CN114574470B

CN114574470B - Signal peptide for improving nattokinase secretion efficiency and application thereof

Info

Publication number: CN114574470B
Application number: CN202210326831.2A
Authority: CN
Inventors: 牟海津; 辜自强; 宁晨; 田明
Original assignee: Weihai Dipusen Biology Technology Co ltd
Current assignee: Weihai Dipusen Biology Technology Co ltd
Priority date: 2022-03-30
Filing date: 2022-03-30
Publication date: 2022-10-28
Anticipated expiration: 2042-03-30
Also published as: CN115948310A; CN114574470A; CN115948309A

Abstract

The invention relates to a signal peptide for improving the secretion efficiency of nattokinase and application thereof, belonging to the technical field of genetic engineering. 5 signal peptides are obtained by modifying the signal peptide and screening the modified signal peptide, and the sequence is shown as SEQ ID N0.1-5. The bacillus subtilis nattokinase high expression strain is constructed, and the activity of protease can be improved.

Description

Signal peptide for improving nattokinase secretion efficiency and application thereof

Technical Field

The invention relates to a signal peptide for improving the secretion efficiency of nattokinase and application thereof, belonging to the technical field of genetic engineering.

Background

Nattokinase (NK, EC 3.4.21.62) is an alkaline serine protease produced by fermentation of bacillus natto using the nutrients in soybeans during fermentation of natto. The enzyme has high-efficiency and safe thrombolysis capability, and the research on the heat tide in the field of cardiovascular and cerebrovascular diseases is promoted. Before this, the commonly used clinical thrombolytic drugs mainly include streptokinase, urokinase and plasminogen activator, but these drugs have the disadvantages of high cost, low safety and short drug effect, and are not suitable for large-scale production. The nattokinase has obvious advantages in the aspects of cost and safety, particularly can directly act on fibrin, greatly improves the thrombolysis efficiency, and has unique superiority and wide development prospect compared with the thrombolytic drug. In addition, the nattokinase is proved to have remarkable effects on blood pressure and blood fat regulation, has research values on the aspects of fatigue resistance, bacteria resistance, oxidation resistance, endocrine regulation and the like, is a high-potential dietary supplement, and has great development significance in the food industry.

Nakamura et al (1992) cloned the nattokinase gene for the first time by the accurate shotgun method at that time, and deeply analyzed the primary structure thereof, and determined the whole gene sequence of the enzyme, and from this time researchers began to modify and optimize the nattokinase gene or its host by means of genetic engineering from the molecular level. Liu Bei domain and the like catch a nattokinase original gene from bacillus natto and construct a temperature-induced expression plasmid pESX-1 in E.coli JF1125, thereby realizing the effective expression of nattokinase in escherichia coli. In addition, other hosts, such as Pichia pastoris, bacillus licheniformis, spodoptera frugiperda, and the like, achieve efficient expression of nattokinase.

In order to improve exogenous expression level of nattokinase, researchers have also carried out a plurality of work on expression element optimization. For example, wu et al tried to start the transcriptional expression of nattokinase by using a promoter (Paprv), and modified the core-35 and-10 sequences thereof by point mutation, and found that the mutation modification of the-10 region improves the enzyme activity by 1.36 times. Original genes and key elements thereof are modified on the molecular level, and the enzyme activity of the nattokinase can be obviously improved.

Disclosure of Invention

The invention aims to solve the technical problem of providing a signal peptide for improving the secretion efficiency of nattokinase and application thereof.

The invention also aims to provide an amino acid sequence of the signal peptide for improving the secretion efficiency of the nattokinase, and the sequence is shown as SEQ ID N0.1-5.

The invention also provides a recombinant expression vector containing any one of the genes of the signal peptide for improving the secretion efficiency of the nattokinase.

The invention also provides a recombinant gene engineering bacterium containing any one of the signal peptides for improving the secretion efficiency of nattokinase. The strain is bacillus subtilis, preferably bacillus subtilis WB800.

The invention also provides application of the recombinant expression vector in producing nattokinase.

The invention also provides application of the recombinant gene engineering bacteria in producing nattokinase.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention constructs an expression vector combining various signal peptides and nattokinase coding genes on the basis of the original nattokinase signal peptide gene with the nucleotide sequence of SEQ ID N0.6, and screens out the signal peptide capable of improving the secretion of the nattokinase by combining a high-flux screening method. Meanwhile, an expression vector combining the signal peptide and the nattokinase zymogen coding gene is constructed, so that the secretory expression of the nattokinase zymogen is improved.

2. The invention provides a nucleotide fragment, a signal peptide with strong secretion expression activity, which can realize high expression of nattokinase and particularly provides an effective element for expressing a nattokinase gene by using bacillus subtilis.

3. The invention can increase the secretion of natto kinase and further improve the enzyme activity by fusing the signal peptide.

4. The invention improves the secretion expression of the nattokinase zymogen by fusing the signal peptide.

Drawings

FIG. 1 is a schematic diagram of plasmid PSPX plasmid construction in example 1;

FIG. 2 is an electrophoretogram of the amplified signal peptide 1 fragment in example 1; wherein lane M: marker DNA; lane 1 is a fragment of the constructed signal peptide 1;

FIG. 3 is an electrophoretogram of a fragment of amplified signal peptide 2 in example 1; wherein lane M: marker DNA; lane 1 is a fragment of the constructed signal peptide 2;

FIG. 4 is an electrophoretogram of the signal peptide 3 fragment amplified in example 1; wherein lane M: marker DNA; lane 1 is a fragment of the constructed signal peptide 3;

FIG. 5 is an electrophoretogram of the amplified signal peptide 4 fragment in example 1; wherein lane M: marker DNA; lane 1 is a fragment of the constructed signal peptide 4;

FIG. 6 is an electrophoretogram of the amplified signal peptide 5 fragment in example 1; wherein lane M: marker DNA; lane 1 is a fragment of the constructed signal peptide 5;

FIG. 7 comparison of the enzyme activities of nattokinase in comparison with the original signal peptide in the signal peptide 1 constructed in example 2;

FIG. 8 comparison of enzyme activities of nattokinase in comparison with that of a signal peptide 2 constructed in example 2;

FIG. 9 comparison of the enzymatic activities of nattokinase comparing signal peptide 3 constructed in example 2 with that of the original signal peptide;

FIG. 10 comparison of the enzymatic activities of nattokinase in the signal peptide 4 constructed in example 2 compared with the original signal peptide;

FIG. 11 comparison of the enzymatic activities of nattokinase in comparison with the original signal peptide in the signal peptide 5 constructed in example 2.

Detailed Description

The techniques used in the following examples for molecular biology experiments, including PCR amplification, plasmid extraction, DNA fragment ligation, gel electrophoresis, etc., are described in detail in molecular cloning, A laboratory Manual (third edition) (Sambrook J, russell DW, janssen K, argentine J. Huang Peyer et al, 2002, beijing: science publishers). Commercial strains can be purchased from the bacillus subtilis WB800, and the bacillus subtilis WB800 used in the embodiment is deposited in a microbiological laboratory applied to the food institute of China university of oceans; the nucleotide and amino acid sequences mentioned in this example are shown in Table 1.

Bacillus subtilis N2 (Bacillus subtilis) is screened by the food institute of China university in oceanic university in a microbiological laboratory and is preserved in China general microbiological culture Collection center in 3 months and 30 days in 2020, with the preservation number of CGMCC No.19541 and the preservation address of Beijing City, chaoyang district, west Chen, no.1 Hospital No. 3.

Example 1 screening of Signal peptide for efficient secretory expression of Nattokinase

By optimizing the signal peptide of the nattokinase, constructing expression vectors combining various signal peptides with nattokinase coding genes and combining a high-throughput screening method, the signal peptide for improving the secretion effect of the nattokinase to different degrees is obtained from cloning, and the method specifically comprises the following steps:

(1) Constructing a pP43NMK-NKF vector: extracting Bacillus subtilis N2 genome DNA (Tiangen, bacterial genome extraction kit), and performing PCR amplification on the Bacillus subtilis N2 genome DNA by using two sections of artificially synthesized fragments SEQ ID N0.8 and SEQ ID N0.9 to obtain nattokinase gene; two segments of artificially synthesized fragments SEQ ID N0.10 and SEQ ID N0.11 are used for carrying out PCR amplification from pP43NMK to obtain a linearized pP43NMK fragment; the recombinant Cloning vector pP43NMK-NKF is constructed by recombination and connection of a nattokinase gene (SEQ ID N0.7) and a linearized pP43NMK fragment by using DNA seamless Cloning (Clon Express II One Step Cloning Kit).

(2) Constructing a signal peptide deletion type linearized vector PSP0: primers SEQ ID N0.12 and SEQ ID N0.13 are designed, based on pP43NMK-NKF, linear vector amplification is carried out, and the amplification system is 50 ul: 2X Phanta Max Buffer (Mg) ²⁺ Plus) 25. Mu.l, dNTP mix (2.5 mM each) 1. Mu.l, upstream and downstream primers (10. Mu.M) 2. Mu.l each, template 1. Mu.l, phanta Max Super-Fidelity DNA Polymerase 1. Mu.l, ddH ₂ O28. Mu.l. The PCR amplification procedure was as follows: 30sec at 95 ℃ and 15sec (1 cycle) at 95 ℃; 15sec at 55 ℃ and 8min at 72 ℃ (33 cycles); 72 ℃ for 10min (1 cycles). The PSP0 linearized vector fragment was prepared. The linearized vector was digested with Fast Digest DpnI, and the treated fragment was purified using a purification kit (

Cycle Pure Kit D6492) and then recovered for later use.

(3) Fishing and purifying the signal peptide:

primers (1) SEQ ID N0.14, SEQ ID N0.15 (2) SEQ ID N0.16, SEQ ID N0.17 (3) SEQ ID N0.18, SEQ ID N0.19 (4) SEQ ID N0.20, SEQ ID N0.21 (5) SEQ ID N0.2, SEQ ID N0.23; the first pair of primers (SEQ ID N0.14, SEQ ID N0.15) corresponds to signal peptide 1, and the design concept is to mutate the first base of the original signal peptide nucleotide sequence (SEQ ID N0.6) from G to A, and then add three bases of ATG at the end of the nucleotide sequence; the 2 nd pair of primers corresponds to the signal peptide 2, the 4 th pair of primers corresponds to the signal peptide 4, and the 5 th pair of primers corresponds to the signal peptide 5, the design concept is that the number of positive charges AT the N end of the signal peptide is increased by designing the primers except that the first base of the nucleotide sequence of the original signal peptide is mutated from G to A, and three bases of AT G are added AT the tail of the nucleotide sequence; the 3 rd pair of primers corresponds to signal peptide 3, and the concept is to design primers to increase the length of the signal peptide core H region, except for mutating the first base of the original signal peptide nucleotide sequence from G to A, and adding three bases of ATG at the end of the nucleotide sequence. The amino acid sequence of the signal peptide is shown as SEQ ID N0.24.

Taking a bacillus subtilis WB800 genome as a template, selecting a gradient PCR mode to hook a target gene, and amplifying the target gene in a system of 50 mul: 5 × PrimeSTAR Buffer (Mg) ²⁺ Plus) 10. Mu.l, dNTP mix (2.5 mM each) 5. Mu.l, upstream and downstream primers (10. Mu.M) 2. Mu.l each, template 1. Mu.l, primeSTAR HS DNA Polymerase (2.5U/. Mu.L) 1. Mu.l, ddH ₂ O29. Mu.l. The gradient PCR amplification procedure was as follows: 30sec at 95 ℃ and 15sec (1 cycle) at 95 ℃; 15sec at 62 ℃, 30sec at 72 ℃, 15sec at 95 ℃ (5 cycles); 6 ℃ 15sec,72 ℃ 30sec,95 ℃ 15sec (5 cycles); 15sec at 58 ℃ and 30sec at 72 ℃ (25 cycles s); 72 ℃ for 7min (1 cycle). The correct band was recovered by purification by electrophoresis analysis of the nucleic acid to determine molecular weight (see FIGS. 2-6) for use as the desired fragment.

(4) Constructing and verifying a signal peptide-guided nattokinase cloning vector PSPX: diluting according to the DNA concentration of the obtained purified signal peptide fragment and the PSP0 vector to ensure that the molar ratio of the linearized vector to the target fragment is 1. Monoclonal sequencing validation of vector PSPX (see FIG. 1) constructed with the pP43NMK vector universal primer pair was performed.

(5) Construction and verification of a signal peptide expression vector: activating correctly sequenced transformant and extracting plasmid: (A)

Plasmid DNA Mini Kit D6942 Kit) to obtain natto kinase cloning vector containing different signal peptides. The nattokinase cloning vector was electrotransformed into Bacillus subtilis WB800 and verified by SDS-PAGE and fibrin plate enzyme production.

Example 2 Signal peptide-mediated detection of Nattokinase expression levels

(1) Inoculating the positive transformant into 5mL liquid LB (Kana), incubating, extracting plasmid sequencing, inoculating the transformant with the correct plasmid into a TB culture medium (Kana), performing shake culture at 37 ℃ and 180r/min for 48h, and centrifuging the fermentation liquor at 8000r/min for 10min to obtain enzyme liquid.

(2) Referring to ultraviolet spectrophotometry established by the Japan Nattokinase Association, enzyme activity is defined as follows: 1 Unit (1 FU) is defined as the supernatant at OD under the conditions specified in the procedure ₂₇₅ The absorbance of (b) was increased by 0.01 enzyme amount per minute.

(3) And (3) measuring the enzyme activity of the natto kinase: compared with the nattokinase original signal peptide SEQ ID N0.6, 5 signal peptides which can improve the activity of the nattokinase are screened out, wherein the signal peptides are respectively signal peptides 1 coded by the amino acid sequence SEQ ID N0.1, and the enzyme activity of the nattokinase can be increased from 22.3FU/mL to 171FU/mL (see figure 7); the signal peptide 2 coded by the SEQ ID N0.2 amino acid sequence can increase the enzyme activity of the nattokinase from 22.3FU/mL to 207.7FU/mL (see figure 8); the signal peptide coded by the SEQ ID N0.3 amino acid sequence can increase the enzyme activity of the nattokinase from 22.3FU/mL to 208.3FU/mL (see figure 9); the signal peptide coded by the SEQ ID N0.4 amino acid sequence can increase the enzyme activity of the nattokinase from 22.3FU/mL to 225.4FU/mL (see figure 10); the signal peptide encoded by the amino acid sequence of SEQ ID N0.5 can increase the enzyme activity of nattokinase from 22.3FU/mL to 235.2FU/mL (see FIG. 11).

TABLE 1 all nucleotide and amino acid sequences used in this example

Sequence listing

<110> Wihai diproson Biotech Ltd

<120> signal peptide for improving secretion efficiency of nattokinase and application thereof

<160> 24

<170> SIPOSequenceListing 1.0

<210> 6

<211> 30

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Arg Ser Lys Lys Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu

1 5 10 15

Ile Phe Thr Met Ala Phe Ser Asn Met Ser Val Gln Ala Ala

20 25 30

<210> 2

<211> 33

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Lys Lys Arg Lys Arg Arg Asn Phe Lys Arg Phe Ile Ala Ala Phe

1 5 10 15

Leu Val Leu Ala Leu Met Ile Ser Leu Val Pro Ala Asp Val Leu Ala

20 25 30

Ala

<210> 3

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Lys Lys Ile Trp Ile Gly Met Leu Ala Ala Ala Val Leu Leu Leu

1 5 10 15

Met Val Pro Lys Val Ser Leu Ala Asp Ala Ala

20 25

<210> 4

<211> 40

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Glu Glu Arg Ser Gln Arg Arg Lys Lys Lys Arg Lys Leu Lys Lys

1 5 10 15

Trp Val Lys Val Val Ala Gly Leu Met Ala Phe Leu Val Ile Ala Ala

20 25 30

Gly Ser Val Gly Ala Tyr Ala Ala

35 40

<210> 5

<211> 44

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Met Phe Arg Leu Phe His Asn Gln Gln Lys Ala Lys Thr Lys Leu Lys

1 5 10 15

Val Leu Leu Ile Phe Gln Leu Ser Val Ile Phe Ser Leu Thr Ala Ala

20 25 30

Ile Cys Leu Gln Phe Ser Asp Asp Thr Ser Ala Ala

35 40

<210> 24

<211> 29

<212> PRT

<213> Bacillus natto (Bacillus natto)

<400> 24

Val Arg Ser Lys Lys Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu

1 5 10 15

Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gln Ala

20 25

<210> 7

<211> 1146

<212> DNA

<213> Bacillus natto (Bacillus natto)

<400> 7

gtgagaagca aaaaattgtg gatcagcttg ttgtttgcgt taacgttaat ctttacgatg 60

gcgttcagca acatgtctgt gcaggctgcc ggaaaaagca gtacagaaaa gaaatacatt 120

gtcggattta agcagacaat gagtgccatg agttccgcca agaaaaagga tgttatttct 180

gaaaaaggcg gaaaggttca aaagcaattt aagtatgtta acgcggccgc agcaacattg 240

gatgaaaaag ctgtaaaaga attgaaaaaa gatccgagcg ttgcatatgt ggaagaagat 300

catattgcac atgaatatgc gcaatctgtt ccttatggca tttctcaaat taaagcgccg 360

gctcttcact ctcaaggcta cacaggctct aacgtaaaag tagctgttat cgacagcgga 420

attgactctt ctcatcctga cttaaacgtc agaggcggag caagcttcgt tccttctgaa 480

acaaacccat accaggacgg cagttctcac ggtacgcatg tcgccggtac gattgccgct 540

cttaataact caatcggtgt tctgggcgta gcgccaagcg catcattata tgcagtaaaa 600

gtgcttgatt caacaggaag cggccaatat agctggatta ttaacggcat tgagtgggcc 660

atttccaaca atatggatgt tatcaacatg agccttggcg gacctactgg ttctacagcg 720

ctgaaaacag tagttgataa agcggtttcc agcggtatcg tcgttgctgc cgcagccgga 780

aacgaaggtt catccggaag cacaagcaca gtcggctacc ctgcaaaata tccttctact 840

attgcagtag gtgcggtaaa cagcagcaac caaagagctt cattctccag cgtaggttct 900

gagcttgatg taatggctcc tggcgtgtcc atccaaagca cacttcctgg aggcacttac 960

ggcgcttata acggaacgtc catggcgact cctcacgttg ccggagcagc agcgctaatt 1020

ctttctaagc acccgacttg gacaaacgcg caagtccgtg atcgtttaga aagcactgca 1080

acatatcttg gaaactcttt ctactatgga aaagggttaa tcaacgtaca agcagctgca 1140

caataa 1146

<210> 8

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aacacatgcc tcagctgcag gccggaaaaa gcagtacaga aaag 44

<210> 9

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tggtgctcga gtgcggccgc ttgtgcagct gcttgtacgt tg 42

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

caccaccacc accaccactg 20

<210> 11

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gtgtacattc ctctcttacc tataatggta cc 32

<210> 12

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gccggaaaaa gcagtacaga aaagaaata 29

<210> 13

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gtgtacattc ctctcttacc tataatggta cc 32

<210> 14

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ggtaagagag gaatgtacac atgagaagca aaaaattgtg gatc 44

<210> 15

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

tctgtactgc tttttccggc agcagcctgc gcagacatg 39

<210> 16

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ggtaagagag gaatgtacac atgaaaaaaa gaaagaggcg aaac 44

<210> 17

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tctgtactgc tttttccggc agctgctagt acatcggctg gc 42

<210> 18

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ggtaagagag gaatgtacac atgaagaaaa tttggattgg aatgc 45

<210> 19

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

tctgtactgc tttttccggc agcggcatcc gcgagac 37

<210> 20

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ggtaagagag gaatgtacac atggaagaac gatcacagcg c 41

<210> 21

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

tctgtactgc tttttccggc agcagcatag gcgccgac 38

<210> 22

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

ggtaagagag gaatgtacac atgtttcgat tgtttcacaa tcagc 45

<210> 23

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

tctgtactgc tttttccggc agcagcgctt gtatcatcgg 40

<210> 1

<211> 87

<212> DNA

<213> Bacillus natto (Bacillus natto)

<400> 1

gtgagaagca aaaaattgtg gatcagcttg ttgtttgcgt taacgttaat ctttacgatg 60

gcgttcagca acatgtctgc gcaggct 87

Claims

1. The recombinant genetic engineering bacteria for producing the nattokinase are characterized by being obtained by transferring recombinant plasmids into bacillus subtilis WB800, wherein the recombinant plasmids comprise genes of signal peptides shown as SEQ ID N0.5 and nattokinase genes derived from bacillus subtilis N2 shown as SEQ ID N0.7, and the genes of the signal peptides are fused to the N end of the nattokinase genes.

2. The use of the recombinant genetically engineered bacterium of claim 1 in the production of nattokinase.