CN107937374A - The Nattokinase that a kind of heat endurance improves - Google Patents
The Nattokinase that a kind of heat endurance improves Download PDFInfo
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- CN107937374A CN107937374A CN201711376822.XA CN201711376822A CN107937374A CN 107937374 A CN107937374 A CN 107937374A CN 201711376822 A CN201711376822 A CN 201711376822A CN 107937374 A CN107937374 A CN 107937374A
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- nattokinase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21062—Subtilisin (3.4.21.62)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention discloses the Nattokinase that a kind of heat endurance improves, belong to protein engineering.Nattokinase the Q59E N218D and N218D that the present invention obtains, heat endurance has compared to protoenzyme to be increased substantially.Nattokinase Q59E N218D and N218D provided by the invention will preferably tackle technique high temperature heating link to a certain extent, and the action time in body can be extended, improve application value of the Nattokinase as treatment cardiovascular and cerebrovascular disease oral drugs.
Description
Technical field
The present invention relates to the Nattokinase that a kind of heat endurance improves, belong to protein engineering field.
Background technology
Nattokinase (NK, EC3.4.21.62) is japanese traditional food natto during the fermentation by Bacillus subtilis natto from Traditional Japanese Food
(Bacillusnatto) a kind of serine protease produced, compared to commercially available thrombolytic drug, has the effect of efficient thrombus dissolving,
It is most potential fibrinolytic protein enzyme.And the research to Nattokinase heat endurance is rarely reported.Nattokinase heat endurance compared with
Difference, for pure enzyme liquid in 55 DEG C of processing, half-life period is only 12min or so, seriously limits its application in the industrial production.
In order to promote exploitation and application of the Nattokinase as thrombolytics, it is badly in need of one kind and can guarantee that it in technique productions high temperature
Enzyme activity is not lost in link, and extends the Nattokinase of its action time in body.
The content of the invention
First purpose of the present invention is to provide a kind of stability-enhanced Nattokinase mutant, the mutant be by
One or two amino acid sites of Nattokinase of the amino acid sequence as shown in SEQ ID NO.1 are mutated.
In one embodiment of the invention, it is characterised in that it by amino acid sequence is SEQ ID that the mutant, which is,
The 59th of Nattokinase shown in NO.1, the 218th one or two amino acid sites are mutated.
In one embodiment of the invention, the mutant is for shown in SEQ ID NO.1 by amino acid sequence
The asparagine of the 218th of Nattokinase carries out sporting aspartic acid.
In one embodiment of the invention, the mutant is for shown in SEQ ID NO.1 by amino acid sequence
The glutamine of the 59th of Nattokinase carries out sporting glutamic acid, while the asparagine of the 218th is mutated
For aspartic acid.
Second object of the present invention is to provide a kind of recombinant bacterium for expressing Nattokinase mutant.
In one embodiment of the invention, the construction method of the recombinant bacterium is:By the base of Nattokinase mutant
Because being connected on carrier pET24a, with e. coli bl21 position host, recombinant bacterium is built.
Third object of the present invention is to provide a kind of method for expressing Nattokinase mutant, comprises the following steps that:
(1) recombinant bacterium is inoculated in the LB culture mediums containing kanamycins, 35-38 DEG C, the training of 150-250r/min shaken overnights
Support and obtain seed liquor.
(2) expression that seed liquor is inoculated in the concentration containing kanamycins respectively by the inoculum concentration that mass fraction is 1-2% is trained
Support in base, 35-38 DEG C, 150-250r/min shaken cultivations to OD600During to 0.6-0.8, derivant IPTG is added, in 15-18 DEG C
Induce the expression of 18-24h Nattokinase mutant.
In one embodiment of the invention, the concentration of the kanamycins is 80-120 μ g/mL.
In one embodiment of the invention, the derivant IPTG to 0.05-0.2mM.
In one embodiment of the invention, the derivant IPTG to 0.1mM.
In one embodiment of the invention, the expression culture medium is TB culture mediums, and the TB nutrient media componentses are:
Tryptone 10-14g/L, yeast extract 20-26g/L, glycerine 3-8g/L, KH2PO42.0-2.6g/L, K2HPO4·
3H2O15.8-16.8g/L, CaCl20.15-0.25g/L, surplus are water.
In one embodiment of the invention, the purge process of the Nattokinase:After bacterial cell disruption being recombinated, collect
Supernatant, after supernatant membrane filtration, at 2-6 DEG C, with the isolated pure enzyme liquid of Nattokinase mutant of nickel column centrifugal column.
Beneficial effects of the present invention:The thermostabilization of Nattokinase mutant Q59E-N218D and N218D provided by the invention
Property increases substantially, and half-life period of the Q59E-N218D at 55 DEG C extends to 32.38min by the 11.9min of wild enzyme,
Half-life period at N218D55 DEG C extends to 28min by the 11.9min of wild enzyme.Therefore, mutation physical efficiency provided by the invention is more preferable
Reply technique high temperature heating link, and the action time in body can be extended, improve Nattokinase applies valency
Value.
Brief description of the drawings
The wild enzymes of Fig. 1 (WT) and single mutant (Q59E, N218D), the heat endurance of double-mutant (Q59E-N218D) are bent
Line.It is incubated at 55 DEG C, in due course sampling, measures it respectively and remain enzyme activity..
Embodiment
LB nutrient media componentses are:Peptone 8-12g/L, yeast extract 4-6g/L, NaCl 8-12g/L
TB nutrient media componentses are:Tryptone 10-14g/L, yeast extract 20-26g/L, glycerine 3-8g/L,
KH2PO42.0-2.6g/L, K2HPO4·3H2O 15.8-16.8g/L, CaCl20.15-0.25g/L
The definition of enzyme activity:In 25 DEG C, pH8.0, concentration of substrate 0.4mM, 1min, 1 μm of o lAAPF is changed into 1 μ
Enzyme amount needed for molpNA is defined as an enzyme activity unit.
Enzyme activity determination method:(contain 0.1mMCaCl in 100mM pH8.0Tris-HCl buffer solutions2), with artificial synthesized
Suc-AAPF-pNA is substrate, reacts 3min at 25 DEG C, and generation paranitroanilinum (pNA) is measured according to the absorption value of 405nm
Concentration mensuration enzyme activity.
Heat endurance determines:Wild enzyme and Q59E-N218D are stored in 55 DEG C of metal baths, sampling, measures its in due course
Remain enzyme activity.
The structure of 1 Nattokinase single mutant of embodiment
(1) structure of mutant Q59E
Using pET24a-pro-NK as masterplate, with primer shown in table 1 under the conditions of shown in table 4, PCR obtains carrying coding
The recombinant vector of Q59E mutant genes, plasmid are named as pET24a-pro-NK/Q59E.
1 mutant Q59E primers of table
PCR product is analyzed and identified by 1.0% agarose gel electrophoresis;It is again that PCR product is pure with DNA purification kits
Change, finally use QuichCutTmDpnI digests 5h in 37 DEG C and removes the template plasmid to methylate, is transferred to BL21 escherichia coli hosts,
Recombinant bacterium is named as BL21/pET24a-pro-NK/Q59E.
(2) structure of mutant N218D
Using pET24a-pro-NK as masterplate, with primer shown in table 2 under the conditions of shown in table 4, PCR obtains carrying coding
The recombinant vector of N218D mutant genes, plasmid pET24a-pro-NK/N218D.
2 mutant N218D primers of table
PCR product is analyzed and identified by 1.0% agarose gel electrophoresis;It is again that PCR product is pure with DNA purification kits
Change, finally use QuichCutTmDpnI digests 5h in 37 DEG C and removes the template plasmid to methylate, is transferred to BL21 escherichia coli hosts,
Recombinant bacterium is named as BL21/pET24a-pro-NK/N218D.
The structure of 2 Nattokinase double-mutant of embodiment
Using small amount plasmid extraction kit upper step extraction pET24a-pro-NK/Q59E plasmids to specifications, then with
PET24a-pro-NK/Q59E is template, and with primer shown in table 3 under the conditions of shown in table 4, PCR obtains carrying coding Q59E-
The recombinant vector of N218D double-mutant genes.
3 mutant Q59E-N218D primers of table
The full plasmid PCR amplification reaction system of table 4
Pcr amplification reaction condition is:
PCR product is analyzed and identified by 1.0% agarose gel electrophoresis;It is again that PCR product is pure with DNA purification kits
Change, finally use QuichCutTmDpnI digests 5h in 37 DEG C and removes the template plasmid to methylate, is transferred to BL21 escherichia coli hosts,
Recombinant bacterium is named as BL21/pET24a-pro-NK/Q59E-N218D.
The expression of 3 Nattokinase single mutant of embodiment and double-mutant
(1) by recombination bacillus coli BL21/pET24a-pro-NK/Q59E-N218D, BL21/pET24a-pro-NK/
Q59E, BL21/pET24a-pro-NK/N218D, are inoculated in the LB culture mediums that 5mL kanamycins concentration is 100 μ g/mL respectively,
37 DEG C, 200r/min shaken overnight cultures obtain seed liquor.
(2) it is 100 μ g/mL's seed liquor to be inoculated in concentration containing kanamycins respectively by the inoculum concentration of mass fraction 1%
In 100mLTB expression culture mediums, 37 DEG C, 200r/min shaken cultivations to OD600During to 0.6-0.8, derivant IPTG is added extremely
0.1mM, 18 DEG C of induction 20h collect thalline, and bacterium is received in the rotating speed centrifugation of 5000g.
(3) restructuring thalline is dissolved in 20mL combination buffer solutions (50mmol/LNa2HPO4、50mmol/LNaH2PO4、
500mmol/LNaCl, 20mmol/L imidazole), ultrasonication, 13000g centrifugation 25min, 0.22 μm of filter membrane mistake of supernatant
Filter.With the combination buffer solution balance columns of 10 times of column volumes, non-specific adsorption is washed away with the combination buffer solution of 5 times of column volumes
Albumen, respectively with the elution buffer of 50 and 100mmol/L imidazoles elution albumen (repeating to elute), collect sample and simultaneously use SDS-
PAGE is analyzed and identified.
The thermal stability analysis of 4 Nattokinase single mutant of embodiment and double-mutant
Heat endurance determines:Wild enzyme, Q59E, N218D, Q59E-N218D are stored in 55 DEG C of metal baths, taken in due course
Sample, measures it and remains enzyme activity.As shown in Figure 1, finding that wild half life of enzyme is 11.9min, the complete deactivation in 50min is single prominent
Become Q59E half-life period as 4.8min, the complete deactivation in 30min, and single mutation N218D half-life period is 28min, is protected in 50min
35% or so enzyme activity is stayed, half-life period of the Q59E-N218D at 55 DEG C is 32.38min, and complete deactivation also remains with 50min
40% or so enzyme activity, it can be seen that, double-mutant Q59E-N218D and single mutant N218D relative to wild enzyme thermostabilization
Property is significantly improved.
The dynamic analysis of 5 Nattokinase single mutant of embodiment and double-mutant
Kinetic constant determines:Using artificial synthesized tetrapeptide suc-AAPF-pNA as substrate, substrate concentration range for 5 ×
10-5To 10-3M, according to paranitroanilinum standard curve, according to the absorption value of 405nm, so as to calculate under each concentration of substrate
Enzyme's reaction speeding, then Km and Vmax are obtained by the fittings of software GraphPadPrism 5.
The dynamic analysis of 5 enzyme of table
As shown in Table 5, although its heat endurance of single mutant N218D is increased substantially compared to wild-type enzyme, but it compares
Enzyme activity and catalytic efficiency (kcat/KM) compared to wild enzyme decline 34% and 40% or so respectively;Single mutant Q59E its compare enzyme
It is living to improve about 53% compared to wild enzyme, but its heat endurance is poor;Double-mutant Q59E-N218D combines both comprehensive
Property is closed, its specific enzyme activity and catalytic efficiency are close to wild-type enzyme, and its heat endurance increases substantially.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various change and modification, therefore the protection model of the present invention
Enclosing be subject to what claims were defined.
Sequence table
<110>Southern Yangtze University
<120>The Nattokinase that a kind of heat endurance improves
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 275
<212> PRT
<213>It is artificial synthesized
<400> 1
Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala Pro Ala Leu
1 5 10 15
His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp
20 25 30
Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val Arg Gly Gly Ala
35 40 45
Ser Phe Val Pro Ser Glu Thr Asn Pro Tyr Gln Asp Gly Ser Ser His
50 55 60
Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly
65 70 75 80
Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu
85 90 95
Asp Ser Thr Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu
100 105 110
Trp Ala Ile Ser Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly
115 120 125
Pro Thr Gly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser
130 135 140
Ser Gly Ile Val Val Ala Ala Ala Ala Gly Asn Glu Gly Ser Ser Gly
145 150 155 160
Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala
165 170 175
Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val
180 185 190
Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr
195 200 205
Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr
210 215 220
Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr
225 230 235 240
Trp Thr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr
245 250 255
Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala
260 265 270
Ala Ala Gln
275
<210> 2
<211> 828
<212> DNA
<213>It is artificial synthesized
<400> 2
gcgcaatctg ttccttatgg catttctcaa attaaagcgc cggctcttca ctctcaaggc 60
tacacaggct ctaacgtaaa agtagctgtt atcgacagcg gaattgactc ttctcatcct 120
gacttaaacg tcagaggcgg agcaagcttc gttccttctg aaacaaaccc ataccaggac 180
ggcagttctc acggtacgca tgtcgccggt acgattgccg ctcttaataa ctcaatcggt 240
gttctgggcg tagcgccaag cgcatcatta tatgcagtaa aagtgcttga ttcaacagga 300
agcggccaat atagctggat tattaacggc attgagtggg ccatttccaa caatatggat 360
gttatcaaca tgagccttgg cggacctact ggttctacag cgctgaaaac agtagttgat 420
aaagcggttt ccagcggtat cgtcgttgct gccgcagccg gaaacgaagg ttcatccgga 480
agcacaagca cagtcggcta ccctgcaaaa tatccttcta ctattgcagt aggtgcggta 540
aacagcagca accaaagagc ttcattctcc agcgtaggtt ctgagcttga tgtaatggct 600
cctggcgtgt ccatccaaag cacacttcct ggaggcactt acggcgctta taacggaacg 660
tccatggcga ctcctcacgt tgccggagca gcagcgctaa ttctttctaa gcacccgact 720
tggacaaacg cgcaagtccg tgatcgttta gaaagcactg caacatatct tggaaactct 780
ttctactatg gaaaagggtt aatcaacgta caagcagctg cacaataa 828
<210> 3
<211> 30
<212> DNA
<213>It is artificial synthesized
<400> 3
gaaacaaacc catacgagga cggcagttct 30
<210> 4
<211> 30
<212> DNA
<213>It is artificial synthesized
<400> 4
gtgagaactg ccgtcctcgt atgggtttgt 30
<210> 5
<211> 30
<212> DNA
<213>It is artificial synthesized
<400> 5
acttacggcg cttatgacgg aacgtccatg 30
<210> 6
<211> 30
<212> DNA
<213>It is artificial synthesized
<400> 6
cgccatggac gttccgtcat aagcgccgta 30
Claims (10)
1. a kind of stability-enhanced Nattokinase mutant, it is characterised in that it by amino acid sequence is SEQ that the mutant, which is,
The 59th of Nattokinase shown in ID NO.1, the 218th one or two amino acid sites are mutated.
2. mutant according to claim 1, it by amino acid sequence is receiving shown in SEQ ID NO.1 that the mutant, which is,
The asparagine mutation of the 218th of beans kinases is aspartic acid.
3. mutant according to claim 1, it is characterised in that it by amino acid sequence is SEQ ID that the mutant, which is,
The glutamine of the 59th of the Nattokinase shown in NO.1 sports glutamic acid, while the asparagine of the 218th is carried out
Sport aspartic acid.
4. express the recombinant bacterium of any mutant of claim 1-3.
5. the construction method of recombinant bacterium according to claim 4:The gene of Nattokinase mutant is connected to carrier
On pET24a, with e. coli bl21 position host, recombinant bacterium is built.
6. the method for recombinant bacterium expression Nattokinase mutant according to claim 4, it is characterised in that the tool of the method
Body step is as follows:
(1) recombinant bacterium is inoculated in the LB culture mediums containing kanamycins, 35-38 DEG C, 150-250r/min shaken overnight cultures obtain
To seed liquor.
(2) seed liquor is inoculated in the expression culture medium of the concentration containing kanamycins respectively by the inoculum concentration that mass fraction is 1-2%
In, 35-38 DEG C, 150-250r/min shaken cultivations to OD600During to 0.6-0.8, derivant IPTG is added, in 15-18 DEG C of induction
The expression of 18-24h Nattokinase mutant.
7. the according to the method described in claim 6, it is characterized in that, derivant IPTG to 0.05-0.2mM.
8. according to the method described in claim 6, it is characterized in that, the expression culture medium is TB culture mediums, the TB is cultivated
Base component is:Tryptone 10-14g/L, yeast extract 20-26g/L, glycerine 3-8g/L, KH2PO42.0-2.6g/L
K2HPO4·3H2O 15.8-16.8g/L, CaCl20.15-0.25g/L, surplus are water.
9. the Nattokinase mutant that the method according to claim 6-8 obtains.
10. the Nattokinase mutant according to right wants 9 is being prepared for treating answering in cardiovascular and cerebrovascular diseases medicament
With.
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WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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