CN103937830B - A kind of recombinant bacterium of efficient secretory expression Nattokinase - Google Patents
A kind of recombinant bacterium of efficient secretory expression Nattokinase Download PDFInfo
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Abstract
The invention discloses a kind of recombinant bacterium that can efficient secretory expression Nattokinase, can significantly improve the output of Nattokinase. Recombinant bacterium of the present invention is the Recombinant Pichia pastoris bacterium that contains two above Nattokinase Pro-NK genes of copy and Hac1p regulatory factor gene, and this recombinant bacterium is at shake flask fermentation after 96 hours, and the enzyme work of Nattokinase can reach 470IU/mL.
Description
Technical field
The present invention relates to a kind of recombinant bacterium, particularly a kind of restructuring that can efficient secretory expression NattokinaseBacterium.
Background technology
Nattokinase (Nattokinase, NK) is a kind of withered by Bacillus natto or natto in soybean isoflavone by natto strainThe serine protease that grass bacillus (Bacillussubtilisnatto) produces. 1980, Japan must seeFirst the professor of foreign firm finds that this enzyme can dissolve artificial thrombus, has then studied the mechanism of action of this enzyme, and by itCalled after Nattokinase.
Since nineteen ninety-five domestic first section of report about natto and Nattokinase, existing many sections of articles so farDeliver, and research and development are gone forward side by side simultaneously. At present the character to Nattokinase (nattokinase gene sequence,Protein amino acid sequence, activated centre and catalytic center, pI, temperature and pH stability, optimal reaction temperatureThe thrombolysis mechanism of degree and pH, inhibitor and activator and Nattokinase comprises the 1. direct thrombolysis of NattokinaseEffect; 2. activate prourokinase and become urokinase; 3. activate vascular endothelial cell and produce t-PA; 4. natto swashsThe indirect thrombolytic effect of enzyme, by degraded and inactivation PAI-1, increase fibrinolysis activity etc. has all been illustrated, Er QieyiThis enzyme is carried out to separation and purification, in vivo and in vitro thrombolysis animal experiment, acute toxicity test and treatment cerebral infarctionThe aspect such as preliminary clinical testing carried out a large amount of research work.
At present, Nattokinase has obtained research extensively and profoundly in Japan, and existing 10 Yu Jia major companies of Japan are rawThe multiple product taking Nattokinase as main component of output all goes on the market, the pharmacy of the international doctor of U.S. health technologyThe Nattokinase capsule of limited company also appears on the market, and Korea S and Korea also have similar products to come out. At meState, existing many manufacturers produce Nattokinase capsule products, but cause commercially available because production bacterial strain output is too lowNattokinase health products are compared with Japanese imported product, and unit of activity differs greatly. Therefore be necessary nattoKinases is produced bacterial strain and is transformed, and significantly to increase bacterial strain output, improves final products quality.
Compared with traditional breeding method, rely on genetic engineering means, use type bost as Escherichia coli, withered grass gemmaIt is in the short time, to improve Nattokinase that bacillus, saccharomyces cerevisiae and Pichia pastoris etc. are produced recombinant natookinaseThe shortcut of output. So far, the Nattokinase that Escherichia coli, bacillus subtilis, saccharomyces cerevisiae are expressedCase be not very successful. In recent years, Pichia pastoris was with its safety, efficient secretory expression, at medicine andIn the expression of important foodstuffs enzyme preparation, obtain increasing application, therefore, produced with Pichia pastorisNattokinase has one of method of application prospect most by being. Have at present some and use Pichia anomala expression nattoKinase whose report, the people such as Luo Lixin at Pichia pastoris GS115, show Nattokinase mature peptide gene in KM71Reach merit, the enzyme activity in fermented supernatant fluid is that the yellow will of 120U/mL(is vertical, Luo Lixin, Ling Junjian etc. receiveThe structure of beans kinase gene recombinant expression carrier and stability ［ J ］ thereof. ACAD J GCP, 2000,16 (4):265-267). Wang Jiangbo etc. by full Nattokinase peptide gene in Pichia pastoris GS115, KM71, SMD1168Successful expression comparative analysis (Wang Jiangbo, Xu Fang, Zhang Jingfang. Nattokinase protogene is in Pichia pastorisSecreting, expressing ［ J ］. China brewages, 2008,19 (196): 40-41). Cai Litao etc. are by NK Gene cloningIn yeast expression vector pHBM905A, methanol induction is expressed, and result SDS-PAGE result showsNK successful expression, fibrin plate experiment Explicit Expression product have good fibrinolytic (Cai Litao,Xu Xiang, Wang Tingting etc. expression and purification and the antibody preparation ［ J ］ of nattokinase gene in Pichia pastoris. ChinaBiochemical drug magazine, 2010 (1)). But in above-mentioned article, the expression of Nattokinase still cannot meetThe demand of industrialization, still need to do further optimization and lifting for producing bacterial strain.
Summary of the invention
The object of the present invention is to provide a kind of recombinant bacterium that can efficient secretory expression Nattokinase.
To achieve these goals, technical scheme of the present invention is as follows:
The recombinant vector of Nattokinase Pro-NK gene, this recombinant vector is by plural NattokinaseAfter Pro-NK gene insertion carrier for expression of eukaryon, obtain.
Further preferred recombinant vector is that plural Nattokinase Pro-NK gene is inserted very in the same wayAfter nuclear expression carrier, obtain.
The coded sequence of above-mentioned Nattokinase Pro-NK gene is as shown in SEQ ID NO.3.
Each Nattokinase Pro-NK gene has an its corresponding promoter.
Above-mentioned promoter is alcohol oxidase I promoter (PAOX1) or formaldehyde dehydrogenase promoter (PFLD1）。
The recombinant vector of preferred Nattokinase Pro-NK gene, the carrier for expression of eukaryon of selecting is pAO815Expression vector; This pAO815 expression vector is by pPICZ α carrier SacI and EcoRI are carried out to two enzymesCutting the 1000bp fragment obtaining obtains with the same pAO815 carrier connection institute digesting with SacI and EcoRI;Each Nattokinase Pro-NK gene is expressed under an its corresponding alcohol oxidase I promoter, phaseBetween adjacent Nattokinase Pro-NK gene, be in series with successively alcohol oxidase I terminator, alcohol oxidase I startupSon and α-mating factor signal peptide sequence.
This recombinant vector is build by the following method and obtain:
(1) artificial synthetic or pcr amplification Nattokinase Pro-NK gene, adds XhoI in its 5' end upstreamRestriction enzyme site, EcoRI restriction enzyme site is added in 3' end downstream; By XhoI and the two enzymes of EcoRI for this genetic fragmentCut after processing and connect with the same pAO815 carrier through XhoI and the digestion of EcoRI double digestion, obtain containing listThe recombinant vector pAO α-PNK of copy Nattokinase Pro-NK gene;
Above-mentioned synthetic method is preferably pcr amplification method, and its concrete steps are as follows: extract natto bacillus subtilisThe chromosomal DNA of bacterium (Bacillussubtilissubspnatto), using it as template, with in sequence tableUpstream and downstream primer shown in SEQIDNO.1 and SEQIDNO.2 carries out PCR, obtains target DNA sheetSection;
(2) with recombinant expressed year that obtains in BamHI and BglII double digestion digestion process step (1)Body pAO α-PNK, reclaims the fragment that obtains 2589bp, connects α-PNK into recombinant expression carrier pAOBamHI site, obtain the recombinant expression carrier that contains two copies Nattokinase Pro-NK genepAOα-PNK-2；
(3) use the same method that the rest may be inferred, obtain containing and be greater than two copy Nattokinase Pro-NK basesThe recombinant vector pAO α-PNK-n of cause, wherein n represents copying of Nattokinase Pro-NK gene in recombinant vectorShellfish number, n > 2.
A recombinant bacterium for efficient secretory expression Nattokinase is to contain two Nattokinases more than copyThe Recombinant Pichia pastoris bacterium (Pichiapastoris) of Pro-NK gene and Hac1p regulatory factor gene.
Above-mentioned Pichia Pastoris is Pichia Pastoris GS115.
Above-mentioned Nattokinase Pro-NK gene is to lead by the recombinant vector of above-mentioned Nattokinase Pro-NK geneEnter in described Pichia Pastoris, obtain containing two Nattokinase Pro-NK genes more than copyRecombinant Pichia pastoris bacterium.
Continue by Hac1p regulatory factor gene be by constitutive expression carrier import to contain two copies withOn the Recombinant Pichia pastoris bacterium of Nattokinase Pro-NK gene in, the recombinant bacterium obtaining can produceHac1p regulatory factor, has further improved the secretory volume of Nattokinase;
Above-mentioned constitutive expression carrier is preferably constitutive expression carrier pGAPZ-Hac1p, this constitutive expressionCarrier is to build and obtain by Hac1p regulatory factor gene being inserted in pGAPZA carrier.
" constitutive expression carrier " herein, represents the not tune of the modulated factor of foreign gene whereinControl, can continual expression alien gene under suitable condition. Above-mentioned carrier pGAPZ-Hac1p adoptsWith constitutive promoter PGAP, genes of interest wherein does not need induction to express.
Above-mentioned Hac1p regulatory factor gene source is in yeast or mould.
The coded sequence of above-mentioned Hac1p regulatory factor gene is as shown in SEQ ID NO.5.
The above-mentioned method that recombinant vector is imported in Pichia yeast is electrotransformation.
The present invention also provides recombinant bacterium the answering in health products processing of above-mentioned efficient secretory expression NattokinaseWith and application in preparation treatment cerebral infarction medicine.
The invention has the beneficial effects as follows: by the constructed recombinant vector of the present invention, the recombinant bacterium obtaining existsIn shaking flask, the yield level of secreting, expressing Nattokinase can reach every milliliter of 470IU(and detects according to urokinase activityMethod is measured), significantly improve than prior art. Nattokinase prepared by recombinant bacterium of the present invention, can be processed intoThe active ingredient of cerebral infarction medicine is treated in health products or conduct.
Brief description of the drawings
Fig. 1 is the structure schematic diagram of secreted expression carrier pAO815alpha.
Fig. 2 is the collection of illustrative plates of recombinant expression carrier pAO α-PNK.
Fig. 3 is the collection of illustrative plates of recombinant expression carrier pAO α-PNK-2.
Fig. 4 is the collection of illustrative plates of constitutive expression carrier pGAPZ-Hac1p.
Detailed description of the invention
Below in conjunction with specific embodiment, the invention will be further described, but not thereby limiting the inventionProtection domain.
The culture medium using in following embodiment and the formula of buffer solution are as follows:
LB(Luria-Bertani) culture medium: 10g/L sodium chloride, 10g/L peptone, 5g/L dusty yeast,pH7.4-7.6；
LLB culture medium: 5g/L sodium chloride, 10g/L peptone, 5g/L dusty yeast, pH7.4-7.6;
BMGY fluid nutrient medium: 10g/L yeastex extract, 20g/L peptone, 20 points of 121 DEG C of sterilizingsZhong Hou, adds 13.4g/LYNB, 4 × 10 when temperature is down to room temperature-4G/L biotin, 10g/L glycerine;
BMMY inducing culture: 10g/L yeastex extract, 20g/L peptone, 20 points of 121 DEG C of sterilizingsZhong Hou, adds 13.4g/LYNB, 4 × 10 when temperature is down to room temperature-4G/L biotin and 0.5%(V/V) firstAlcohol;
YPD culture medium: 10g/L yeast extract, 20g/L peptone, 20g/L glucose;
YPD flat board: 10g/L dusty yeast, 20g/L peptone, 20g/L glucose, 15g/L agarose;
MD flat board: 13.4g/LYNB, 4 × 10-4G/L biotin, 20g/L glucose, 15g/L agarose;
200mmol/L trichloroacetic acid: take 3.268g trichloroacetic acid, be settled to 100mL after being dissolved in water;
STE buffer solution: 10mmol/LTris-Cl, pH8.0,0.1mmol/LNaCl, 1mmol/LEDTA,pH8.0；
TE buffer solution: 10mmol/LTris-Cl, pH8.0,0.1mmol/LNaCl, 1mmol/LEDTA,pH8.0；
50mmol/L borate buffer solution: add 9g sodium chloride at 19.07g sodium tetraborate, add water after dissolving fixedHold to 1000mL, with boron acid for adjusting pH be 8.5.
Experiment material in following embodiment, if no special instructions, all can purchase by conventional commercial sources. The method adopting in following embodiment, if no special instructions, is conventional method.
Embodiment 1, the structure of recombinant expression carrier that contains Nattokinase Pro-NK gene
(1) extracting of Bacillus subtilis natto (Bacillussubtilissubspnatto) chromosomal DNA
By the Bacillus subtilis natto of-70 DEG C of preservations, (deposit number is CGMCC1.1086, purchased from ChinaCommon micro-organisms culture presevation administrative center) spore suspension or inclined-plane mycelium be seeded to 5mLLB cultivateIn base, 37 DEG C of concussions are cultivated 48 hours. Get 3mL bacterium liquid centrifugal, collect mycelium, with 500 μ LSTETwice of buffer solution washing mycelium. Then with 500 μ LSTE, mycelium is fully suspended, add lysozyme to itFinal concentration is 2mg/mL, and now thallus suspension liquid becomes translucent glue, then adds the 2%SDS of 250 μ L(Sodiumdodecylsulfate, sodium hexadecyl sulfate), concussion mixes. Then add 250 μ L neutralityPhenol/chloroform mixed solution fully mixes, and wherein the volume ratio of phenol, chloroform is 25:24. 10000r/minAfter centrifugal 5 minutes, supernatant is transferred in new Eppendorf pipe, adds RNase to its final concentration to be20-40 μ g/mL, 37 DEG C are incubated 30 minutes. Then adopt identical method, with neutral phenol/chloroform repetitionExtracting 3-4 time, exists until two-phase interface no longer includes albuminate, then removes the aqueous solution twice with chloroform extractingIn phenol. To the sodium acetate and the isopyknic isopropyl alcohol that add 1/10 volume 3mol/L in the aqueous solution, mixedEven rear room temperature is placed 30 minutes. Centrifugal 5 minutes of 10000r/min, with 70% ethanol washing, precipitation once,After precipitation vacuum is drained, redissolve in 100-200 μ LTE buffer solution, obtain Bacillus subtilis natto dyeingBody DNA, sets it as synthetic Nattokinase Pro-NK gene PCR reaction template used.
(2) structure of intermediate carrier
As shown in Figure 1, pPICZ α carrier (purchased from Invitrogen company) is used to SacI/EcoRI double digestion,Obtain the fragment of 1000bp, small fragment (is purchased with the same pAO815 carrier digesting with SacI/EcoRIFrom Invitrogen company) connect, obtain the excretion vector pAO815alpha based on pAO815, shouldP in expression vectorAOX1Strong promoter can regulate and control to insert the expression of genes of interest, and contains α-matingSignal peptide sequence, can make the destination gene expression inserting is exocytosis albumen.
(3) pcr amplification of Nattokinase Pro-NK gene
According to the Nattokinase Pro-NK gene (GenBank in Bacillus subtilis natto source in GenBankNumbering: AF368283.1) design PCR primer, upstream and downstream primer is respectively as SEQ ID NO.1Shown in SEQIDNO.2, the nucleotide sequence of Nattokinase Pro-NK gene is as SEQ IDShown in NO.3.
Upstream primer: 5'-GACTCTCGAGAAAAGAGCCGGAAAAAGCAGTACA-3'
Downstream primer: 5'-GGTCGAATTCTATTATTGTGCAGCTGCTT-3'
There is one in α-mating of excretion vector pAO815alpha factor signal peptide-coding sequence downstreamXhoI restriction enzyme site (CTCGAG, corresponding amino acid is Leu-Glu), thereafter immediately following a Kex2 albumenEnzyme recognition site (AAAAGA, corresponding amino acid sequence Lys-Arg), therefore, the design of upstream primer isBefore Nattokinase Pro-NK gene, introduce XhoI site and Kex2 protease recognition site sequence. Pass throughThis design, after Nattokinase Pro-NK gene directly can being connected to signal peptide sequence, albumen turns over like thisAfter translating, under the effect of Pichia pastoris self Kex2 protease, can finally give expression to compared with destination proteinThe maturation protein that amino acid is constant, and can not introduce extra amino acid residue because of the reason of adding restriction enzyme site.Downstream primer comprises terminator codon TAT, and introduces an EcoRI restriction enzyme site (GAATTC).
The Bacillus subtilis natto chromosomal DNA obtaining taking step (1) is as template, add upstream primer,Downstream primer, PrimestarDNA polymerase (purchased from TaKaRa company) and dNTP mixture, carry outPCR reaction, reaction condition is as follows: predeformation 3min at 98 DEG C; Cyclic amplification 30 times: 98 DEG C of 15s,55 DEG C of 15s, 72 DEG C of 70s; Finally extend 90s at 72 DEG C. The product obtaining after amplification is through order-checking qualification,Obtain the genetic fragment of size for 1080bp, the coded sequence that it contains Nattokinase Pro-NK gene, andHave XhoI restriction enzyme site in the 5' of this sequence end upstream, there is EcoRI restriction enzyme site in 3' end downstream.
(4) structure of the recombinant expression carrier that contains single copy Nattokinase Pro-NK gene
By the PCR product of step (3) gained with after XhoI and EcoRI double digestion, with same through XhoIPostdigestive excretion vector pAO815alpha is connected with EcoRI double digestion, obtains containing single copy and receivesThe recombinant expression carrier pAO α-PNK of beans kinases Pro-NK gene, the collection of illustrative plates of this carrier as shown in Figure 2.
The recombinant expression carrier pAO α obtaining-PNK is transformed to bacillus coli DH 5 alpha competent cell, with containingThere is the LB Screening of Media of 10 μ g/mL ampicillins to go out required recon and carry out sequencing analysis, processOrder-checking qualification, in the recombinant vector obtaining, the sequence of Insert Fragment and structure were all correct, by this recombinant expressed yearBody called after pAO α-PNK.
(5) structure of the recombinant expression carrier that contains two copies nattokinase gene
By BamHI and BglII double digestion for the recombinant expression carrier pAO α obtaining in step (4)-PNK,Reclaim the BamHI-PNK-BglII fragment of 2589bp, connect into the BamHI of expression vector pAO α-PNKSite, get final product contain two copies nattokinase gene expression vector pAO α-PNK-2, the collection of illustrative plates of this carrierAs shown in Figure 3.
The recombinant expression carrier pAO α obtaining-PNK-2 is transformed to bacillus coli DH 5 alpha competent cell, useThe LB Screening of Media that contains 10 μ g/mL ampicillins goes out required recon and carries out sequencing analysis, warpCross order-checking qualification, in the recombinant vector obtaining, the sequence of Insert Fragment and structure are all correct, and this is recombinant expressedCarrier called after pAO α-PNK-2.
(6) structure of constitutive expression carrier pGAPZ-Hac1p
First manually synthetic Hac1p regulatory factor gene, its coded sequence is as sequence table SEQ IDNO.5 instituteShow, and hold EcoRI of each introducing and NotI site at the 5' of its sequence end and 3', enzyme is cut after processing, willHac1p gene is connected to equally the pGAPZA carrier of cutting through EcoRI and NotI enzyme (purchased from InvitrogenCompany) upper, obtain pGAPZ-Hac1p constitutive expression carrier, the collection of illustrative plates of this carrier is as shown in Figure 4.
The recombinant expression carrier pGAPZ-Hac1p obtaining is transformed to bacillus coli DH 5 alpha competent cell, useThe LLB Screening of Media that contains 25 μ g/mL bleomycins (Zeocin) goes out required recon and checks orderAnalyze, through order-checking qualification, in the recombinant vector obtaining, the sequence of Insert Fragment and structure are all correct, by thisRecombinant expression carrier called after pGAPZ-Hac1p.
Preparation and the Function detection thereof of the yeast strain of embodiment 2, expression Nattokinase
1. express the preparation of the bacterial strain of Nattokinase
(1) electricity transforms the preparation of pichia pastoris phaff cell
Inoculation Pichi strain GS115(is purchased from Invitrogen company, CA.18100) in 500mLYPDIn culture medium, 30 DEG C, 200r/min are cultured to OD600=1.3-1.5. Under 4 DEG C, 4500r/min conditionWithin centrifugal 5 minutes, collect thalline, use respectively 1 of the aqua sterilisa of 500mL, 250mL precooling and 20mL precoolingThe each washing of mol/L sorbierite once. Wash at every turn and all within centrifugal 5 minutes, collect thalline at 4 DEG C, 4500r/min afterwards,Finally suspend with the 1mol/L sorbierite of 1mL precooling, obtain electric shock competent cell.
(2) preparation of the Recombinant Pichia pastoris bacterial strain that contains recombinant expression carrier pAO α-PNK
Get recombinant expression carrier pAO α-PNK approximately 10 about μ g that obtain in embodiment 1, cut with StuI enzymeLinearisation, by ethanol precipitation, reclaims linear DNA and is dissolved in 10 μ L sterilized waters. By above-mentioned linearisationDNA mixes with 80 μ LGS115 competent cells, proceeds in the 0.2cm electric shock cup of precooling. According to makeElectricity consumption conversion instrument (GenepulserXCellTM, purchased from BIO-RAD company) shock by electricity. Electric shock finishesAfter add immediately 1mL precooling 1mol/L sorbierite to electric shock cup, then by full the solution in electric shock cupPortion is transferred in an aseptic centrifuge tube, directly gets 200-300 μ L bacterium liquid coating MD flat board. Be inverted training for 30 DEG CSupport 2-4 days until bacterium colony occurs, obtain single copy bacterial strain of expressing Nattokinase, called after GSPNK.
(3) preparation of the Recombinant Pichia pastoris bacterial strain that contains recombinant expression carrier pAO α-PNK-2
The method providing according to step (2), by the recombinant expression carrier obtaining in embodiment 1PAO α-PNK-2 carries out linearisation and electric conversion processing, obtains finishing red containing the restructuring of Nattokinase two copies geneYeast, obtains the two copies bacterial strain of expressing Nattokinase, called after GS2PNK after plate screening.
(4) contain expression vector pAO α-PNK-2 and constitutive expression carrier pGAPZ-Hac1p simultaneouslyThe preparation of Recombinant Pichia pastoris bacterial strain
Get the constitutive expression carrier pGAPZ-Hac1p approximately 10 μ g that obtain in embodiment 1, cut with AvrII enzymeLinearisation, transforms recombinant bacterial strain GS2PNK according to the method providing in step (2) by its electricity, electric shock knotAfter bundle, add immediately the 1mol/L sorbierite of 1mL precooling to electric shock cup, then by the solution in electric shock cupAll be transferred in an aseptic centrifuge tube, bathe 2 hours in 30 DEG C of temperature, then get 500 μ L bacterium liquid and be coated onOn the YPD flat board that contains 50 μ g/mL bleomycins (Zeocin). Be inverted for 30 DEG C and cultivate 2-4 days until bacteriumDrop out now, by bacterium colony, PCR identifies, can obtain the recombinant yeast pichia pastoris containing the Hac1p protein regulation factorBacterial strain, called after GS2PNK-H.
2. Function detection
(1) fermented and cultured of recombinant bacterium
Recombinant bacterial strain GSPNK obtained above, GS2PNK and GS2PNK-H are inoculated in respectively to 25mLIn BMGY fluid nutrient medium, be arranged three groups in parallel, under 30 DEG C, the condition of 200r/min, shaking table is cultured toOD600=10-20, centrifugal 5 minutes of 3000r/min collects thalline, abandons supernatant, washs bacterium with sterile purified waterBody 1-2 time. Thalline is diluted to OD with BMMY inducing culture600=10, with 4 layers of gauzes replacement cottonPlug, in 30 DEG C, the cultivation of 200r/min shaking table, adding methyl alcohol to its final concentration every day is 0.5%(V/V).
(2) mensuration that ectoenzyme is lived
Recombinant bacterium is cultivated after 4 days 30 DEG C of inductions, draws zymotic fluid 1mL in centrifuge tube, 10000r/Centrifugal 10 minutes of min, draws supernatant for subsequent use in test tube. In another test tube, add 50mmol/(Fibrinogen, purchased from Sigma, article No. is T86 for L borate buffer solution 1.4mL and fibrinogen solution30, specification is that 100mg/ props up) 0.4mL, in the thermostat water bath of 37 DEG C, heat after 5 minutes, addFibrin ferment (Thrombin, purchased from Sigma, article No. is T4648, specification is that 1000U/ props up) 0.1mL,Mix. In 37 DEG C of water-baths, be incubated 10 minutes, then add 0.1mL sample solution, mixed for 5 seconds, insulation60 minutes, in the time of insulation the 20th minute and the 40th minute, take out respectively vibration. After insulation finishes, toward examinationIn pipe, add 200mmol/L trichloroacetic acid 2mL, then put into water-bath insulation 20 minutes, 15000r/Centrifugal 5 minutes of min. Get supernatant and measure light absorption value at 275nm place. Add the enzyme liquid of the deactivation of same amountMake blank, the processing method of blank is the same with the processing method of sample. Every group all establish 3 parallelSample, final measurement result is the mean value of 3 measured values.
The enzyme unit definition alive of Nattokinase is: at 37 DEG C, and every decomposition 1 in the borate buffer solution that pH is 8.5The needed enzyme amount of fibrin of mg is an enzyme activity unit IU.
Test result is as shown in table 1, after induction is fermented 96 hours, and GSPNK, GS2PNK and GS2PNThe average enzyme of the shake flask fermentation liquid of K-H recombinant bacterial strain live can reach respectively 180,230 and 470IU/mL(with urineKinases is measurement standard), illustrate that the increase of copy number has significantly improved Nattokinase expression, importingCome from after the Hac1p regulatory factor gene of saccharomyces cerevisiae, enzyme is lived and is further promoted again, has proved this energy factorCan make the secreting, expressing level of Nattokinase significantly be strengthened.
The Nattokinase enzyme (IU/ml) alive of the different restructuring yeast strains of table 1
Claims (8)
1. a recombinant bacterium for efficient secretory expression Nattokinase, is characterized in that, described recombinant bacterium is simultaneouslyThe restructuring Pasteur that contains two above Nattokinase Pro-NK genes of copy and Hac1p regulatory factor gene is finishedRed saccharomycete (Pichiapastoris);
Described Nattokinase Pro-NK gene imports to heavily by the recombinant vector of Nattokinase Pro-NK geneIn group Pichia Pastoris;
The recombinant vector of described Nattokinase Pro-NK gene is by plural Nattokinase Pro-NKAfter gene insertion carrier for expression of eukaryon, obtain.
2. the recombinant bacterium of efficient secretory expression Nattokinase according to claim 1, is characterized in that,Each Nattokinase Pro-NK gene is expressed under an its corresponding promoter.
3. the recombinant bacterium of efficient secretory expression Nattokinase according to claim 2, is characterized in that,Described promoter is alcohol oxidase I promoter or formaldehyde dehydrogenase promoter.
4. the recombinant bacterium of efficient secretory expression Nattokinase according to claim 3, is characterized in that,Each Nattokinase Pro-NK gene is expressed under an its corresponding alcohol oxidase I promoter, phaseBetween adjacent Nattokinase Pro-NK gene, be in series with successively alcohol oxidase I terminator, alcohol oxidase I startupSon and α-mating factor signal peptide sequence.
5. the recombinant bacterium of efficient secretory expression Nattokinase according to claim 1, is characterized in that,Described carrier for expression of eukaryon is pAO815 expression vector; Described pAO815 expression vector is by by pPICZ αCarrier carries out with SacI and EcoRI 1000bp fragment that double digestion obtains and disappears with SacI and EcoRI with sameThe pAO815 carrier of changing connects gained.
6. the recombinant bacterium of efficient secretory expression Nattokinase according to claim 1, is characterized in that,Described Hac1p regulatory factor gene is to import to two nattos more than copy by constitutive expression carrierIn the Recombinant Pichia pastoris bacterium of kinases Pro-NK gene.
7. the recombinant bacterium of efficient secretory expression Nattokinase according to claim 6, is characterized in that,Described constitutive expression carrier is constitutive expression carrier pGAPZ-Hac1p, and this constitutive expression carrier is logicalCross and Hac1p regulatory factor gene is inserted into structure in pGAPZA carrier and obtains.
8. the recombinant bacterium of efficient secretory expression Nattokinase according to claim 6, is characterized in that,Described Hac1p regulatory factor gene source is in yeast or mould.
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| CN104531833A (en) * | 2014-12-18 | 2015-04-22 | 天津市百奥生物技术有限公司 | Nattokinase activity measurement method |
| CN106085934B (en) * | 2016-06-12 | 2019-10-29 | 武汉康复得生物科技股份有限公司 | Food-grade Nattokinase expresses bacterium |
| CN107723253B (en) * | 2017-09-29 | 2021-10-15 | 天津昕因达生物技术有限公司 | Double-plasmid co-transformed exogenous gene high-expression genetic engineering bacterium |
| CN109082434A (en) * | 2018-09-14 | 2018-12-25 | 深圳上泰生物工程有限公司 | A kind of pichia vector and preparation method thereof and recombinant pichia yeast strain |
| CN109182313B (en) * | 2018-10-08 | 2022-09-06 | 南京福斯弗瑞生物科技有限公司 | Nattokinase and construction and production method of expression vector thereof |
| CN114375304A (en) | 2019-07-11 | 2022-04-19 | 克莱拉食品公司 | Protein composition and food thereof |
| US12096784B2 (en) | 2019-07-11 | 2024-09-24 | Clara Foods Co. | Protein compositions and consumable products thereof |
| EP4100535A4 (en) * | 2020-02-04 | 2024-01-24 | Clara Foods Co. | Systems and methods for high yielding recombinant microorganisms and uses thereof |
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| CN102965292A (en) * | 2012-12-10 | 2013-03-13 | 江南大学 | Glucose oxidase secretion enhanced bacterial strain and application thereof |
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| 未折叠蛋白反应细胞内信号传导新途径;山松等;《生命科学》;20010228;第13卷(第1期);第34-36、5页 * |
| 纳豆激酶基因在巴斯德毕赤酵母中的表达;罗立新等;《华南理工大学学报（自然科学版）》;20030228;第31卷(第2期);第1-4页 * |
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