CN110564708A

CN110564708A - Recombinant phospholipase D and application thereof in synthesis of phosphatidylserine or other phospholipids

Info

Publication number: CN110564708A
Application number: CN201910996555.9A
Authority: CN
Inventors: 毛相朝; 张海洋; 孙建安; 薛长湖
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2019-10-19
Filing date: 2019-10-19
Publication date: 2019-12-13
Anticipated expiration: 2039-10-19
Also published as: CN110564708B

Abstract

The invention provides a recombinant phospholipase D, which is a novel PLD obtained by screening through an escherichia coli surface display technology high-throughput screening technology and a DNA gene recombination technology. The amino acid sequence of the recombinant phospholipase D provided by the invention is SEQ ID NO. 1. The recombinase PLDr34 provided by the invention has the advantages that the transesterification activity is improved by 3.24 times, the conversion rate is 80.3%, the selectivity is 86.8%, and the recombinase PLDr34 is an ideal catalyst for synthesizing Phosphatidylserine (PS), DHA-phosphatidylserine (DHA-PS) and other rare phospholipids.

Description

Recombinant phospholipase D and application thereof in synthesis of phosphatidylserine or other phospholipids

Technical Field

The invention belongs to the technical field of functional enzyme screening, and particularly relates to recombinant phospholipase D and application thereof in synthesis of phosphatidylserine, DHA-phosphatidylserine or other phospholipids.

Background

Phospholipids are widely present in biological tissue structures and are important components of cell membranes, and are composed of a hydrophilic phosphate group head and a hydrophobic fatty acid chain tail. Currently, 4 main groups have been identified: ethanolamine, inositol, serine, and choline. These groups form the most important phospholipids, namely Phosphatidylethanolamine (PE), Phosphatidylinositol (PI), Phosphatidylserine (PS) and Phosphatidylcholine (PC). PC is the most predominant natural phospholipid, however, it does not meet human nutritional or other needs, and synthesis of phospholipids of a particular configuration from natural phospholipids has important industrial application value.

the research in recent years shows that human body supplements a certain amount of PS, which is helpful for enhancing the mobility of cell membranes, improving enzyme activity, enhancing cognitive ability and delaying senility. Alzheimer's Disease (AD) is one of the important diseases affecting the quality of life of the elderly, the cause of which is related to the deterioration of brain function, and improving the lipid composition of damaged nerve membranes, especially increasing the content of PS in nerve cell membranes, which is one of the effective methods for preventing and treating AD. PS can regulate the activity of human body by regulating the content of stress hormone, and when the content of stress hormone in the body is reduced, PS can stimulate nerve to make it secrete hormone; when the content of stress hormone is increased, the stress hormone can inhibit the organism and relieve emotional stress. In addition, PS can reduce the loss of amino acids in the muscle and protect the function of the muscle membrane. The fatty acid side chains of phospholipids also have a significant impact on phospholipid activity, and for some time in the past, krill oil has been one of the most nutritionally valuable and healthy foods that people can eat, with phospholipids extracted from krill oil accounting for about 40% of the lipids, and Winther et al have found that 58% of PC contains n-3PUFAs in at least one location, while 10% of PC contains n-3PUFAs in two locations, with the highest n-3PUFAs being EPA and DHA, with small amounts of stearidonic acid and docosapentaenoic acid (DPA). DHA-phosphatidylserine (DHA-PS) and DHA-phosphatidylethanolamine (DHA-PE) are more efficient at transporting DHA to the brain than DHA-phosphatidylcholine (DHA-PC).

Later researches show that PS, PC, PE and the like have similar structures and can be mutually converted, so that the researches on preparing functional phospholipid by an enzyme method are carried out continuously. Because the enzyme catalysis method has high selectivity and good performance under mild conditions, the method for synthesizing functional phospholipid by biological catalysis is significant, and products which are not easy to obtain by chemical catalysis are easier to obtain.

The reactions for enzyme-catalyzed phospholipid modification mainly include hydrolysis, transesterification, transphosphatidylation, and the like. Phospholipase D (PLD) can catalyze phosphatidyl group to perform base exchange reaction with other hydroxyl compounds to prepare novel phospholipid, and phospholipid modified by enzyme method is improved in nutritive value, emulsifying property, taste and color, so that application range and value of phospholipid are greatly widened.

Although PLD exhibits good catalytic potential, as a cytotoxic protein, the lower yield limits their prospects for industrial applications. The formation of inclusion bodies is another factor limiting the industrial application of PLD, and inclusion body proteins generally require the addition of chemical reagents such as urea and guanidine chloride for renaturation in order to form correctly folded soluble proteins, usually overnight incubation and shaking/stirring, and even microwave heating as an auxiliary means, which is very disadvantageous for the production or application of PLD. The screening of high-activity PLD is still the key of phospholipase modification.

Disclosure of Invention

The invention provides a recombinant phospholipase D, which is a novel PLD obtained by screening an escherichia coli surface display technology high-throughput screening technology and a DNA gene recombination technology, and is used for preparing PS by using an enzyme-catalyzed transesterification reaction; thereby overcoming the defects of low enzyme activity and low expression level of the prior PLD.

the recombinant phospholipase D provided by the invention comprises:

1) An enzyme having the amino acid sequence of SEQ ID NO. 1;

2) An enzyme derived from 1) having 90% or more homology with the sequence of 1);

Preferably, the homology of the enzyme in 2) is 95% or more;

More preferably, the homology is 99% or more;

A specific sequence of the gene encoding the recombinant phospholipase D of the invention is SEQ ID NO 2

In another aspect, the present invention also provides a recombinant expression vector for transforming/transfecting into a host cell to recombinantly express the recombinant phospholipase D;

one embodiment of the recombinant expression vector is specifically described as pET28a-PLDr34 carrying a gene encoding recombinant phospholipase D;

The invention also provides a recombinant engineering bacterium, which carries the recombinant expression vector;

The recombinant engineering bacteria are applied to the preparation of the recombinant phospholipase D through recombinant expression.

The invention also provides the application of the recombinant phospholipase D in preparing phospholipid;

The phospholipid is preferably Phosphatidylserine (PS) and DHA-phosphatidylserine (DHA-PS).

The transesterification activity of recombinase PLDr34 provided by the invention is improved by 3.24 times, the conversion rate is 80.3%, the selectivity is 86.8%, and the recombinase PLDr34 is an ideal catalyst for synthesizing PS, DHA-PS and other rare phospholipids.

drawings

FIG. 1: agarose gel electrophoresis picture of PLD recombination process in the invention;

FIG. 2: a whole-cell enzyme activity screening chart based on escherichia coli surface display;

FIG. 3: a screened recombinase high-density fermentation enzyme activity determination diagram;

FIG. 4: results of transesterification catalyzed by high activity PLDr 34;

FIG. 5: the result of the reaction for catalyzing and synthesizing DHA-PS by the high-activity PLDr34 is shown in the figure.

Detailed Description

The technical solution of the present invention is further illustrated by the following specific examples.

Example 1: screening and preparation of recombinant PLD

1. PCR amplification of phospholipase D fragments

Firstly, amplifying two kinds of PLD proteins obtained by an applicant by a PCR method, namely S.griseofusus strain PLDsg/G213S and S.roseus, PLDsr, with the size of about 1500-1600 bp, wherein the amino acid sequence of PLDsg/G213S is SEQ ID NO. 3, and the sequence of a coding gene is SEQ ID NO. 4; roseus, the amino acid sequence of PLDsr is SEQ ID NO. 5, and the sequence of coding gene is SEQ ID NO. 6. The PCR product was then recovered and the concentration of both fragments was determined using a microanalyzer. Two equal amounts of amplified PCR product solutions were mixed and cut into fragments with DNsae I, and the cut products were detected by agarose gel electrophoresis (FIG. 1A). According to the materials and the method, 0.3 mu L of DNase I (diluted to 1U/. mu.L, diluted at present) is used for enzyme digestion at 15 ℃ for 2min, the effect is best, the fragment size is between 100 and 200bp, and the expected requirements are met. Cutting the gel and recovering the nucleic acid fragment with the fragment size of 100-200 bp.

2. Primer-free PCR amplification of phospholipase D fragment

Taking products recovered after enzyme digestion as templates, and performing primer-free PCR with the templates serving as primers. The PCR product is detected by 1% agarose gel electrophoresis, and an electrophoretogram shows that a band is successfully amplified by primer-free PCR, the brightness appears between 500 and 3000bp, a bright band of about 1500 to 2000bp is recovered by cutting gel, and the product is named as pld-1 (figure 1B).

3. PCR amplification of phospholipase D fragment with primer

Pld-1 is taken as a template, and primers of (i) AT-sg-F and AT-sg-R, (ii) AT-sg-F and AT-sr-R, (iii) AT-sr-F and AT-sg-R, and (iv) AT-sr-F and AT-sr-R are taken as primers to amplify to obtain a full-length mutation library. The PCR products were analyzed by agarose gel electrophoresis, and the objective products were recovered by cutting the gel and named pld 1-1, pld 1-2, pld 2-1, pld 2-2, respectively (FIG. 1C).

The 4 recombinant fragments are connected with the successfully constructed Escherichia coli surface display vector pAT-PLD in a seamless splicing mode. The ligation products were first transformed into DH 5. alpha. competent cells, all colonies grown were washed with sterile water, transferred to LB tubes, mixed plasmids were extracted and transformed into E.coli BL21(DE3) competent cells, plated on a PLD screening plate containing kanamycin, and the grown clones were restreaked in preparation for the next screening.

4. screening of high-Activity Strain

150 well grown clones from the previous round of solid plates were inoculated into 5mL tubes at OD₆₀₀when the value is about 0.6, 0.1mM IPTG is added for induction for 4h, and after freezing and centrifuging for 10min at 5000r/min, the whole cell thallus is taken to determine the hydrolysis activity, and the result is shown in figure 2. Among them, 8 mutants 12, 34, 35, 61, 62, 64, 72 and 88 had significantly improved enzyme activity compared to the control group.

The hydrolytic activity of PLD was used as a screening standard, and positive clones from the first round of screening were cultured in high-density medium ZYP-5052 for the second round of screening. Finally, the enzyme activity of recombinase Recom-35 is improved by 1.15 times, and the enzyme activities of Recom-12, Recom-34 and Recom-88 are respectively improved by 0.15-0.86 times by comparison of the enzyme activities (figure 3).

the recombinase from the previous round of screening was subjected to sequencing analysis, wherein the entire open reading frame of Recom-34 (recombinase PLDr34) of the present invention is 1617bp (SEQ ID NO:2) and consists of 538 amino acids (SEQ ID NO: 1). Recom-34 fuses the N-terminal 105 amino acids of the PLDsg, and the 105 th to 500 th amino acids are consistent with the PLDsr, wherein the sequence consistency of Recom-34 and PLDsg is 83.27%, and the sequence consistency of PLDsr is 88.10%. The sequence is submitted to an NCBI database for sequence information analysis, the similarity of the sequence information and PLD of Streptomyces sp.WM6372 in the database is highest, the consistency is 89.40 percent, the enzyme has longer evolutionary distance with several reported Streptomyces, and the enzyme is a novel enzyme.

Example 2: recombinant PLDs enzyme synthesis of PS and DHA-PS

In order to verify the capability of the recombinant PLDs enzyme and parent enzyme in the invention for synthesizing PS, a biphase reaction system is adopted to synthesize PS by taking phosphatidylcholine PC and L-serine as substrates. The parental enzymes PLDsr and PLDsg/G213S were used as controls, Reom-12, Recom-34 and Recom-35 were used as experimental groups, and the same weight of enzyme powder, 1M L-serine and 50mM CaCl₂Dissolved in 1mL HAc-NaAc buffer (20mM, pH 6.0), and the organic phase contained PC (dissolved in anhydrous diethyl ether)20 mg/mL). Optimizing the enzymatic reaction time, after reacting for 6 hours, centrifuging to take an organic phase, and detecting the reaction result by using thin layer chromatography and HPLC.

The results are shown in a of FIG. 4, in which lanes 1 from left to right are the substrates phosphatidylcholine PC, lanes 2 and 3 are the parent enzymes PLDsg/G213S and PLDsr, and lanes 4-6 are the recombinases Reom-12, Recom-34 and Recom-35, respectively. The results showed that under the same reaction conditions, the phospholipid fraction of the control group PLDsg/G213S was 31.3% Phosphatidic Acid (PA), 43.9% PC and 24.8% PS, respectively, the transesterification enzyme activity was 0.05U/mg (. mu.mol/h/mg), and the parent enzyme PLDsr could not catalyze the transesterification reaction. The recombinase Recom-34 catalyzes the transesterification reaction, with a PS content of 80.3% of the total phospholipids, producing only 12.2% of PA as hydrolysis by-product and 7.4% of substrate residue. Although recombinase Recom-35 reacts most rapidly and no PC is detected in the total phospholipid fraction, 27% of PA by-product is produced and the conversion rate reaches 73%. Therefore, the recombinant enzyme Recom-34 is preferably selected for the synthesis of PS in the present invention. Compared with the parent enzyme PLDsg/G213S, the recombinant enzyme Recom-34 has greatly improved enzymatic performance in terms of reaction speed and reaction efficiency.

The recombinase Recom-34 is used for DHA-PS synthesis, the result is shown in FIG. 5, and the 2 nd lane is the reaction result, which shows that the recombinase Recom-34 of the invention can be used for DHA-PS synthesis.

the invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention fall within the scope of the invention.

sequence listing

<110> China oceanic university

<120> a recombinant phospholipase D and its application in synthesis of phosphatidylserine or other phospholipids

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Arg Leu Asp Ala Gly Ala Asp Asp Pro Ala Gly Trp Leu Leu Gln Thr

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Asp His Pro Val Ser Asp Val Asp Leu Ala Leu Thr Gly Pro Ala Ala

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Ala Leu Ala Thr Lys Leu Ala Asp Gly Val Lys Val Arg Ile Val Val

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agcgatccgg caaatcgtgg tgcagttggt agtggcggtt atagccagat taagagcctg 1260

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gccttttata tcggcagcaa aaatctgtac ccggcatggc tgcaggattt tggctatatt 1500

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Leu Phe Ala Ala Val Pro Ala Ser Ala Ala Pro Ala Ala Gly Ser Gly

1 5 10 15

Pro Ala Ala Pro Ala Pro His Leu Asp Ala Val Glu Gln Thr Leu Arg

20 25 30

Gln Val Ser Pro Gly Leu Glu Gly Gln Val Trp Glu Arg Thr Gly Gly

35 40 45

Asn Val Leu Asp Ala Ser Thr Pro Gly Gly Gly Asp Trp Leu Leu Gln

50 55 60

Thr Pro Gly Cys Trp Gly Asp Asp Thr Cys Ala Ala Arg Pro Gly Thr

65 70 75 80

Glu Arg Leu Leu Ala Lys Met Thr Glu Asn Val Ser Arg Ala Thr Arg

85 90 95

Thr Val Asp Ile Ser Thr Leu Ala Pro Phe Pro Asn Gly Ala Phe Gln

100 105 110

Asp Ala Ile Val Ala Gly Leu Lys Ser Ser Ala Ala Arg Gly Asn Lys

115 120 125

Leu Thr Val Arg Val Leu Val Gly Ala Ala Pro Ile Tyr His Met Asn

130 135 140

Val Leu Pro Ser Lys Tyr Arg Asp Glu Leu Val Ala Lys Leu Gly Ala

145 150 155 160

Asp Ala Arg Asn Val Asp Leu Asn Val Ala Ser Met Thr Thr Ser Lys

165 170 175

Thr Ser Phe Ser Trp Asn His Ser Lys Leu Leu Val Val Asp Gly Gln

180 185 190

Ser Val Ile Thr Gly Gly Ile Asn Asp Trp Lys Asp Asp Tyr Leu Glu

195 200 205

Thr Ala His Pro Val Ala Asp Val Asp Leu Ala Leu Arg Gly Pro Ala

210 215 220

Ala Ala Ser Ala Gly Arg Tyr Leu Asp Glu Leu Trp Ser Trp Thr Cys

225 230 235 240

Gln Asn Arg Asn Asn Ile Ala Gly Val Trp Phe Ala Ser Ser Asn Gly

245 250 255

Thr Ala Cys Met Pro Ala Met Ala Lys Asp Thr Ala Pro Ala Ala Pro

260 265 270

Pro Ala Ala Pro Gly Asp Val Pro Ala Ile Ala Val Gly Gly Leu Gly

275 280 285

Val Gly Ile Lys Arg Ser Asp Pro Ser Ser Ala Phe Arg Pro Thr Leu

290 295 300

Pro Ser Ala Ala Asp Thr Lys Cys Val Val Gly Leu His Asp Asn Thr

305 310 315 320

Asn Ala Asp Arg Asp Tyr Asp Thr Val Asn Pro Glu Glu Ser Ala Leu

325 330 335

Arg Thr Leu Ile Ser Ser Ala Lys Gly His Ile Glu Ile Ser Gln Gln

340 345 350

Asp Val Asn Ala Thr Cys Pro Pro Leu Pro Arg Tyr Asp Ile Arg Val

355 360 365

Tyr Asp Ala Leu Ala Ala Arg Met Ala Ala Gly Val Lys Val Arg Ile

370 375 380

Val Val Ser Asp Pro Ala Asn Arg Gly Ala Val Gly Ser Gly Gly Tyr

385 390 395 400

Ser Gln Ile Lys Ser Leu Ser Glu Ile Ser Asp Thr Leu Arg Asp Arg

405 410 415

Leu Ala Leu Leu Thr Gly Asp Gln Gly Ala Ala Lys Ala Thr Met Cys

420 425 430

Ser Asn Leu Gln Leu Ala Thr Phe Arg Ser Ser Lys Ser Pro Thr Trp

435 440 445

Ala Asp Gly His Pro Tyr Ala Gln His His Lys Val Val Ser Val Asp

450 455 460

Asp Ser Ala Phe Tyr Ile Gly Ser Lys Asn Leu Tyr Pro Ala Trp Leu

465 470 475 480

Gln Asp Phe Gly Tyr Val Val Glu Ser Pro Ala Ala Ala Ala Gln Leu

485 490 495

Asn Ala Gln Leu Leu Ala Pro Gln Trp Arg Tyr Ser Arg Ala Thr Ala

500 505 510

Thr Ile Asp His Glu Arg Ser Leu Cys Gln Ala

515 520

<210> 6

<211> 1572

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ctgttcgccg ccgtcccggc ctcggcggcc ccggcggccg gctccggtcc cgccgccccg 60

gcgccccacc tcgacgcggt ggagcagact ctccgccagg tttcccccgg cctcgaaggc 120

caggtgtggg aacgcaccgg cggcaacgtc ctcgacgcgt cgacgcccgg cggcggcgac 180

tggctgctgc agacccccgg ttgttggggc gacgacacgt gcgccgctcg cccggggacc 240

gagcggctgc tcgcgaagat gacggagaac gtctcccggg cgacgcgtac cgtcgacata 300

tcgaccctgg cgccgtttcc caacggcgcg ttccaggacg cgatcgtcgc gggcctcaag 360

tcctcggccg cgcgcgggaa caagctcacg gtgcgcgtgc tggtcggcgc cgcccccatc 420

taccacatga acgtcctgcc gtcgaagtac cgggacgagc tcgtcgccaa gctgggcgcg 480

gacgcccgca acgtcgacct caacgtcgcc tcgatgacga cctcgaagac gtcgttctcc 540

tggaaccact ccaagctgct cgtggtcgac ggccagtcag tgatcaccgg cggcatcaac 600

gactggaagg acgactacct ggagaccgcc cacccggtcg ccgacgtcga cctcgccctg 660

cgcgggccgg ccgccgcctc cgccggccgc tacctggacg agctgtggtc ctggacctgc 720

cagaacagga acaacatcgc cggcgtctgg ttcgcctcct ccaacggcac cgcctgcatg 780

cccgcgatgg ccaaggacac cgcccccgcc gcacccccgg ccgcccccgg cgacgtaccg 840

gccatcgccg tcggcggact cggcgtcggc atcaagcgaa gcgatccctc ctccgccttc 900

cggcccaccc tgcccagcgc cgccgacacc aagtgcgtcg tcggcctcca cgacaacacg 960

aacgccgacc gcgactacga cacggtcaac cccgaggaga gcgccctgcg gacgctgatc 1020

tccagcgcga aggggcacat cgagatctcc cagcaggacg tcaacgcgac ctgcccgccc 1080

ctgccgcgct acgacatccg cgtctacgac gccctcgccg cacggatggc ggcaggagtc 1140

aaggtgcgca tcgtcgtcag cgaccccgcg aaccgcggag ccgtgggcag cggcggctac 1200

tcgcagatca agtcgctctc ggagatcagc gacaccctgc gcgaccgtct cgccctgctg 1260

accggagacc agggcgcggc gaaggccacg atgtgctcga acctccagct ggccaccttc 1320

cgcagctcga agagcccgac ctgggccgac ggccacccgt acgcgcagca ccacaaggtg 1380

gtctccgtcg acgactcggc cttctacatc ggctccaaga acctctaccc ggcgtggctc 1440

caggacttcg ggtacgtggt ggagagcccg gcggccgcgg cccagctgaa cgcccagctc 1500

ctggccccgc agtggcggta ctcccgcgcg acggccacga tcgaccacga acgctccctc 1560

tgccaggcct ga 1572

Claims

1. A recombinant phospholipase D, comprising:

1) An enzyme having the amino acid sequence of SEQ ID NO. 1;

The homology is 95% or more,

The homology is more than 99%.

2. A gene encoding the recombinant phospholipase D of claim 1.

3. The gene of claim 2, wherein the nucleotide sequence of the gene is SEQ ID NO 2.

4. A recombinant expression vector for transforming/transfecting a host cell to recombinantly express the recombinant phospholipase D of claim 1.

5. The recombinant expression vector of claim 4, wherein the recombinant expression vector is the pET28a-PLDr34 vector carrying a gene encoding the recombinant phospholipase D of claim 1.

6. a recombinant engineered bacterium carrying the recombinant expression vector of claim 4 or 5.

7. the use of the recombinant engineered bacterium of claim 6 for the recombinant expression production of the recombinant phospholipase D of claim 1.

8. Use of the recombinant phospholipase D of claim 1 in the preparation of a phospholipid.

9. A method for producing a phospholipid, wherein the phospholipid or DHA-phospholipid is produced by using the recombinant phospholipase D according to claim 1.

10. the method of claim 9, wherein the phospholipid is phosphatidylserine or DHA-phosphatidylserine.