CN107058204A - One plant can be with the bacillus subtilis bacterial strain of efficient secretion Nattokinase and high-purity Nattokinase preparation technology - Google Patents
One plant can be with the bacillus subtilis bacterial strain of efficient secretion Nattokinase and high-purity Nattokinase preparation technology Download PDFInfo
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Abstract
One plant can be with the bacillus subtilis strain of efficient secretion Nattokinase, Bacillus subtilis natto NK aprN genes are expanded using round pcr, and according to bacillus subtilis codon-bias optimize starting 30 amino acid codon, recombinant expression plasmid pHT01 aprN are built, through restricted digestion, PCR amplifications and sequence verification its correctness.PHT01 aprN are imported by bacillus subtilis 168 (Bacillus subtilis 168) by electric shocking method, engineering bacteria is obtained using chlorampenicol resistant screening.After optimization of fermentation conditions, through IPTG induced expressions, highest enzyme activity is that highest enzyme activity reaches 7778U/ml in 289U/ml, high density fermentation zymotic fluid in shaking table culture zymotic fluid, and it is 981731U/g to prepare high-purity Nattokinase enzyme preparation highest enzyme activity through affinity column chromatography.
Description
Technical field
It is more particularly to a kind of efficient the present invention relates to the technique that a kind of microbial strains and its fermentation prepare high-purity enzyme preparation
The technique secreted the bacillus subtilis bacterial strain of Nattokinase and prepare high-purity Nattokinase enzyme preparation.
Background technology
Natto is that soya bean passes through the fermented bean products food that natto withered grass bar fermentation of bacillus is made.Nattokinase (NK)
It is a kind of serine protease produced by Bacillus subtilis natto, is present in natto.NK is removed has strong in vivo and in vitro
Outside FA, the effects such as also promoting blood flow, anti-platelet aggregation and hypotensive.NK in 1980 first
By that must see that foreign firm doctor has found, its relative molecular mass (Mr) is 27000, and isoelectric point is 8.6 ± 0.3.The heart as caused by thrombus
Vascular diseases seriously endanger human health, and current thrombolytics includes injection class degraded medicine, and (urokinase, streptokinase and tectotype are fine
Fibrillarin activator) and class loading plasmase albumen (Nattokinase and Lumbrokinase) two major classes.Inject class degraded medicine
Thing highly relies on the level of in-vivo tissue plasminogen, there is certain toxicity, half-life short and cost height.And NK belongs to day
Right tunning, it is safe, with internal long action time, it is cheap, have prevention effect and be easy to large-scale production etc.
Advantage, has developed into health food, and its clinical practice has obtained U.S. FDA accreditation.
The yield and purity of NK industrialized productions are improved, it is significant to treatment thrombus disease.Using optimal startup
With signal peptide, from the bacterial strain of protease deficiency with reduce NK degradeds or directly transformation NK gene orders etc. technological means with
Reach the purpose for improving NK yield.Solubility expression can be achieved in NK in bacillus subtilis, and Wu S etc. construct withered grass gemma
Bacillus engineering bacteria, and NK expression quantity is added 136% by optimal startup, up to 1999U/ml (flat band method).Other are thin
Bacterium, also by genetic engineering fermentation NK such as Escherichia coli, bacillus licheniformis, streptococcus lactis and saccharomycete.But these
The problem of technology needs to solve there is also some, such as goes out inactive inclusion body in expression in escherichia coli；In Other Engineering
Although in bacterium NK can solubility expression, production of enzyme is low, and purifying removal process is complicated, and be further improved engineering bacteria structure
Make.
Nishito Y et al. are it has been proved that natto fermentation Pseudomonas is in a subspecies for bacillus subtilis.Bacillus subtilis
Bacterium no pathogenicity, without obvious codon preference, possess comprising the mechanism such as transcription, translation, protein folding and secretion, usually
It is used as engineered strain carrier construction.This experiment early stage checking B.subtilis 168 contains NK gene orders, but can not express
The NK of activity.Using molecular biology method, according to B.subtilis 168 Characteristic of Codon Usage, optimize NK-aprN
The sequence of 30 amino acid before gene code, and six histidine marks are added, using pHT01 as expression vector, successfully build work
Highest enzyme activity is up to 289U/ml after journey bacterium, optimization of fermentation conditions, and highest enzyme activity reaches 7778U/ in high density fermentation zymotic fluid
Ml, prepares high-purity Nattokinase enzyme preparation highest enzyme activity for 981731U/g through affinity column chromatography, is nattokinase gene engineering
Further the industrialized production of research and Nattokinase lays the foundation.
The content of the invention
It is an object of the invention to overcome the shortcomings of that natural strain fermentation performance can be swashed there is provided one plant with efficient secretion natto
The bacillus subtilis engineered strain and engineered strain of enzyme are used for the technique for preparing high-purity Nattokinase enzyme preparation of fermenting.This research
The aprN genes of Bacillus subtilis natto are expanded using round pcr, and it is excellent according to the codon-bias of bacillus subtilis
The codon of 30 amino acid of starting is changed, has constructed recombinant expression plasmid pHT01-aprN, expanded through restricted digestion, PCR
Its correctness with sequence verification.PHT01-aprN is imported by (the Bacillus of bacillus subtilis 168 by electric shocking method
Subtilis 168), obtain engineering bacteria using chlorampenicol resistant screening.Through IPTG induced expressions, shake flask fermentation culture highest enzyme
Living is that highest enzyme activity reaches 7778U/ml in 289U/ml, high density fermentation zymotic fluid, and it is sharp to prepare high-purity natto through affinity column chromatography
Enzyme enzyme preparation highest enzyme activity is 981731U/g.
To solve the above problems, the present invention uses following technical scheme：
One plant can be with the bacillus subtilis engineered strain of efficient secretion Nattokinase, it is characterised in that the bacterial strain
Classification And Nomenclature is：Bacillus subtilis (Bacillus subtilis), deposit number is CGMCC No：13496, depositary institution
For China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address is Chaoyang District, Beijing City north
The institute 3 of occasion West Road 1, preservation date is on December 26th, 2016.
The invention discloses the method for the bacillus subtilis engineered strain of construction efficient secretion Nattokinase, Shaking culture
Fermentating enzyme-producing condition and enzyme activity determination method, are operated as follows：
Step one：The amplification and optimization of NK-aprN genetic fragments
Using the bacterium solutions of B.subtilis 168 as the masterplate of first time amplification, with F listed by table 11-R1Primer pair is expansion for the first time
Increase primer, gel electrophoresis checking；Glue reclaim product is second of PCR amplification masterplate, with F listed by table 12-R1Primer pair is second
Secondary amplimer, gel electrophoresis checking；Second of glue reclaim product is that third time PCR expands masterplate, with F listed by table 13-R2HisDraw
Thing is to for third time amplimer, amplifying the aprN genetic fragments for optimizing preceding 30 codons.
Step 2：Target gene is cloned
The NK genetic fragments that step one is amplified are connected with pMD19-T carriers, and connection product is converted using thermal shock method
Into E.coli DH5 α competence, Amp resistant panels are coated with.
Step 3：The structure of recombinant plasmid and checking
Plasmid pHT01 and the cloned plasmids double digestion of the positives bacterium of extraction step two, with glue reclaim kit recovery purifying
Purpose fragment, T4 ligase low temperature stays overnight enzyme company, product Transformed E .coli DH5 α competent cells.After bacterium colony PCR identifications, take
Correct positive colony activation culture, extracts plasmid order-checking.
Step 4：The structure of engineering bacteria and identification
B.subtilis 168 competent cell is prepared, the plasmid electricity extracted in step 4 is turned into competent cell.With
The 5-8 single bacterium colony grown on machine picking chlorampenicol resistant flat board, bacterium colony PCR identifications take correct positive colony activation training
Support, extract plasmid order-checking.
Step 5：Engineering bacterium fermentation
Recombinant seed liquor is activated, using chloramphenicol as antibiotic, seed liquor switching fermentation medium culture, OD600
25 DEG C to 30 DEG C are cooled in 0.8-1.0, final concentration of 0.2-1.0mM IPTG Fiber differentiations are added.
Step 6：The measure of Nattokinase enzyme activity
The present invention detects Nattokinase enzyme activity using tetra-peptide substrate method.Make the standard curve of paranitroanilinum.NK activity
Unit is defined as the content of NK release 1nmol nitroanilines per minute.
The invention also discloses the fermentating enzyme-producing condition of engineering bacteria high density fermentation and the system of high-purity Nattokinase enzyme preparation
Standby technique, is operated as follows：
(1) high density fermentation
Inoculation：Activated spawn, prepares primary seed solution and secondary seed solution under 37 DEG C, 200rpm condition of culture, plants age
For 12h secondary seed solution fermentation medium (the final concentration of 5 μ g/mL of chloramphenicol, defoamer are seeded to according to 10% inoculum concentration
0.01%).Fermentation medium (1L)：Glucose 28.5g, glycerine 57g, yeast extract 60g, pyridinedicarboxylic acid 0.167g,
NH4Cl 3g、K2HPO4·3H2O 1g、MnSO4·H2O 0.032g、FeSO4·7H2O 0.05g、CoCl2·6H2O 0.01g、
ZnCl2 0.01g、MgSO4·7H2O 2g、CaCl2·2H2O 5g, moisturizing to 1000ml.
Fermentation：Stir speed (S.S.) is initially set to 800rpm, and throughput is 12L/min.Arrange parameter, controls the temperature in tank to be
37 DEG C, dissolved oxygen amount be the 30%, pH 7.0 of zymotic fluid, according to dissolved oxygen situation adjustment stir speed (S.S.) and throughput.
Protein induced expression：Sampling detection OD600, 28 DEG C are cooled to logarithm early stage, IPTG is added and starts induction fermentation.
Feed supplement：Supplemented medium (1L)：Glycerine 250g, yeast extract 100g, moisturizing to 1000ml.Detect zymotic fluid
OD600With the Expenditure Levels of glycerine in fermentation medium, a feed supplement and continuous feeding mode feed-batch culture is respectively adopted.
Terminate fermentation：After fermented and cultured 24-28h, fermentation is terminated.
(2) preparation of high-purity Nattokinase enzyme preparation
The chilled collected after centrifugation supernatant of zymotic fluid, (NH4)2SO4Precipitation remove foreign protein and precipitate NK, ni-sepharose purification,
Desalination is simultaneously concentrated plus the step such as cryoprotector and freeze-drying, and high-purity nattokinase enzyme preparation, Nattokinase enzyme activity is made
Reach 981713U/g.
The present invention has following beneficial effect：The Nattokinase engineering bacteria of bacillus subtilis is constructed, realizes that natto swashs
Enzyme is up to 289 U/ml in its extracellular expression, optimized fermentation culture conditions, enzyme activity, can be further used for industrializing liquid fermentation
Production.
Brief description of the drawings
The NK-aprN gene PCR amplimers of table 1.
The NK-aprN gene PCR amplification systems of table 2.
Fig. 1 agarose gel electrophoresis identification NK-aprN gene PCR amplification electrophoretograms.
Fig. 2 agarose gel electrophoresis identification aprN gene clonings PCR amplification electrophoretograms.
Fig. 3 agarose gel electrophoresis identifies pMD19-T：AprN plasmid enzyme restriction electrophoretograms.
Fig. 4 agarose gel electrophoresis identifies pHT01 plasmid enzyme restriction electrophoretograms.
Fig. 5 agarose gel electrophoresis identifies pHT01：AprN plasmid construction electrophoretograms.
Fig. 6 agarose gel electrophoresis identification engineering bacteria builds electrophoretogram.
Fig. 7 paranitroanilinum standard curves.
Fig. 8 engineering bacterias enzyme activity and cell growth figure.
Fig. 9 NK high density fermentations OD600, enzyme activity.
Embodiment
Embodiments of the invention are elaborated below：The present embodiment is carried out lower premised on technical solution of the present invention
Implement, give detailed embodiment and process, but protection scope of the present invention is not limited to following embodiments.It is following to implement
The experimental method of unreceipted actual conditions in example, generally according to normal condition, for example Sambrook equimoleculars are cloned：Laboratory hand
Volume (New York:Cold Spring Harbor Laboratory Press, 1989) described in condition, or according to manufacture
Condition proposed by manufacturer.
Embodiment 1
Step one：The amplification and optimization of NK-aprN genetic fragments
LB fluid nutrient mediums：10g/L peptones, 10g/L sodium chloride, 5g/L yeast extracts.
LB solid mediums：LB fluid nutrient mediums, 15g/L agaroses.
Amp solution：100mg/mL.
Nattokinase gene sequence (GI is included according to GenBank:608796180) it is, close according to Host Strains B.subtitle
Numeral preference (http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgiSpecies=1423),
Using biosoftware oligo design primers (table 1), expand aprN genes and optimize its preceding 30 amino acid codes.Primer
By Sheng Gong bioengineering, Co., Ltd synthesizes.
After the bacterial strains of B.subtilis 168 activation of preservation, it is inoculated according to 2% inoculum concentration in LB culture mediums, 37
DEG C, 200rpm culture 12h.The method that the clone of target gene uses over-lapping PCR, i.e., using bacterium solution as the first time
PCR (German Eppendrof companies) expands masterplate, glue reclaim (the German Eppendrof companies) production after gel electrophoresis is verified
Thing expands masterplate, then the progress third time PCR amplifications by masterplate of this glue reclaim product as second of PCR.Primer is followed successively by
F1-R1、F2-R1And F3-R2His, expand and optimize the aprN genes for coming from B.subtilis natto, such as Fig. 1.
Amplimer such as table 1, combination primer is followed successively by primer and is followed successively by F1-R1、F2-R1And F3-R2His, amplification system is such as
Table 2, amplification reaction condition：94 DEG C of pre-degenerations 10min, 94 DEG C of denaturation 40s, 54 DEG C of annealing 40s, 72 DEG C of extension 30s, totally 35 are followed
Ring, 72 DEG C of extension 10min.
Step 2：Target gene is cloned
Gene clone method reference《Molecular Cloning:A Laboratory guide》.Kit reclaims and purifies third time pcr amplification product,
Product is connected to plasmid pMD19-T by TA clones.PMD19-T carrier linked systems：The 0.5 μ L of μ L, pMD19-T of DNA 4.5, delay
The μ L of solution I 5 are rushed, add up to 10 μ L, 4 DEG C connect overnight.Connection product is imported into Escherichia coli by heat-shock transformed method to experience
In state, Amp resistant panels (the μ g/mL of final concentration 100) are coated with.Positive colony bacterium colony DNA is extracted, passes through restriction enzyme
Cleavage Map and PCR primer sequence verification, will correctly carry pMD19-T:AprN plasmid-bearing strains be placed in glycerol tube-
80 DEG C of preservations.
Step 3：Structure and the engineering bacteria construction of recombinant plasmid
pMD19-T：AprN is by restriction enzyme EcoR V and Bam HI digestions, the aprN genetic fragments insertion cut
Between pHT01 Sma I and BamHI restriction enzyme sites, connection product is imported by E. coli competent by heat-shock transformed method
In, it is coated with Amp resistant panels (the μ g/mL of final concentration 100).Positive colony bacterium colony DNA is extracted, passes through restriction enzyme enzyme
Atlas analysis and PCR primer sequence verification are cut, pHT01 will be correctly carried:AprN plasmid-bearing strains are placed in -80 DEG C of glycerol tube
Preserve.
Step 4：The structure of engineering bacteria and identification
B.subtilis 168 competent cell prepares the method with electroporated reference Xue.Random picking chloramphenicol
The single bacterium colony grown in resistant panel, extracts positive colony bacterium colony DNA, is analyzed by restriction endonuclease map
With PCR primer sequence verification, pHT01 will be correctly carried:AprN plasmid-bearing strains are placed in -80 DEG C of preservations of glycerol tube.
Step 5：Nattokinase engineering bacterium fermentation：
Fermentation medium：20g/L glucose, 20g/L yeast extracts, 2g/L K2HPO4·3H2O、1g/L MgSO4·
7H2O, 2% glycerine and 1mM pyridinedicarboxylic acids, pH are adjusted to 7.0-8.0.
Engineering bacteria in glycerol tube is taken to be inoculated in 2% inoculum concentration in LB culture mediums (containing 5 μ g/ml chloramphenicol), 200rpm,
37 DEG C of incubated overnights.Seed liquor is inoculated in 25ml fermentation mediums A with 2% inoculum concentration, 37 DEG C, 200rpm shaking table cultures,
To OD600Between 0.8-1.0,25 DEG C are cooled to.Final concentration of 0.5mM derivant IPTG is accessed, in the different fermentations time
Determine the OD of bacterium solution600With NK enzyme activity, such as Fig. 8.
Step 6：Tetra-peptide substrate method detects enzyme activity
The standard curve of paranitroanilinum is made first：Prepare paranitroanilinum sample solution, concentration is respectively 0.02,
0.03rd, 0.04,0.06 and 0.08mmol, the light absorption value of various concentrations nitroaniline sample solution is determined under 405nm, is made with water
For blank control.4 DEG C of zymotic fluid, 8000r/min centrifugation 1min, supernatant are diluted to various concentrations as detection sample liquid.Inspection
Survey method is improved on the basis of document：Take 100 μ L samples dilutions and the 50 final concentration of 0.5mM of μ L four peptide substrates (D-
Val-Leu-Lys- paranitroanilinum) mix and 1min is reacted under 37 DEG C of water-baths, the glacial acetic acid for adding 75 μ L 50% is terminated instead
Should, reaction solution light absorption value is determined under 405 nm.NK active units are defined as NK 1 nmol nitroanilines of release per minute
Content.
Embodiment 2
Inoculation：Activated spawn, prepares primary seed solution and secondary seed solution under 37 DEG C, 200rpm condition of culture, plants age
For 12h secondary seed solution fermentation medium (the final concentration of 5 μ g/mL of chloramphenicol, defoamer are seeded to according to 10% inoculum concentration
0.01%).Fermentation medium (1L)：Glucose 28.5g, glycerine 57g, yeast extract 60g, pyridinedicarboxylic acid 0.167g,
NH4Cl 3g、K2HPO4·3H2O 1g、MnSO4·H2O 0.032g、 FeSO4·7H2O 0.05g、CoCl2·6H2O 0.01g、
ZnCl2 0.01g、MgSO4·7H2O 2g、CaCl2·2H2O 5g, moisturizing to 1000ml.
Fermentation：Stir speed (S.S.) is initially set to 800rpm, and throughput is 12L/min.Arrange parameter, controls the temperature in tank to be
37 DEG C, dissolved oxygen amount be the 30%, pH 7.0 of zymotic fluid, according to dissolved oxygen situation adjustment stir speed (S.S.) and throughput.
Protein induced expression：Detect OD600, work as OD60028 DEG C are cooled to during equal to 10 and adds final concentration of 0.2-1.0mM
IPTG carries out protein induced expression.
Feed supplement：Supplemented medium (1L)：Glycerine 250g, yeast extract 100g, moisturizing to 1000ml.Continuous feeding is trained
Support, setting feed regimes are：Ferment 3h, OD600=10 start feed supplement, and 11-14h feed rates are 250mL/h feed supplements 4h, 15-
18h is again with 170mL/h speed feed supplement 4h, 19-20h finally with 170mL/h speed feed supplement 2h.
Terminate fermentation：After fermented and cultured 24h, fermentation is terminated.
After testing, fermentation 20h enzyme activity can reach 7778.02IU/mL (RSD%=0.8), OD600For 173.5 ± 0.85,
See Fig. 9.
Embodiment 3
Zymotic fluid takes supernatant through 4 DEG C, 9000r/m refrigerated centrifuge 10min；By finely ground (NH4)2SO4According to 20%
Saturation degree is added in fermented supernatant fluid, is preserved and is stayed overnight at 4 DEG C, 10000r/m centrifugation 30min, discards precipitation；Continue in fermentation
In clear liquid plus ammonium sulfate is to 60% saturation degree, preserves and stay overnight at 4 DEG C, 10000r/m centrifuges 30min, abandoning supernatant, the rate of recovery
Reach 72%.
Embodiment 4
Balance nickel post：The nickeliferous fillers of 1mL are drawn into purification column, are first cleaned 2 times with 8 times of volumes of deionized water, then with 8 times
Volume combination liquid is cleaned three times.
It is incubated：When nearly being flow to end with reference to liquid, 60mL fermented supernatant fluids are added, 4h is incubated to 4 DEG C of vertical rotary refrigerators, carries
The NK of Histag labels is combined with filler.
Rinsing：Lower end drains the liquid not combined with nickel post, and the rinsing liquid for adding 10 times of column volumes washes foreign protein, on ice
Vertical reversion 5min, is repeated 4 times.
Elution：Filler middle is vertically taken in liquid-transfering gun absorption 1.5mL eluents fully to overhang filler, is stood on ice
2min, collects efflux.Repeat elution 10 times or so.
Eluent use 5KD super filter tube, 300g centrifugation, using pure water as dilution, with reach concentration Nattokinase and
The purpose of desalination.Finite concentration cryoprotector is added into concentration Nattokinase solution, freeze-drying is made high-purity and received
Beans kinases enzyme preparation, after testing Nattokinase enzyme activity reach 981713U/g.
The NK-aprN gene PCR amplimers of table 1
Note：Underline to expand with wave part table and repeat；Black matrix overstriking simultaneously underlines part and represents restriction enzyme site；
Italic underlines part and represents to add six histidines.
The NK-aprN gene PCR amplification systems of table 2
| Component | Volume (μ L) |
| Genomic DNA template | 2 |
| Primer-F | 1 |
| Primer-R | 1 |
| dNTP | 2 |
| 10×Buffer | 2.5 |
| Taq archaeal dna polymerases | 0.25 |
| H2O | 16.25 |
| Total | 25 |
Claims (9)
1. one plant can be with the bacillus subtilis engineered strain of efficient secretion Nattokinase, it is characterised in that point of the bacterial strain
Class is named as：Bacillus subtilis (Bacillus subtilis), deposit number is CGMCC No：13496, depositary institution is
China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address is the Chaoyang District, Beijing City North Star
The institute 3 of West Road 1, preservation date is on December 26th, 2016.
2. one plant can be with the construction method of the bacillus subtilis engineered strain of efficient secretion Nattokinase, it is characterised in that bag
Include following steps：
Step one：The amplification and optimization of NK-aprN genetic fragments
Using the bacterium solutions of B.subtilis 168 as the masterplate of first time amplification, with F listed by table 11-R1Primer pair is drawn for amplification for the first time
Thing, gel electrophoresis checking；Glue reclaim product is second of PCR amplification masterplate, with F listed by table 12-R1Primer pair expands for second
Primer, gel electrophoresis checking；Second of glue reclaim product is that third time PCR expands masterplate, with F listed by table 13-R2HisPrimer pair is
Third time amplimer, amplifies the aprN genetic fragments for optimizing preceding 30 codons；
Step 2：Target gene is cloned
The NK genetic fragments that step one is amplified are connected with pMD19-T carriers, and connection product is transformed into using thermal shock method
In E.coli DH5 α competence, Amp resistant panels are coated with；
Step 3：The structure of recombinant plasmid and checking
Plasmid pHT01 and the cloned plasmids double digestion of the positives bacterium of extraction step two, with glue reclaim kit recovery purifying purpose
Fragment, T4 ligase low temperature stays overnight enzyme company, and product Transformed E .coli DH5 α competent cells after bacterium colony PCR identifications, take correct
Positive colony activation culture, extract plasmid order-checking；
Step 4：The structure of engineering bacteria and identification
B.subtilis 168 competent cell is prepared, the plasmid electricity extracted in step 4 is turned into competent cell, chosen at random
The 5-8 single bacterium colony grown in extracting chloromycetin resistant panel, bacterium colony PCR identifications, takes correct positive colony activation culture, extracts
Plasmid order-checking.
3. according to claim 2 can be with the structure side of the bacillus subtilis engineered strain of efficient secretion Nattokinase
Method, it is characterised in that also include：
Step 5：Engineering bacterium fermentation
Recombinant seed liquor is activated, using chloramphenicol as antibiotic, seed liquor switching fermentation medium culture, inoculum concentration 1-
7.5%, OD60025 DEG C to 30 DEG C are cooled in 0.8-1.0, final concentration of 0.2-1.0mM IPTG Fiber differentiations are added.
4. according to claim 2 can be with the structure side of the bacillus subtilis engineered strain of efficient secretion Nattokinase
Method, amplification system such as table 2, amplification reaction condition：94 DEG C of pre-degenerations 10min, 94 DEG C of denaturation 40s, 54 DEG C of annealing 40s, 72 DEG C are prolonged
30s is stretched, totally 35 circulations, 72 DEG C of extension 10min.
5. according to claim 3 can be with the structure side of the bacillus subtilis engineered strain of efficient secretion Nattokinase
Method, it is characterised in that chlorampenicol resistant screening is carried out with 5 μ g/ml chloramphenicol.
6. according to claim 3 can be with the structure side of the bacillus subtilis engineered strain of efficient secretion Nattokinase
Method, it is characterised in that the fermentation medium is：20g/L glucose, 20g/L yeast extracts, 2g/L K2HPO4·3H2O、
1g/L MgSO4·7H2O, 2% glycerine and 1mM pyridinedicarboxylic acids, pH are adjusted to 7.0-8.0.
7. according to claim 3 can be with the structure side of the bacillus subtilis engineered strain of efficient secretion Nattokinase
Method, it is characterised in that fermentation culture conditions are：Inoculum concentration 7.5%, OD600For 0.8 when start induction, 30 DEG C of inducing temperature,
IPTG induced concentrations are 0.5mM.
8. it is a kind of high-purity with can be prepared with the bacillus subtilis engineered strain of efficient secretion Nattokinase described in claim 1
The technique of Nattokinase, it is characterised in that including：
(1) high density fermentation
Inoculation：Activated spawn, prepares primary seed solution and secondary seed solution under 37 DEG C, 200rpm condition of culture, and kind age is 12h
Secondary seed solution be seeded to fermentation medium according to 10% inoculum concentration, the final concentration of 5 μ g/mL of chloramphenicol, defoamer are
0.01%；
Fermentation：Stir speed (S.S.) is initially set to 800rpm, and throughput is 12L/min；Arrange parameter, it is 37 to control temperature in tank
DEG C, dissolved oxygen amount be the 30%, pH 7.0 of zymotic fluid, according to dissolved oxygen situation adjustment stir speed (S.S.) and throughput；
Protein induced expression：Sampling detection OD600, 28 DEG C are cooled to logarithm early stage, IPTG is added and starts induction fermentation；
Feed supplement：Supplemented medium：Glycerine 250g, yeast extract 100g, moisturizing to 1000ml；Detect zymotic fluid OD600And fermentation
The Expenditure Levels of glycerine in culture medium, are respectively adopted a feed supplement and continuous feeding mode feed-batch culture；
Terminate fermentation：After fermented and cultured 24-28h, fermentation is terminated；
(2) preparation of high-purity Nattokinase enzyme preparation
The chilled collected after centrifugation supernatant of zymotic fluid, (NH4)2SO4Precipitation removes foreign protein and precipitates NK, ni-sepharose purification, desalination
And concentrate plus the step such as cryoprotector and freeze-drying, high-purity nattokinase enzyme preparation is made.
9. the technique according to claim 8 for preparing high-purity Nattokinase, it is characterised in that the fermentation medium：Portugal
Grape sugar 28.5g, glycerine 57g, yeast extract 60g, pyridinedicarboxylic acid 0.167g, NH4Cl 3g、K2HPO4·3H2O 1g、
MnSO4·H2O 0.032g、FeSO4·7H2O 0.05g、CoCl2·6H2O 0.01g、ZnCl2 0.01g、MgSO4·7H2O
2g、CaCl2·2H2O 5g, moisturizing to 1000ml.
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