Application of phospholipase D mutant in biosynthesis of phosphatidylserine

Abstract

The invention provides a phospholipase D mutant and application thereof, compared with an amino acid sequence SEQ ID NO.2, four sites of Y6570, D6595, R6661 and D6669 in the amino acid sequence SEQ ID NO.2 are respectively subjected to single mutation, pairwise combined mutation, three combined mutation or one of four combined mutation; the novel phospholipase D mutant industrial enzyme is used for synthesizing and preparing L-phosphatidylserine. The phospholipase D mutant enzyme constructed by the invention has the characteristics of low enzyme cost, short conversion time, simple process operation and the like, and has a wide prospect of large-scale industrial application.

Description

Technical Field

[0001] The present invention relates to a novel phospholipase D and a mutant thereof, in particular to an industrial enzyme and a mutant thereof for synthesizing L-phosphatidylserine by a bioenzymatic method, and belongs to the technical field of bioenzyme engineering. Background Art

[0002] L-Phosphatidylserine (PS) is present in bacteria , yeast ,plant, Mammals thing An important Membrane phospholipids Phosphatidylserine is also known as compound neuraminic acid. It is abbreviated as PS and is extracted from the residue of natural soybean oil. Cell membrane of Active substances , especially in brain cells. Its main function is to improve Nerve cells Function, adjustment Nerve impulses conduction, improve brain memory function, because it has a strong Lipophilicity After absorption, it can quickly pass through the blood-brain barrier and enter the brain, soothing vascular smooth muscle cells and increasing blood supply to the brain.

[0003] At present, there are three main methods for obtaining L-phosphatidylserine. The first is direct extraction, that is, extraction from soybeans, peanuts (CN102964379A) ​​and animal viscera and brains (CN1583766A, CN1562081). This method requires a large amount of methanol and water, and the purity of the purified product is not high; the second is that as described in CN201310312735.3, dangerous chemicals such as phosphorus oxychloride are required, which is difficult to separate and purify, and the overall yield is not high; at the same time, the environmental pollution is serious; the third is the currently more popular bioenzymatic method for preparation, which mainly has two ways: the first is a two-phase method, as described in JP2001186898, which is a reaction of an organic phase and a water mixed system, with a conversion rate of only 60.80%, and a high reaction cost. The second is a homogeneous method, which mainly increases the amount of water in the reaction system to 60-95%, and then utilizes a homogenizing device to improve the product conversion rate, such as JP2002051794, CN200810017261.9, but the product purification is relatively difficult. Another method is to utilize γ-valerolactone and 2-methyltetrahydrofuran (CN102676600) as solvents to carry out enzyme catalytic conversion, and the yield of phosphatidylserine can reach more than 90%, but the solvent treatment of this method is more troublesome. Therefore, how to improve the catalytic activity of PLD enzyme has gradually become a hot spot of people's concern, but there is no report on the successful biosynthesis of L-phosphatidylserine by PLD mutant enzymes. Summary of the invention

[0004] The invention discloses a kind of glycine fermentation bacillus from soybean Bacillus glycinifermentans ) and its mutant genes, and provide a method for constructing an in vitro heterologous expression system of the enzyme and a method for constructing an enzyme mutant, as well as a method for using the enzyme and its mutant as a biocatalyst to prepare L-phosphatidylserine.

[0005] The nucleotide sequence of phospholipase D (PLD) is shown in SEQ ID No.1; the amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.2.

[0006] The gene sequence of phospholipase D (PLD) was synthesized by Jiangsu GenScript Biotechnology Co., Ltd., and EcoT14 and EcoT22 restriction endonuclease sites were added at both ends of the coding region. The target gene fragment was digested by restriction endonucleases EcoT14 and EcoT22, and then connected, transformed and screened with the pET28a(+) vector that had been digested by the same double enzymes. The positive plasmid PLD-pET28a(+) obtained by screening was transferred into the BL21(DE3) host bacteria, thereby constructing an in vitro heterologous expression system of PLD.

[0007] The construction of the mutant of the PLD enzyme is obtained by the technical means of directed evolution. Specifically, the mutant is obtained by using directed technologies such as error-prone PCR, DNA rearrangement, semi-rational design and three-dimensional structure simulation. More specifically, the present invention carries out directed evolution of the enzyme by three-dimensional structure simulation technology. The three-dimensional structure of the PLD enzyme is simulated by homology modeling, and one or more possible sites related to catalysis are predicted by using the energy minimum principle and molecular docking technology, and then saturated site-directed mutagenesis (NNK) is performed on these sites to screen out mutants with significantly improved activity.

[0008] The present invention predicts that the sites that may be related to catalysis and substrate binding are Y6570, D6595, R6661, and D6669 through three-dimensional structure simulation technology. These four sites are subjected to site-directed saturation mutagenesis respectively.

[0009] in,

[0010] Forward primer for Y6570 site mutation:

[0011] CGCCATGCGCGCGCGGGCNNNKAAAACCCTGGAATCGTTTTG,

[0012] Reverse Primer:

[0013] CAAAACGATTCCAGGGTTTTMNNGCCCGCGCGCGCATGGCG;

[0014] Forward primer for D6595 site mutation:

[0015] CGAAAAATATGGCGTGGGCGNNKTGGGATTGCCCGGAAGCG,

[0016] Reverse Primer:

[0017] CGCTTCCGGGCAATCCCAMNNCGCCACGCCATATTTTTCG;

[0018] Forward primer for R6661 site mutation:

[0019] GCCGCCGCCGCCATGCGNNKGCGGGCTATAAAACCCTGGAAG,

[0020] Reverse Primer:

[0021] CTTCCAGGGTTTATAGCCCGCMNNCGCATGGCGGCGGCGGC;

[0022] Forward primer for mutation at site D6669:

[0023] CGAAAGATATGGCGTTGGCGNNKTGGGATTGCCAGGAAGCG,

[0024] Reverse Primer:

[0025] CGCTTCCGGGCAATCCCAMNNCGCCACGCCATACTTTTCG;

[0026] Then, high pressure liquid chromatography (HPLC) is used to screen the mutants. More specifically, when the tyrosine (Y) at site 6570 mutates to proline (P), the catalytic activity of the mutant is improved relative to the wild-type enzyme, and when the aspartic acid (D) at site 6595 mutates to methionine (M), the enzyme activity of the mutant is improved relative to the wild-type enzyme. When the arginine (R) at site 6661 mutates to serine (S), the enzyme activity of the mutant is improved. When the aspartic acid (D) at site 6669 mutates to histidine (H), the enzyme activity of the mutant is improved. When the above four sites are subjected to single mutation, two-two combined mutation, three combined mutations or four combined mutations, the catalytic activity of the mutant is further improved relative to the single mutant. According to existing public knowledge, any gene can be operated or modified, connected to various expression vectors, transformed into suitable host cells, and induced under appropriate conditions to overexpress the target protein. Therefore, the vector for expressing the PLD enzyme and its mutants can be pET or pCW or pUC, etc., and the expression host can be Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Streptomyces, Bacillus subtilis, etc.

[0027] Beneficial Effects of the Invention

[0028] 1) The enzyme mutant and coenzyme regeneration system involved in the present invention can convert 20% of the substrate into L-phosphatidylserine within 8 hours at 40°C, with a conversion rate of >95%.

[0029] The reaction conditions are mild, there are almost no by-products, the energy cycle system is stable, and it has broad prospects for industrial application. DETAILED DESCRIPTION

[0030] The present invention is described in detail below with reference to the following examples. The embodiments are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention.

[0031] In the examples, the experimental methods without specifying specific conditions are usually carried out according to conventional conditions, such as the methods described in Molecular Cloning Experimental Manual (J. Sambrook, DW Russell, translated by Huang Peitang, Wang Jiaxi, Zhu Houchu, etc., third edition, Beijing: Science Press, 2002).

[0032] The phospholipase D mutant of the present invention is produced in a recombinant microbial cell, which is one of an Escherichia coli strain, a Bacillus subtilis strain, a Streptomyces strain, a saccharomyces cerevisiae or a Pichia pastoris.

[0033] Example 1 Mutant library construction and high-throughput screening method:

[0034] In order to improve the activity of wild-type PLD enzyme, a random mutant library was constructed by error-prone PCR using the recombinant expression vector pET28a(+)-RT-PLD as the DNA template. 2+ and Ca 2+ The concentration and the concentration of dCTP and dTTP oligonucleotides are adjusted to make the base mismatch rate of the mutant library 0.5%, that is, to ensure that one mutant has 1 to 3 amino acids mutated. The specific process of constructing the mutant library is as follows.

[0035] The error-prone PCR reaction conditions are: first pre-denaturation at 95°C for 5 minutes; then denaturation at 94°C for 30 seconds, annealing at 55°C for 1 minute, and 72°C for 1.5 minutes, for a total of 30 cycles; and finally extension at 72°C for 10 minutes.

[0036] The error-prone PCR product obtained above was excised from the gel, recovered and purified, connected with the prokaryotic expression vector pET28a(+), and then transformed into recombinant genetic engineering bacteria to obtain a mutant library with a large library capacity.

[0037] Error-prone PCR reaction system and conditions:

[0038] Error-prone PCR reaction system:

[0039]

[0040] Screening mutant library:

[0041] The high-throughput screening method of the phospholipase D mutant library in the present invention adopts the NADPH determination method, which has a maximum absorption peak at 262nm, uses 10g / L lecithin as a substrate to screen mutants, and measures its absorbance at 262nm by an ELISA instrument. The smaller the value, the higher the enzyme activity, indicating that the ability to tolerate high-concentration substrates is stronger, and the mutant is a beneficial mutant.

[0042] About 20,000 clones were screened from the mutant library, and 100 mutants with obvious numerical changes were obtained. Then, these 100 mutants were screened by shaking flask. The specific process is: these 100 mutants were inoculated into a 500ml shaking flask containing 100ml LB medium for fermentation and induction, and their catalytic activity of 10g / L lecithin was determined by colorimetry to obtain the optimal mutant, named SEQ ID NO.2. The sequencing results showed that the mutant sequences were: Y6570P, D6595M, R6661S, D6669H

[0043] Example 2 Mutant cultivation and screening:

[0044] The phospholipase D (PLD) gene fragment was synthesized by Jiangsu GenScript Biotechnology Co., Ltd. and recombined into the pUC18 vector. After double digestion with restriction endonucleases EcoT14 and EcoT22 at 40°C for 4 hours, it was separated by 1% agarose gel electrophoresis and the gel was cut and recovered. It was then connected to the expression vector pET28a(+) that had been double digested at 16°C overnight. The ligation solution was transformed into OmniMAX competent cells, and colony PCR screening and sequencing verification were performed to obtain the positive recombinant plasmid PLD-pET28a(+). The positive recombinant plasmid PLD-pET28a(+) was transformed into the expression host bacteria BL21(DE3) to obtain the prokaryotic expression strain PLD-pET28a(+) / BL21(DE3), which was used as the primary strain for subsequent directed evolution and fermentation.

[0045] After the plasmid obtained by the above mutation was transformed into the BL21 (DE3) host bacteria, it was spread on the LB solid medium containing 30μg / ml kanamycin, and inverted and cultured at 37℃ overnight. Then, a single clone was picked from the plate and placed in a 96-well plate for culture. The overnight cultured bacterial solution was transferred to a 96-well plate containing fresh LB medium, and after shaking culture at 37℃ and 220rpm for 4h, IPTG with a final concentration of 0.1mM was added for induction, and cultured at 30℃ overnight. The bacterial cells were collected by centrifugation at 4℃ and 5000rpm for 10min, and the crude enzyme solution or lyophilized powder was prepared after ultrasonic disruption for use.

[0046] Substrate concentration 10g / L, 5mM pH 7.0 sodium phosphate buffer, 5mM sodium hexametaphosphate, 5mM magnesium chloride, 10mM calcium chloride, 10% of the crude enzyme solution prepared above, placed at 37°C, 220rpm stirring reaction. Samples were taken at 2h and 10h for HPLC detection.

[0047] The clones with significantly improved substrate conversion rates at 2h and 10h were expanded and cultured, and then sequenced to verify the mutation. The sequencing results showed that the mutation sites contained in the clones with significantly improved mutant enzyme activity were as follows: when the tyrosine (Y) at site 6570 mutated to proline (P), the catalytic activity of the mutant was improved relative to the wild-type enzyme, and when the aspartic acid (D) at site 6595 mutated to methionine (M), the mutant enzyme activity was improved relative to the wild-type enzyme. When the arginine (R) at site 6661 mutated to serine (S), the mutant enzyme activity was improved. When the aspartic acid (D) at site 6669 mutated to histidine (H), the mutant enzyme activity was improved. Subsequently, these sites were mutated in pairs, three or four combinations, and activity detection found that the catalytic activity of some sites after combined mutation was significantly improved compared with single-point mutations.

[0048] Example 3 Biocatalysis of double-site mutants:

[0049] Dissolve 5g of substrate soybean lecithin in 100ml 5mM pH7.0 sodium phosphate buffer. After the substrate is completely dissolved, add 5mM pH7.0 sodium phosphate buffer, 5mM sodium hexametaphosphate, 5mM magnesium chloride, 10mM calcium chloride, and 0.2g PLD mutant (D6595M, R6661S) lyophilized powder. Place the reaction solution in a 40°C constant temperature water bath and mechanically stir the reaction. After 10h of reaction, perform HPLC detection, and the substrate conversion rate is >90%. After purification, the purity of the phosphatidylserine is greater than 80%.

[0050] Example 4 Biocatalysis of three-site mutants:

[0051] Dissolve 5g of substrate soybean lecithin in 100ml 5mM pH7.0 sodium phosphate buffer. After the substrate is completely dissolved, add 5mM pH7.0 sodium phosphate buffer, 5mM sodium hexametaphosphate, 5mM magnesium chloride, 10mM calcium chloride, and 0.2g PLD mutant (Y6570P, D6595M, R6661S) lyophilized powder. The reaction solution is placed in a 40°C constant temperature water bath and mechanically stirred for reaction. After 10h of reaction, HP LC detection is performed, and the substrate conversion rate is >95%. After purification, the purity of phosphatidylserine is greater than 85%.

[0052] Example 5 Biocatalysis of four-site mutants:

[0053] Dissolve 5g of substrate soybean lecithin in 100ml 5mM pH7.0 sodium phosphate buffer. After the substrate is completely dissolved, add 5mM pH7.0 sodium phosphate buffer, 5mM sodium hexametaphosphate, 5mM magnesium chloride, 10mM calcium chloride, and 0.2g PLD mutant (Y6570P, D6595M, R6661S, D6669H) lyophilized powder. The reaction solution is placed in a 40°C constant temperature water bath and mechanically stirred for reaction. After 10h of reaction, HP LC detection is performed, and the substrate conversion rate is >98%. After purification, the purity of phosphatidylserine is greater than 90%.

[0054] Complement(6386-6787)Id:KMM56815.1

[0055] SEQ ID NO.1:

[0056] MKKVKNRWIIALCAVGIHLSIGSVYAWSVFTNPLAERFGWSLSQISLTFSIAILFLGLSAAFLGHFVEKHGPRKAGMLAAFFFGTGVTFSGVAVQLGSLPMLYLFYGVFGGIGLGVGYITPVSSLVKWFPDRRGLATGLAIMGFGFAALISGPVMQALIVTAGIANTFFILGICYFLIMLCSSLYLSPPETDWQPKGLSSEKSKRPRSTDLSQLTANEAVKTRRFYYLWLMLFINVTCGIAIISIASPLAQESAGMSAAGAAALVGVLGAFNGLGRIGWASISDYIGRPNTYTAFFAAQLIAFPLLPFMTDHIMFSIFMALIYTCYGGGFAAIPAYIGDLFGTKQLGAIHGYMLTAWAAAGLAGPMFSSWIRDVTGSYSQSLAVFAVLFGIGLCLSIFIRADMKQLRKKADIRFSDSVKG

[0057] SEQ ID NO.2:

[0058] MKKVKNRWIIALCAVGIHLSIGSVYAWSVFTNPLAERFGWSLSQISLTFSIAILFLGLSAAFLGHFVEKHGPRKAGMLAAFFFGTGVTFSGVAVQLGSLPMLYLFYGVFGGIGLGVGYITPVSSLVKWFPDRRGLATGLAIMGFGFAALISGPVMQALIVTAGIANTFFILGICYFLIMLCSSLPLSPPETDWQPKGLSSEKSKRPRSTMLSQLTANEAVKTRRFYYLWLMLFINVTCGIAIISIASPLAQESAGMSAAGAAALVGVLGAFNGLGSIGWASISHYIGRPNTYTAFFAAQLIAFPLLPFMTDHIMFSIFMALIYTCYGGGFAAIPAYIGDLFGTKQLGAIHGYMLTAWAAAGLAGPMFSSWIRDVTGSYSQSLAVFAVLFGIGLCLSIFIRADMKQLRKKADIRFSDSVKG

Claims

1. A phospholipase D (PLD) mutant, characterized in that: The amino acid sequence of the mutant is compared with the amino acid sequence SEQ ID NO.2, and Y6570, D6595, R6661, and D6669 in the amino acid sequence SEQ ID NO.2 are subjected to single mutation, two-by-two combined mutation, or three or four combined mutations.

2. The phospholipase D mutant according to claim 1, characterized in that: The tyrosine (Y) at position Y6570 in the amino acid sequence SEQ ID NO.2 was mutated to proline (P), the aspartic acid (D) at position D6595 was mutated to methionine (M), the arginine (D) at position R6661 was mutated to serine (S), and the aspartic acid (D) at position D6669 was mutated to histidine (H).

3. The phospholipase D mutant according to claim 1, characterized in that: The tyrosine (Y) at position Y6570 in the amino acid sequence SEQ ID NO.2 was mutated to proline (P).

4. The phospholipase D mutant according to claim 1, characterized in that: The aspartic acid (D) at position D6595 in the amino acid sequence SEQ ID NO.2 was mutated to methionine (M).

5. The phospholipase D mutant according to claim 1, characterized in that: The arginine (D) at position R6661 in the amino acid sequence SEQ ID NO.2 was mutated to serine (S).

6. The phospholipase D mutant according to claim 1, characterized in that: The aspartic acid (D) at position D6669 in the amino acid sequence SEQ ID NO.2 was mutated to histidine (H).

7. The phospholipase D mutant according to claim 1, characterized in that: The phospholipase D mutant is produced in a recombinant microbial cell, which is one of an Escherichia coli strain, a Bacillus subtilis strain, a Saccharomyces cerevisiae strain or a Pichia pastoris strain.

8. A method for preparing the phospholipase D mutant according to claim 1, comprising the following steps: 1) Using the PLD-pET28a(+) recombinant plasmid as a template, site-directed saturation mutagenesis was performed on four sites, Y6570, D6595, R6661, and D6669. Forward primer for Y6570 site mutation: CGCCATGCGCGCGCGGGCNNKAAAACCCTGGAATCGTTTTG, reverse primer: CAAAACGATTCCAGGGTTTTMNNGCCCGCGCGCGCATGGCG; Forward primer for mutation at site D6595: CGAAAAATATGGCGTGGCGNNKTGGGATTGCCCGGAAGCG, reverse primer: CGCTTCCGGGCAATCCCAMNNCGCCACGCCATATTTTTCG; Forward primer for R6661 site mutation: GCCGCCGCCGCCATGCGNNKGCGGGCTATAAAACCCTGGAAG, Reverse primer: CTTCCAGGGTTTATAGCCCGCMNNCGCATGGCGGCGGCGGC; Forward primer for mutation at site D6669: CGAAAGATATGGCGTGGCGNNKTGGGATTGCCAGGAAGCG, reverse primer: CGCTTCCGGGCAATCCCAMNNCGCCACGCCATACTTTTCG; 2) Mutant culture: After the plasmid obtained by the above mutation is transformed into the BL21 (DE3) host bacteria, it is spread on the LB solid medium containing 30 μg / ml kanamycin, and inverted and cultured at 37°C overnight. Then, a single clone is picked from the plate and placed in a 96-well plate for culture; the overnight cultured bacterial liquid is transferred to a 96-well plate containing fresh LB medium, and after shaking culture at 37°C and 220 rpm for 4 hours, IPTG with a final concentration of 0.1 mM is added for induction, and cultured at 30°C overnight; the bacterial cells are collected by centrifugation at 4°C and 5000 rpm for 10 minutes, and the crude enzyme solution is obtained after ultrasonic disruption, or it is prepared into lyophilized powder; 3) Screening of mutants: substrate concentration 10 g / L, 5 mM pH 7.0 sodium phosphate buffer, 5 mM sodium hexametaphosphate, 5 mM magnesium chloride, 10 mM calcium chloride, added to the crude enzyme solution prepared above at a ratio of 10%, placed at 37°C, 220 rpm stirring reaction; samples were taken at 2h and 10h for HPLC detection; sequencing results showed that the mutation sites contained in the clones with significantly improved mutant enzyme activity were as follows: when the tyrosine (Y) at position Y6570 mutated to proline (P), the aspartic acid (D) at position D6595 mutated to methionine (M), the arginine (D) at position R6661 mutated to serine (S), and the aspartic acid (D) at position D6669 mutated to histidine (H), the PLD enzyme activity increased 11.2 times.

9. Use of the phospholipase D mutant industrial enzyme as claimed in claim 1 for catalyzing the synthesis of L-phosphatidylserine.

10. A method for synthesizing L-phosphatidylserine by catalysis of the phospholipase D mutant industrial enzyme as claimed in claim 7, comprising the following steps: 1) Dissolve 1 g of substrate lecithin in 100 ml of 5 mM pH 7.0 sodium phosphate buffer; 2) After the substrate described in step 1) is completely dissolved, 5 mM sodium hexametaphosphate, 5 mM magnesium chloride, 10 mM calcium chloride, and 0.2 g of phospholipase D mutant industrial enzyme freeze-dried powder are added; 3) placing the reaction solution obtained in step 2) in a constant temperature water bath at 40° C. and mechanically stirring the reaction; after reacting for 10 hours, HPLC detection was performed, and the substrate conversion rate was >95%; after purification, L-phosphatidylserine was obtained, and the purity of the target product was greater than 90%.

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