) 2μL
upstream primer P1 (20. mu. mol/L) 5μL
Downstream primer P6 (20. mu. mol/L) 5μL
Recombinant plasmid pUC-T-plDm209 2μL
LA TaqDNA polymerase 0.5μL
ddH2O 10.5μL
Total volume 50μL
PCR2, reaction system 50 μ L, consisting of:
Figure GDA0003012420260000101
Figure GDA0003012420260000111
the PCR1 and PCR2 amplification programs were set up as:
a. pre-denaturation at 95 deg.C for 5 min;
b. denaturation: 30s at 95 ℃;
c. annealing: 30s at 68 ℃;
d. extension: 45s at 72 ℃;
e.b-d for 10 cycles;
f. extension at 72 ℃ for 10 min.
PCR3, the reaction system is as follows:
2×buffer 25μL
dNTPs(2.5mmol/L each) 2μL
PCR1 product 1μL
PCR2 product 1μL
LA TaqDNA polymerase 0.5μL
ddH2O 20.5μL
Total volume 50μL
The PCR3 amplification program was set up as:
a. pre-denaturation at 95 deg.C for 5 min;
b. denaturation: 30s at 95 ℃;
c. annealing: 45s at 70 ℃;
d. extension: 90s at 72 ℃;
e.b-d for 10 cycles;
f. extension at 72 ℃ for 10 min.
PCR4, the reaction system is as follows:
Figure GDA0003012420260000112
Figure GDA0003012420260000121
the PCR4 amplification program was set up as:
a. pre-denaturation at 95 deg.C for 5 min;
b. denaturation: 30s at 95 ℃;
c. annealing: 45s at 70 ℃;
d. extension: 90s at 72 ℃;
e.b-d for 30 cycles;
f. extension at 72 ℃ for 10 min.
Sequencing is carried out on the finally obtained PCR product (Beijing Hua Dai bioengineering company), and the result shows that the amplified phospholipase D gene segment plDm209-519 with double mutation of Asn209Ile and Asp519Val is obtained, and the sequence is shown in the table 1.
The other mutation steps are continued to be consistent with the steps of the embodiment 2 or/and the embodiment 3, all the mutant primer sequences are shown in the following table, the primers are replaced according to the steps on the basis of the plDm209-519, the point mutation of Asp139Ile, Asp256Thr and Gln388Cys is carried out in sequence according to the following table, and the primers are sent to a sequencing company for sequencing, so that the phospholipase D gene segment plDm139-209 and 256-and 388-519 with 5 amino acid mutation points is confirmed, the amino acid sequence is shown as SEQ ID NO.4, and the nucleotide sequence is shown as SEQ ID NO. 3.
Mutants F-end primer R-terminal primer
plDm139 P7:SEQ ID NO.11 P8:SEQ ID NO.12
plDm209 P3:SEQ ID NO.7 P4:SEQ ID NO.8
plDm256 P9:SEQ ID NO.13 P10:SEQ ID NO.14
plDm388 P11:SEQ ID NO.15 P12:SEQ ID NO.16
plDm519 P5:SEQ ID NO.9 P6:SEQ ID NO.10
Example 4: construction of bacillus subtilis high-activity phospholipase D recombinant strain
1. Construction of expression vector pBSA43
An expression vector pBSA43 is obtained by taking an escherichia coli-bacillus subtilis shuttle cloning vector pBE2 as a framework and cloning a strong bacillus constitutive promoter P43 and a levansucrase signal sequence sacB which can ensure that a recombinant protein is directly secreted into a culture medium. It carries AmprAnd KmrThe gene can utilize ampicillin resistance as a selection marker in escherichia coli, and can utilize kanamycin resistance as a selection marker in bacillus subtilis and bacillus licheniformis.
2. Construction of high-Activity phospholipase D expression vector pBSA43-plDmx
The high-activity phospholipase D gene constructed by overlapping PCR and a bacillus subtilis expression vector pBSA43 are subjected to BamHI and HindIII double enzyme digestion and then are connected to construct a recombinant plasmid pBSA43-plDmx, the recombinant plasmid pBSA43-plDmx is transformed into escherichia coli DH5 alpha competent cells, positive transformants are selected, the plasmids are extracted for enzyme digestion verification and sequencing, and the successful construction is determined, so that the recombinant strain pBSA43-plDmx is obtained.
3. Expression vector pBSA43-plDmx transformation of Bacillus subtilis WB600
Adding 60 mu L of competent cells and 1 mu L (50 ng/. mu.L) of pBSA43-plDmx into a precooled 1mm electric rotating cup, uniformly mixing and carrying out ice bath for 5min, setting parameters (25 mu F, 200 omega, 4.5-5.0ms), shocking once, immediately adding 1mL of recovery medium (LB +0.5mol/L sorbitol +0.5mol/L mannitol), uniformly mixing, sucking into a 1.5mL EP tube, shaking and culturing for 3h at 37 ℃, centrifuging, reserving 200 mu L of recovery, coating on a resistant LB plate, culturing for 24h at 37 ℃, picking up a transformant, extracting a plasmid, carrying out enzyme digestion verification (shown in figure 2), and obtaining the bacillus subtilis recombinant strain WB600/pBSA 43-plDmx.
Example 5: construction of pichia pastoris high-activity phospholipase D free expression recombinant strain
1. Construction of high-activity phospholipase D expression vector pPIC9K-plDmx
Carrying out EcoRI and NotI double enzyme digestion on the overlapped PCR purified product and a pichia pastoris expression vector pPIC9K, then carrying out ligation, transforming the product into escherichia coli DH5 alpha competent cells, and selecting AmprAnd (3) carrying out colony culture on the positive transformant, then upgrading the plasmid, and successfully carrying out enzyme digestion verification (as shown in figure 3) to obtain a recombinant expression vector pPIC 9K-plDmx.
2. Construction of high-activity phospholipase D high-expression recombinant strain
(1) Linearization of plasmid DNA
Before transformation of Pichia pastoris, the recombinant expression plasmid pPIC9K-plDmx was linearized with SacI and SalI restriction enzymes, respectively.
(2) Transformation of linearized plasmid pPIC9K-plDmx into Pichia pastoris
Adding competent cells and linearized plasmid pPIC9K-plDmx into a 1.5mL precooled centrifuge tube, blowing, beating and uniformly mixing, and then adding the mixture into a precooled electric rotor cup;
carrying out ice bath on the transformation cup for 10min, and then carrying out electric transformation;
thirdly, immediately adding 1mL of precooled 1mol/L sorbitol solution into an electrotransfer cup after electric shock, and transferring the electrotransfer solution into a new 1.5mL centrifuge tube;
fourthly, standing and culturing for 1 to 2 hours at the temperature of 30 ℃, and sucking 200 mu L of pichia pastoris GS115 electrotransfer liquid and coating the liquid on an MD culture medium.
(3) Identification of positive transformant and screening of phospholipase D high-yield strain
Culturing an MD flat plate coated with an electrotransfer solution at 30 ℃ for 2-3 d;
secondly, selecting transformants, extracting yeast genomes, diluting by 100 times, and then using the yeast genomes as templates for PCR. Positive transformants were determined by using Pichia pastoris GS115/pPIC9K, which had been transformed with the empty plasmid pPIC9K, as a control.
Thirdly, after positive transformants are determined, high geneticin resistant transformants with large single colony on geneticin resistant plates with different concentrations are picked firstly, and then phospholipase D enzyme activity of the picked transformants is respectively measured, so that a high yield strain GS115/pPIC 9K-plDmx of phospholipase D is obtained.
Example 6: construction of recombinant bacteria for displaying high-activity phospholipase D on pichia pastoris cell surface
1. Construction of recombinant plasmid pPIC9K-Flo-plDmx
Carrying out double enzyme digestion on the overlapped PCR purified product and a pichia pastoris surface display expression vector pPIC9K-Flo through SnaBI and EcoRI, then carrying out ligation, transforming the product into escherichia coli DH5 alpha competence, and selecting AmprAnd (3) carrying out colony culture on the positive transformant, then, obtaining a quality-improved plasmid, and successfully carrying out enzyme digestion verification (as shown in figure 4), thus obtaining the recombinant expression vector pPIC 9K-Flo-plDmx.
2. Construction of recombinant pichia pastoris
After the recombinant expression vector pPIC9K-Flo-plDmx which is verified to be correct by sequencing is linearized by SalI, pichia pastoris GS115 is transformed by an electrical transformation method, and recombinants are screened by an MD plate to obtain pichia pastoris cell surface display high-activity phospholipase D recombinant bacteria GS115/pPIC 9K-Flo-plDmx.
Example 7: expression and preparation of high-activity phospholipase D in bacillus subtilis recombinant bacteria
Inoculating a bacillus subtilis recombinant strain WB600/pBSA43-plDmx into an LB liquid culture medium containing kanamycin (50 mu g/mL), and culturing at 37 ℃ and 220r/min overnight;
transferring the strain to 50mL LB culture medium according to the inoculum size of 1%, culturing at 37 ℃ and 220r/min for 48h to obtain a high-activity phospholipase D crude enzyme solution;
thirdly, precipitating enzyme protein by a fractional salting-out method, collecting protein precipitate, dissolving, dialyzing to remove salt, performing ion exchange chromatography and gel chromatography, and freeze-drying to obtain the high-activity phospholipase D pure enzyme powder.
Example 8: expression and preparation of high-activity phospholipase D in pichia pastoris free expression recombinant bacteria
Firstly, selecting a pichia pastoris recombinant strain GS115/pPIC 9K-plDmx on a YPD flat plate, inoculating the pichia pastoris recombinant strain GS115/pPIC 9K-plDmx into 50ml of YPD liquid culture medium, and culturing the YPD liquid culture medium for 24 hours at the temperature of 30 ℃ at the speed of 250 r/min;
transferring the strain to a BMGY culture medium by 1%, culturing at 30 ℃ for about 24 hours at 250r/min, centrifuging at 4000r/min for 5 minutes to obtain thalli, and transferring the thalli to a BMMY culture medium;
continuing to culture at 30 ℃ for 250r/min, and supplementing 250 mu L of methanol every 24 h. After culturing for 5 days, centrifuging to obtain supernatant to obtain crude enzyme solution of phospholipase D;
fourthly, precipitating the enzyme protein by a fractional salting-out method, collecting the protein precipitate, dialyzing to remove salt after dissolving, and freeze-drying after ion exchange chromatography and gel chromatography to prepare the high-activity phospholipase D pure enzyme powder.
Example 9: preparation of pichia pastoris cell surface display high-activity phospholipase D whole-cell catalyst
Firstly, selecting a pichia pastoris cell surface display high-activity phospholipase D recombinant strain GS115/pPIC9K-Flo-plDmx on a YPD plate, inoculating the pichia pastoris cell surface display high-activity phospholipase D recombinant strain GS115/pPIC9K-Flo-plDmx into 50ml of YPD liquid culture medium, and culturing the YPD liquid culture medium at the temperature of 30 ℃ at the speed of 250r/min for 24 hours;
transferring the strain to a fresh BMGY culture medium by 1%, culturing at 30 ℃ for about 24 hours at 250r/min, centrifuging at 4000r/min for 5 minutes to obtain thalli, and transferring the thalli to a BMMY culture medium;
continuing to culture at 30 ℃ for 250r/min, and supplementing 250 mu L of methanol every 24 h. After culturing for 5 days, centrifugally collecting and taking thalli, washing the thalli for 1-2 times by using membrane water, adding a protective agent, and performing vacuum freeze drying to obtain the pichia pastoris cell surface display high-activity phospholipase D cell catalyst.
Example 10: phospholipase D Activity assay
1. Principle of phospholipase D enzyme activity determination
And (3) performing activity detection by adopting an enzyme-linked colorimetric method: phospholipase D catalyzes and hydrolyzes L-alpha-lecithin to generate choline, the choline generates hydrogen peroxide under the action of choline oxidase, the hydrogen peroxide generates quinoneimine chromogenic substance with 4-aminoantipyrine and phenol under the action of peroxidase, and the light absorption value is 500 nm.
2. Phospholipase D enzyme activity determination method
(1) Lecithin emulsion: 0.345g of lecithin, 2ml of diethyl ether, 3ml of 7.5% TritanX-100,20mlH2and O, fully and uniformly mixing.
(2) Reaction termination solution: 1M Tris-HCl, 0.5M EDTA, pH8.0
Measurement of phospholipase D:
a10 ml test tube was filled with 1.15ml lecithin emulsion, 0.1ml 100mM Tris-HCl, 0.05ml CaCl20.1ml of the crude enzyme solution was reacted in a water bath at 37 ℃ for 10min, followed by adding 0.2ml of the reaction-stopping solution, boiling for 5min, and cooling to room temperature. Subsequently, 4ml of 10mM Tris-HCl containing 2U of choline-containing oxidase, 4U of peroxidase, 2mg of 4-antipyrine, 1mg of phenol, 20mg of Tritan X-100 was added, reacted at 37 ℃ for 20min, followed by measurement of absorbance at 500 nm.
The blank sample was zeroed by replacing the enzyme solution in the reaction with water.
Definition of enzyme activity: the amount of enzyme required for phospholipase D to catalyze hydrolysis of L- α -lecithin to release 1.0 μmol choline within 1min at pH8.0 and T37 ℃.
And (3) measuring the enzyme activity of the phospholipase D, wherein the enzyme activity of the high-activity phospholipase D obtained by fermentation is measured as follows:
Figure GDA0003012420260000151
Figure GDA0003012420260000161
example 11: preparation of phosphatidic acid with high-activity phospholipase D
The substrate is 1g of soybean lecithin (PC content is 90%), the substrate is dissolved in 10ml of phosphate buffer solution with pH7.0, and 3U of high-activity phospholipase D is added into a reaction system per ml, wherein the high-activity phospholipase D is prepared according to the invention in the embodiment 7-9 (the high-activity phospholipase D can be obtained by fermenting any mutant and the addition amount of enzyme powder during catalysis can reach 3U/ml). The reaction temperature was 40 ℃ and the reaction was carried out for 12 hours under the stirring action of a magnetic stirrer, followed by extraction with 30ml of chloroform/methanol (2: 1) to obtain phosphatidic acid, the conversion rate of which to prepare phosphatidic acid was 72.4%.
Example 12: preparation of phosphatidylserine with high-activity phospholipase D
Substrates, 1g of soybean lecithin (PC content 90%) and 2.5g of serine, were dissolved in 5ml of ph5.5 acetic acid-sodium acetate buffer, and mixed until the total volume was 10ml, and 6U of high-activity phospholipase D was added per ml of the reaction system, wherein the high-activity phospholipase D was prepared according to examples 7-9 of the present invention (obtained by fermentation using any mutant, and the amount of enzyme powder added during catalysis was 6U/ml). The reaction temperature was 40 ℃ and the reaction was carried out for 12 hours with stirring by a magnetic stirrer, followed by extraction with 30ml of chloroform/methanol (2: 1) to obtain phosphatidylserine with a conversion of 78.5% for phosphatidylserine.
Sequence listing
<110> Tianjin science and technology university
<120> a novel phospholipase D
<130> 1
<141> 2019-06-18
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1683
<212> DNA
<213> Streptomyces huoshi (Streptomyces halstediiTCCC 21102)
<400> 1
atgatcaagg ttggtggtgt tgctgaccca agatctagac cagaaccacc accatctcag 60
ggtactactt tggctagaac tgttagaact actgctttgg tttccacttc cgctttggct 120
ttgggtttct ctttgttggg tgctactgct actgttccag cttacgctgc tgaagctcca 180
actccacatt tggactctgt tgagcagact ttgagacagg tttctccagg tttggaaggt 240
tccgtttggg aaagaactgc tggtaacaga ttgggttctt ccactccagg tggtgctgat 300
tggttgttgc aaactccagg ttgttggggt gatgctgctt gtactgatag accaggttct 360
agaagattgt tggacaagat gagacaggac attgctggtg ctagacagac tgttgacatc 420
tccactttgg ctccatttcc aaacggtggt taccaggatg ctatcgttgc tggtttgaaa 480
gagtccgctc agaagggaaa cagattgaag gttagaatca tggttggtgc tgctccaatc 540
taccactcta ctgttattcc atcctcctac agagatgagt tgttggctaa gttgggtcca 600
gctgctgctg ctaacatcac tttgaacgtt gcttccatga ctacttccaa gacttccttc 660
tcttggaacc actccaagtt gatcgttgtt gacggtggtt ccgttatcac tggtggtatc 720
aactcttgga aggacgacta cttggacact actcacccag tttctgacgt tgacttggct 780
ttgtctggtc ctgctgctgg ttctgctggt agatacttgg attctttgtg ggactggact 840
tgtagaaaca agggtaactg gtcctccgtt tggttcgctg cttcttctgg tgctgactgt 900
atgccagctt tgccaagacc agctactcct gaaggtggtg gtgatgttcc tgctttggct 960
gttggtggtt tgggtgttgg tattagacag aacgacccta cttcctcatt cagaccagtt 1020
ttgcctactg ctggtgacac taagtgtggt attggtgttt ccgacaagac taacgctgac 1080
agagactacg acactgttaa cccagaagaa tccgctttga gagctttggt ttcttccgct 1140
acttcccaca ttgagatttc ccaacaagac gttcacgcta cttgtccacc attgccaaga 1200
tacgaggtta gattgtacga cgctttggct gctaagttgg tttccggtgt taaggttaga 1260
atcgttgttt ccgacccagc taacagaggt actattggat ctggtggtta ctcccagatc 1320
aagtccttgt ctgaagtttc cgatgctttg agaggtagag ttactgcttt gactggtgac 1380
ggtggtagag ctagaactgc tttgtgtgag aacttgcagt tggctacttt cagagcttct 1440
gacaagccaa cttgggctga tggtaagcca tacgctcaac accacaaatt ggtttctgtt 1500
gacggatccg ctttctacat cggatccaag aacttgtacc catcctggtt gcaagacttc 1560
ggttacgttg ttgaatctcc tgccgctgct gctcagttga gaactgattt gttggatcct 1620
cagtggagat actcccaggc tacagctact tacgattacg ctagaggttt gtgtcagggt 1680
taa 1683
<210> 2
<211> 560
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<213> Streptomyces huoshi (Streptomyces halstediiTCCC 21102)
<400> 2
Met Ile Lys Val Gly Gly Val Ala Asp Pro Arg Ser Arg Pro Glu Pro
1 5 10 15
Pro Pro Ser Gln Gly Thr Thr Leu Ala Arg Thr Val Arg Thr Thr Ala
20 25 30
Leu Val Ser Thr Ser Ala Leu Ala Leu Gly Phe Ser Leu Leu Gly Ala
35 40 45
Thr Ala Thr Val Pro Ala Tyr Ala Ala Glu Ala Pro Thr Pro His Leu
50 55 60
Asp Ser Val Glu Gln Thr Leu Arg Gln Val Ser Pro Gly Leu Glu Gly
65 70 75 80
Ser Val Trp Glu Arg Thr Ala Gly Asn Arg Leu Gly Ser Ser Thr Pro
85 90 95
Gly Gly Ala Asp Trp Leu Leu Gln Thr Pro Gly Cys Trp Gly Asp Ala
100 105 110
Ala Cys Thr Asp Arg Pro Gly Ser Arg Arg Leu Leu Asp Lys Met Arg
115 120 125
Gln Asp Ile Ala Gly Ala Arg Gln Thr Val Asp Ile Ser Thr Leu Ala
130 135 140
Pro Phe Pro Asn Gly Gly Tyr Gln Asp Ala Ile Val Ala Gly Leu Lys
145 150 155 160
Glu Ser Ala Gln Lys Gly Asn Arg Leu Lys Val Arg Ile Met Val Gly
165 170 175
Ala Ala Pro Ile Tyr His Ser Thr Val Ile Pro Ser Ser Tyr Arg Asp
180 185 190
Glu Leu Leu Ala Lys Leu Gly Pro Ala Ala Ala Ala Asn Ile Thr Leu
195 200 205
Asn Val Ala Ser Met Thr Thr Ser Lys Thr Ser Phe Ser Trp Asn His
210 215 220
Ser Lys Leu Ile Val Val Asp Gly Gly Ser Val Ile Thr Gly Gly Ile
225 230 235 240
Asn Ser Trp Lys Asp Asp Tyr Leu Asp Thr Thr His Pro Val Ser Asp
245 250 255
Val Asp Leu Ala Leu Ser Gly Pro Ala Ala Gly Ser Ala Gly Arg Tyr
260 265 270
Leu Asp Ser Leu Trp Asp Trp Thr Cys Arg Asn Lys Gly Asn Trp Ser
275 280 285
Ser Val Trp Phe Ala Ala Ser Ser Gly Ala Asp Cys Met Pro Ala Leu
290 295 300
Pro Arg Pro Ala Thr Pro Glu Gly Gly Gly Asp Val Pro Ala Leu Ala
305 310 315 320
Val Gly Gly Leu Gly Val Gly Ile Arg Gln Asn Asp Pro Thr Ser Ser
325 330 335
Phe Arg Pro Val Leu Pro Thr Ala Gly Asp Thr Lys Cys Gly Ile Gly
340 345 350
Val Ser Asp Lys Thr Asn Ala Asp Arg Asp Tyr Asp Thr Val Asn Pro
355 360 365
Glu Glu Ser Ala Leu Arg Ala Leu Val Ser Ser Ala Thr Ser His Ile
370 375 380
Glu Ile Ser Gln Gln Asp Val His Ala Thr Cys Pro Pro Leu Pro Arg
385 390 395 400
Tyr Glu Val Arg Leu Tyr Asp Ala Leu Ala Ala Lys Leu Val Ser Gly
405 410 415
Val Lys Val Arg Ile Val Val Ser Asp Pro Ala Asn Arg Gly Thr Ile
420 425 430
Gly Ser Gly Gly Tyr Ser Gln Ile Lys Ser Leu Ser Glu Val Ser Asp
435 440 445
Ala Leu Arg Gly Arg Val Thr Ala Leu Thr Gly Asp Gly Gly Arg Ala
450 455 460
Arg Thr Ala Leu Cys Glu Asn Leu Gln Leu Ala Thr Phe Arg Ala Ser
465 470 475 480
Asp Lys Pro Thr Trp Ala Asp Gly Lys Pro Tyr Ala Gln His His Lys
485 490 495
Leu Val Ser Val Asp Gly Ser Ala Phe Tyr Ile Gly Ser Lys Asn Leu
500 505 510
Tyr Pro Ser Trp Leu Gln Asp Phe Gly Tyr Val Val Glu Ser Pro Ala
515 520 525
Ala Ala Ala Gln Leu Arg Thr Asp Leu Leu Asp Pro Gln Trp Arg Tyr
530 535 540
Ser Gln Ala Thr Ala Thr Tyr Asp Tyr Ala Arg Gly Leu Cys Gln Gly
545 550 555 560
<210> 3
<211> 1683
<212> DNA
<213> Artificial sequence ()
<400> 3
atgatcaagg ttggtggtgt tgctgaccca agatctagac cagaaccacc accatctcag 60
ggtactactt tggctagaac tgttagaact actgctttgg tttccacttc cgctttggct 120
ttgggtttct ctttgttggg tgctactgct actgttccag cttacgctgc tgaagctcca 180
actccacatt tggactctgt tgagcagact ttgagacagg tttctccagg tttggaaggt 240
tccgtttggg aaagaactgc tggtaacaga ttgggttctt ccactccagg tggtgctgat 300
tggttgttgc aaactccagg ttgttggggt gatgctgctt gtactgatag accaggttct 360
agaagattgt tggacaagat gagacaggac attgctggtg ctagacagac tgttatcatc 420
tccactttgg ctccatttcc aaacggtggt taccaggatg ctatcgttgc tggtttgaaa 480
gagtccgctc agaagggaaa cagattgaag gttagaatca tggttggtgc tgctccaatc 540
taccactcta ctgttattcc atcctcctac agagatgagt tgttggctaa gttgggtcca 600
gctgctgctg ctaacatcac tttgatcgtt gcttccatga ctacttccaa gacttccttc 660
tcttggaacc actccaagtt gatcgttgtt gacggtggtt ccgttatcac tggtggtatc 720
aactcttgga aggacgacta cttggacact actcacccag tttctaccgt tgacttggct 780
ttgtctggtc ctgctgctgg ttctgctggt agatacttgg attctttgtg ggactggact 840
tgtagaaaca agggtaactg gtcctccgtt tggttcgctg cttcttctgg tgctgactgt 900
atgccagctt tgccaagacc agctactcct gaaggtggtg gtgatgttcc tgctttggct 960
gttggtggtt tgggtgttgg tattagacag aacgacccta cttcctcatt cagaccagtt 1020
ttgcctactg ctggtgacac taagtgtggt attggtgttt ccgacaagac taacgctgac 1080
agagactacg acactgttaa cccagaagaa tccgctttga gagctttggt ttcttccgct 1140
acttcccaca ttgagatttc ctgccaagac gttcacgcta cttgtccacc attgccaaga 1200
tacgaggtta gattgtacga cgctttggct gctaagttgg tttccggtgt taaggttaga 1260
atcgttgttt ccgacccagc taacagaggt actattggat ctggtggtta ctcccagatc 1320
aagtccttgt ctgaagtttc cgatgctttg agaggtagag ttactgcttt gactggtgac 1380
ggtggtagag ctagaactgc tttgtgtgag aacttgcagt tggctacttt cagagcttct 1440
gacaagccaa cttgggctga tggtaagcca tacgctcaac accacaaatt ggtttctgtt 1500
gacggatccg ctttctacat cggatccaag aacttgtacc catcctggtt gcaagtcttc 1560
ggttacgttg ttgaatctcc tgccgctgct gctcagttga gaactgattt gttggatcct 1620
cagtggagat actcccaggc tacagctact tacgattacg ctagaggttt gtgtcagggt 1680
taa 1683
<210> 4
<211> 560
<212> PRT
<213> Artificial sequence ()
<400> 4
Met Ile Lys Val Gly Gly Val Ala Asp Pro Arg Ser Arg Pro Glu Pro
1 5 10 15
Pro Pro Ser Gln Gly Thr Thr Leu Ala Arg Thr Val Arg Thr Thr Ala
20 25 30
Leu Val Ser Thr Ser Ala Leu Ala Leu Gly Phe Ser Leu Leu Gly Ala
35 40 45
Thr Ala Thr Val Pro Ala Tyr Ala Ala Glu Ala Pro Thr Pro His Leu
50 55 60
Asp Ser Val Glu Gln Thr Leu Arg Gln Val Ser Pro Gly Leu Glu Gly
65 70 75 80
Ser Val Trp Glu Arg Thr Ala Gly Asn Arg Leu Gly Ser Ser Thr Pro
85 90 95
Gly Gly Ala Asp Trp Leu Leu Gln Thr Pro Gly Cys Trp Gly Asp Ala
100 105 110
Ala Cys Thr Asp Arg Pro Gly Ser Arg Arg Leu Leu Asp Lys Met Arg
115 120 125
Gln Ile Ile Ala Gly Ala Arg Gln Thr Val Ile Ile Ser Thr Leu Ala
130 135 140
Pro Phe Pro Asn Gly Gly Tyr Gln Asp Ala Ile Val Ala Gly Leu Lys
145 150 155 160
Glu Ser Ala Gln Lys Gly Asn Arg Leu Lys Val Arg Ile Met Val Gly
165 170 175
Ala Ala Pro Ile Tyr His Ser Thr Val Ile Pro Ser Ser Tyr Arg Asp
180 185 190
Glu Leu Leu Ala Lys Leu Gly Pro Ala Ala Ala Ala Asn Ile Thr Leu
195 200 205
Ile Val Ala Ser Met Thr Thr Ser Lys Thr Ser Phe Ser Trp Asn His
210 215 220
Ser Lys Leu Ile Val Val Asp Gly Gly Ser Val Ile Thr Gly Gly Ile
225 230 235 240
Asn Ser Trp Lys Asp Asp Tyr Leu Asp Thr Thr His Pro Val Ser Thr
245 250 255
Val Asp Leu Ala Leu Ser Gly Pro Ala Ala Gly Ser Ala Gly Arg Tyr
260 265 270
Leu Asp Ser Leu Trp Asp Trp Thr Cys Arg Asn Lys Gly Asn Trp Ser
275 280 285
Ser Val Trp Phe Ala Ala Ser Ser Gly Ala Asp Cys Met Pro Ala Leu
290 295 300
Pro Arg Pro Ala Thr Pro Glu Gly Gly Gly Asp Val Pro Ala Leu Ala
305 310 315 320
Val Gly Gly Leu Gly Val Gly Ile Arg Gln Asn Asp Pro Thr Ser Ser
325 330 335
Phe Arg Pro Val Leu Pro Thr Ala Gly Asp Thr Lys Cys Gly Ile Gly
340 345 350
Val Ser Asp Lys Thr Asn Ala Asp Arg Asp Tyr Asp Thr Val Asn Pro
355 360 365
Glu Glu Ser Ala Leu Arg Ala Leu Val Ser Ser Ala Thr Ser His Ile
370 375 380
Glu Ile Ser Cys Gln Asp Val His Ala Thr Cys Pro Pro Leu Pro Arg
385 390 395 400
Tyr Glu Val Arg Leu Tyr Asp Ala Leu Ala Ala Lys Leu Val Ser Gly
405 410 415
Val Lys Val Arg Ile Val Val Ser Asp Pro Ala Asn Arg Gly Thr Ile
420 425 430
Gly Ser Gly Gly Tyr Ser Gln Ile Lys Ser Leu Ser Glu Val Ser Asp
435 440 445
Ala Leu Arg Gly Arg Val Thr Ala Leu Thr Gly Asp Gly Gly Arg Ala
450 455 460
Arg Thr Ala Leu Cys Glu Asn Leu Gln Leu Ala Thr Phe Arg Ala Ser
465 470 475 480
Asp Lys Pro Thr Trp Ala Asp Gly Lys Pro Tyr Ala Gln His His Lys
485 490 495
Leu Val Ser Val Asp Gly Ser Ala Phe Tyr Ile Gly Ser Lys Asn Leu
500 505 510
Tyr Pro Ser Trp Leu Gln Val Phe Gly Tyr Val Val Glu Ser Pro Ala
515 520 525
Ala Ala Ala Gln Leu Arg Thr Asp Leu Leu Asp Pro Gln Trp Arg Tyr
530 535 540
Ser Gln Ala Thr Ala Thr Tyr Asp Tyr Ala Arg Gly Leu Cys Gln Gly
545 550 555 560
<210> 5
<211> 25
<212> DNA
<213> Artificial sequence ()
<400> 5
atgatcaagg ttggtggtgt tgctg 25
<210> 6
<211> 32
<212> DNA
<213> Artificial sequence ()
<400> 6
ttaaccctga cacaaacctc tagcgtaatc gt 32
<210> 7
<211> 36
<212> DNA
<213> Artificial sequence ()
<400> 7
atggaagcaa cgatcaaagt gatgttagca gcagca 36
<210> 8
<211> 36
<212> DNA
<213> Artificial sequence ()
<400> 8
tgctgctgct aacatcactt tgatcgttgc ttccat 36
<210> 9
<211> 35
<212> DNA
<213> Artificial sequence ()
<400> 9
caacaacgta accgaagact tgcaaccagg atggg 35
<210> 10
<211> 35
<212> DNA
<213> Artificial sequence ()
<400> 10
cccatcctgg ttgcaagtct tcggttacgt tgttg 35
<210> 11
<211> 38
<212> DNA
<213> Artificial sequence ()
<400> 11
ggtgctagac agactgttat catctccact ttggctcc 38
<210> 12
<211> 38
<212> DNA
<213> Artificial sequence ()
<400> 12
ggagccaaag tggagatgat aacagtctgt ctagcacc 38
<210> 13
<211> 40
<212> DNA
<213> Artificial sequence ()
<400> 13
cttggacact actcacccag tttctaccgt tgacttggct 40
<210> 14
<211> 40
<212> DNA
<213> Artificial sequence ()
<400> 14
agccaagtca acggtagaaa ctgggtgagt agtgtccaag 40
<210> 15
<211> 47
<212> DNA
<213> Artificial sequence ()
<400> 15
tacttcccac attgagattt cctgccaaga cgttcacgct acttgtc 47
<210> 16
<211> 47
<212> DNA
<213> Artificial sequence ()
<400> 16
gacaagtagc gtgaacgtct tggcaggaaa tctcaatgtg ggaagta 47

Claims (5)

1. A phospholipase D mutant, characterized in that, based on the phospholipase D amino acid sequence shown in SEQ ID No.2, the following mutations have occurred: gln388 Cys; or Gln388Cys, Asp519 Val.
2. The phospholipase D mutant of claim 1 which encodes the gene.
3. Use of the phospholipase D mutant according to claim 1 or the gene according to claim 2, for the production of phosphatidic acid and phosphatidylserine.
4. A recombinant vector or a recombinant bacterium comprising the gene of claim 2, wherein the expression vector is pBSA43, and the host cell is bacillus subtilis WB 600; or the expression vector is pPIC9K, and the host cell is Pichia pastoris GS 115; alternatively, the expression vector is pPIC9K-Flo and the host cell is Pichia pastoris GS 115.
5. The method for producing a mutant of phospholipase D as set forth in claim 1, comprising the steps of:
(1) carrying out enzyme digestion on the gene of claim 2, and connecting the gene with a vector to obtain a new recombinant vector;
(2) transforming the recombinant vector into a host cell to obtain a recombinant strain, and fermenting the recombinant strain to obtain high-activity phospholipase D;
the expression vector is pBSA43, and the host cell is Bacillus subtilis WB 600; or the expression vector is pPIC9K, and the host cell is Pichia pastoris GS 115; alternatively, the expression vector is pPIC9K-Flo and the host cell is Pichia pastoris GS 115.
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