CN101914476B - Deep sea elastase gene as well as preparation method and application thereof - Google Patents
Deep sea elastase gene as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a deep sea elastase gene as well as a preparation method and an application thereof, belonging to the field of biotechnology. The invention relates to a cryophilic bacterium CF6-2, a cryophilic elastase gene extracted from the cryophilic bacterium CF6-2 and deep sea elastase expressed and translated by the gene. The cryophilic elastase derives from a deep sea low-temperature high-salinity environment. Compared with other kinds of elastase, the cryophilic elastase has higher elastin degradation efficiency in sea water at 0-30 DEG C and can exert functions at normal temperature. The cryophilic elastase is extremely unstable at the temperature above 40 DEG C and is inactivated at middle temperature, so that the safety of the cryophilic elastase is ensured. The cryophilic elastase has cracking activity on various gram-positive bacteria, not only can be applied to the fields of medical treatment, daily chemical industry and the like, but also has great application potential in the fields of marine biomedicine, aquatic product processing and the like.
Description
Technical field
The present invention relates to a kind of deep-sea Pancreatopeptidase E gene and preparation method thereof and application, belong to technical field of biotechnology.
Background technology
Pancreatopeptidase E (Elastase) be a kind of be the proteolytic ferment of characteristic with insoluble hydrolysate property elastin (elastin), at a plurality of proteolytic enzyme monoids,, existence is arranged all in L-Cysteine HCL Anhydrous and the metalloprotease like Tryase.Wherein the Pancreatopeptidase E in the metalloprotease distributes at most, at M4, and M9, all flexible proteolytic enzyme exists in M10 and the M23 family.At present, Pancreatopeptidase E is mainly as the biochemical drug of treatment hyperlipidaemia, control atherosclerosis, and treatment is definite, and is safe and reliable.In addition, Pancreatopeptidase E also can be used for foodstuffs industry.Many animal and plant protein; Protein wastes such as the ligament that more particularly is difficult to handle and eat, Aorta blood vessel, muscle tendon all can be degraded by it, therefore will be used widely at aspects such as the making of agricultural byproducts deep processing, high protein food, can processing.Aspect daily chemical industry, Pancreatopeptidase E is mainly used in the production of makeup.In recent years there is report that Pancreatopeptidase E is added makeup abroad, can promotes, improve cutaneous circulation, and improve the skin lipid metabolism; Strengthen skin, the elasticity of hair, flexibility, delay skin aging reduces wrinkle and pigmentation; Can also promote natural on-off cycles of hair growth, anti-loss etc.
M23 family protein enzyme all contains a zine ion, and its avtive spot is arranged in HXXXD and these two of HXH and the interactional motif of zine ion.Protein structure in this family all is made up of two half folded mutually β buckets, and its reactive site is positioned at the half bigger β barrel shape structure of its C end.The proteolytic enzyme of the M23 family of report all has tangible activity to the Gly-X peptide bond at present.Because of containing 33% Gly in the elastin, so M23 family protein enzyme all has Degradation to elastin.In addition; Owing to contain a large amount of Polysaccharides, peptide complexess in the gram-positive bacteria cell wall; And the peptide bridge in the Polysaccharides, peptide complexes contains a lot of Gly; Particularly 5 amino acid all are Gly in the peptide bridge of streptococcus aureus (Staphylococcus aureus), so M23 family protein enzyme has splitting action to gram-positive microorganism.
The proteolytic enzyme that present M23 family has identified has only 9, all is to be produced by the land bacterium.These enzymes activity at low temperatures are lower, and enzyme is stable under middle high temperature should not inactivation and influenced its security, and these shortcomings have seriously limited the application of M23 family protein enzyme.Derive from the M23 family Pancreatopeptidase E of deep-sea bacterium owing to pass through to low temperature and secular adaptation of hypersaline environment and evolution; Have active at low temperatures high; The distinct advantages of at high temperature suitable inactivation, deep-sea proteolytic enzyme is because the environment uniqueness also possibly have new elastin degradation mechanism in addition.These advantages possibly make the Pancreatopeptidase E of marine source have broad application prospects in fields such as medicine, foods and cosmetics.But because difficulties such as sampling and cultivations, the Pancreatopeptidase E of marine source is also seldom studied at present, does not still derive from the report of the M23 family Pancreatopeptidase E at deep-sea.
Summary of the invention:
The present invention is directed to the deficiency of prior art, the gene (pseudoalterin) and preparation method thereof and application of a kind of deep-sea Pancreatopeptidase E is provided.This Pancreatopeptidase E can be used for elastin is carried out enzymolysis, also can be used for treatment by bacterial inflammation such as staphylococcuses.This enzyme enzyme at low temperatures is alive high, and extremely unstable under the middle high temperature, middle temperature just can make its complete deactivation, thereby has guaranteed its security.The present invention also provides the generation bacterium-cold-adaptive microbe bacterium strain Pseudoalteromonas sp.CF6-2 of this Pancreatopeptidase E.
Terminological interpretation:
1.PCR technology: the chain polymerization enzyme reaction, utilize archaeal dna polymerase, template, primer and dNTP carry out the technology of amplification in vitro specific DNA fragments.
2.Tail round pcr: hot asymmetric chain polymerization enzyme reaction, use the primer of two kinds of different lengthss, thereby make the annealing temperature of two kinds of primers different.Long primer is three nested primerss with known array design, and annealing temperature 58-63 ℃, short primer is any degenerated primer, can with DNA random incorporation, annealing temperature 47-48 ℃.Each circulation of PCR temperature cycle comprises: two high temperature annealing reactions, at this moment have only long primer annealing and extension; A low-temperature annealing reaction, at this moment two kinds of primers are annealed simultaneously and are increased.
3.CDD analysis software: a kind of software that is used for inferring and analyzing the structural domain information of specific amino acids sequence formation that provides on the NCBI website.
Technical scheme of the present invention is following:
One strain cold-adaptive microbe bacterium strain (Pseudoalteromonas sp.) CF6-2, this bacterial strain on July 27th, 2010 were preserved in Chinese typical culture collection center, address: Chinese Wuhan Wuhan University, its culture presevation is numbered CCTCC NO:M2010189.
By the right cold Pancreatopeptidase E gene that above-mentioned cold-adaptive microbe bacterium strain (Pseudoalteromonas sp.) CF6-2 extracts, its nucleotide sequence is shown in SEQ ID NO.1.
A kind of expression vector that contains nucleotide sequence shown in the SEQ ID No.1.
A kind of reconstitution cell that contains above-mentioned expression vector.
By above-mentioned deep-sea Pancreatopeptidase E of fitting cold elastin enzyme gene expression translation, its aminoacid sequence is shown in SEQ ID NO.2.
Cold-adaptive microbe bacterium strain of the present invention (Pseudoalteromonas sp.) CF6-2 separates from South China Sea sediments and obtains.Fitting cold Pancreatopeptidase E gene (pseudoalterin) is the genomic dna that from cold-adaptive microbe bacterium strain (Pseudoalteromonas sp.) CF6-2, extracts.At first separation and purification the cold-adaptive microbe bacterium strain CF6-2 right cold Pancreatopeptidase E (pseudoalterin) of being produced, measured the N terminal sequence of this enzyme.According to its N terminal sequence and M23 family protein enzyme conservative gene sequences Design primer, utilize PCR and Tail round pcr from the genomic dna of cold-adaptive microbe bacterium strain CF6-2, to clone the gene of the right cold Pancreatopeptidase E of coding.Nucleotide sequence to this gene is measured.This gene DNA fragment is 1240bp altogether, and the open reading frame coding that wherein contains a 1212bp is fitted cold Pancreatopeptidase E, and initiator codon is positioned at 1bp, and terminator codon is positioned at 1210bp, 403 amino acid of encoding altogether.Therefore, the precursor of fitting cold Pancreatopeptidase E is one and contains 403 amino acid whose polypeptide that it comprises signal peptide, leading peptide and catalyst structure domain three parts.Ripe Pancreatopeptidase E contains 173 amino acid, is a Pancreatopeptidase E of not reporting as yet with new sequence in the metalloprotease M23 family.
Above-mentioned deep-sea Pancreatopeptidase E is the application in sanitas, anti-inflammation and sterilization medicine and the daily cosmetics in preparation low temperature hypersaline environment.
Beneficial effect of the present invention is following:
(1) right cold Pancreatopeptidase E Pseudoalterin of the present invention derives from the deep sea low temperature hypersaline environment; Compare with other Pancreatopeptidase E; Fit cold Pancreatopeptidase E and in 0～30 ℃ of seawater, have very high elastin degradation efficiency, normal temperature just can play a role down.And extremely unstable more than 40 ℃, middle temperature just can make enzyme deactivation, thereby has guaranteed its security.
(2) right cold Pancreatopeptidase E Pseudoalterin of the present invention has lytic activity to multiple gram-positive microorganism; Except being applied to medical treatment; Daily use chemicals etc. also have huge application potential to aspects such as sea life medicine, processing of aquatic products beyond the aspect.
Description of drawings:
The electrophorogram of the genomic dna of the cold-adaptive microbe bacterium strain CF6-2 of Fig. 1, extraction;
Wherein: 1, the genomic dna of cold-adaptive microbe bacterium strain CF6-2; 2, dna molecular amount marker.
Fig. 2, fit the electrophorogram of the gene fragment of cold Pancreatopeptidase E through PCR and Tail pcr amplification clone's coding;
A, regular-PCR amplification; Wherein: 1, dna molecular amount marker; 2, the dna fragmentation (300bp) of amplification;
B, Tail pcr amplification result; Wherein: 1, dna molecular amount marker; 2, N extreme direction third round PCR product (800bp); 3, C extreme direction third round PCR product (250bp);
C, the right full gene electrophoresis result of cold Pancreatopeptidase E; Wherein: 1, dna molecular amount marker; 2, fit cold Pancreatopeptidase E gene.
The right cold Pancreatopeptidase E electrophorogram of Fig. 3, purifying;
Wherein: M, marker; 1, the right cold Pancreatopeptidase E of purifying; 2, cold-adaptive microbe bacterium strain CF6-2 fermented liquid.
Fig. 4, the hydrolysis site of right cold Pancreatopeptidase E on elastin that utilizes liquid matter couplet to analyze;
Wherein: black arrow be in the M23 family other proteolytic enzyme with fit the site that cold Pancreatopeptidase E can hydrolysis; Red arrow be newfound have only fit the site that cold Pancreatopeptidase E can hydrolysis.
Fig. 5, temperature are to fitting the influence curve figure that cold Pancreatopeptidase E enzyme is lived.
Fig. 6, pH value are to fitting the influence curve figure that cold Pancreatopeptidase E enzyme is lived.
The complete genome sequence of Fig. 7, Pancreatopeptidase E and amino acid sequence coded corresponding relation figure thereof;
Wherein: arrow is signal peptide and leading peptide cleavage site; Asterisk is an avtive spot; Trilateral is and the zinc ion action site.
Embodiment
Below in conjunction with embodiment, the present invention is done further elaboration, but institute of the present invention protection domain is not limited thereto.
Cold-adaptive microbe bacterium strain among the embodiment (Pseudoalteromonas sp.) CF6-2, this bacterial strain on July 27th, 2010 were preserved in Chinese typical culture collection center, address: Chinese Wuhan Wuhan University, its culture presevation is numbered CCTCC NO:M2010189.
Embodiment 1: the clone who fits cold elastin enzyme coding gene
1, the extraction of cold-adaptive microbe bacterium strain (Pseudoalteromonas sp.) CF6-2 genomic dna.
Process for extracting carries out as follows and with reference to hundred Imtech genomes extraction test kit specification sheets:
(1) get 1ml cold-adaptive microbe bacterium strain (Pseudoalteromonas sp.) CF6-2, the centrifugal 30sec of 10000rpm abandons supernatant, collects thalline;
(2) add the resuspended washed cell of 200 μ l damping fluid KB, the centrifugal 30sec of 10000rpm is resuspended in cell concussion or piping and druming among the 200 μ l damping fluid KB after abandoning supernatant;
(3) add 200 μ l and combine liquid CB, fully mixing adds 20 μ l Proteinase K (20mg/ml) solution again, and fully mixing is placed 10min for 70 ℃;
(4) the cooling back adds 100 μ l Virahols, and fully mixing gets mixing solutions;
(5) mixing solutions that step (4) is made adds among the adsorption column AC, and the centrifugal 30sec of 10000rpm outwells the waste liquid in the collection tube;
(6) add 500 μ l inhibitions and remove liquid IR, the centrifugal 30sec of 12000rpm abandons waste liquid;
(7) add 700 μ l rinsing liquid WB, the centrifugal 30sec of 12000rpm discards waste liquid;
(8) add 500 μ l rinsing liquid WB, the centrifugal 30sec of 12000rpm discards waste liquid;
(9) adsorption column AC is put back in the sky collection tube, the centrifugal 2min of 13000rpm removes rinsing liquid;
(10) take out adsorption column AC and put into centrifuge tube, be added on the elution buffer EB100 μ l after the preheating in 65～70 ℃ of water-baths in the middle part of adsorption film, room temperature is placed 3～5min, the centrifugal 1min of 12000rpm; The solution that obtains is added in the centrifugal adsorption column again, and room temperature is placed 2min, the centrifugal 1min of 12000rpm; Get the centrifugal solution that obtains and be cold-adaptive microbe bacterium strain (Pseudoalteromonas sp.) CF6-2 genomic dna solution;
(11) it is for use that cold-adaptive microbe bacterium strain (Pseudoalteromonas sp.) CF6-2 genomic dna solution places 4 ℃ of refrigerators preservations.
2, primer design and synthetic
According to the maturing enzyme N terminal sequence ATFTMNLP and the conserved regions sequence TGPHLHFSL of M23 family that fit cold Pancreatopeptidase E; Design two degenerate primer M1:GCNACNTTYACNATGAAYYTNCC (SEQ ID NO.3); M2:NSWRAARTGNARRTGNGGNCCNGT (SEQ ID NO.4), above-mentioned primer give birth to worker biotech company by Shanghai and synthesize.
3, utilize PCR to fit cold elastin enzyme coding gene partial sequence amplification
(1) being primer with M1 and M2, is template with the genomic dna, carries out pcr amplification; The PCR reaction conditions is: 95 ℃, and 5 minutes; 95 ℃ then, 1 minute; 55 ℃, 1 minute; 72 ℃, 2 minutes, after 30 circulations; 72 ℃, extended 10 minutes;
(2) pcr amplification product is carried out 1% agarose gel electrophoresis, the result obtains the dna fragmentation of a treaty 300bp, and the DNA with Omega company reclaims test kit according to its working instructions recovery amplification of DNA fragments then.
(3) dna fragmentation is connected with cloning vector
The amplification of DNA fragments that recovery is obtained is connected on the pGEMT carrier of Promega company, and step can be with reference to pGEMT carrier working instructions.The ligation system:
Add ddH
2O to 10 μ l
The mixing sample, centrifugal 2sec on whizzer concentrates on the pipe end to sample, connects in 16 ℃ of water-baths and spends the night.
(4) (date of publication: 2005-3-1) the competent method of preparation intestinal bacteria prepares the bacillus coli DH 5 alpha competence for Science Press, ISBN:7030103386 by " molecular cloning experiment guide ".
The reorganization pGEMT carrier that (5) will connect by the heat shock method for transformation on " molecular cloning experiment guide " goes to the bacillus coli DH 5 alpha competence.
(6) bacillus coli DH 5 alpha that transforms is applied to the Mai Kangkai substratum (available from sincere biotech company) that contains 50 μ g/L penbritins; 37 ℃ of incubated overnight; Select white transformant as template; As primer, verify through bacterium colony PCR whether plasmid in the white transformant contains the dna fragmentation of pcr amplification with M1 and M2.
(7) transformant that plasmid is contained the dna fragmentation of pcr amplification is served the sea and is given birth to the order-checking of worker Bioisystech Co., Ltd, obtains the sequence of one section 303bp.
4.Tail PCR proceeds to fit cold elastin enzyme coding gene complete sequence amplification
According to the 303bp sequences Design three-wheel nested primers that obtains in 3:
N-1：CACCAGAACCATTGTTAAAGTC(SEQ?ID?NO.5)，
N-2：AAGTCTAATGAAGAATAAGGATAAC(SEQ?ID?NO.6)，
N-3：GGATAACCTGAGCCAGTGTTTGAA(SEQ?ID?NO.7)；
C-1：ACTTACAATACAATAATGGTGACACTG(SEQ?ID?NO.8)，
C-2：GCCAGGAACGTTGCTAGGTC(SEQ?ID?NO.9)，
C-3：AGGTCGTTATGCGAATAGTTA(SEQ?ID?NO.10)。
Design two degenerate primer N:AAKYRTATG at random again; C:GCAGCGTTA.
For the unknown nucleotide sequence that gets fragment N extreme direction, be primer with N-1 and N earlier, with the genome template, carry out first round PCR, condition is: 94 ℃, 30 seconds; 53 ℃, 1 minute; 72 ℃, 2 minutes; 5 circulations.94 ℃ then, 30 seconds; 28 ℃, 3 minutes; 72 ℃, 2 minutes; 94 ℃, 20 seconds; 40 ℃, 1 minute; 72 ℃, 2 minutes; 10 circulations.94 ℃ then, 20 seconds; 53 ℃, 1 minute; 72 ℃, 2 minutes; 94 ℃, 20 seconds; 53 ℃, 1 minute; 72 ℃, 2 minutes; 94 ℃, 30 seconds; 40 ℃, 1 minute; 72 ℃, 2 minutes; 12 circulations.72 ℃ were extended 10 minutes then.
Be that primer carry out second take turns PCR as template with N-2 and N with gained PCR product again, condition is: 94 ℃, and 20 seconds; 55 ℃, 1 minute; 72 ℃, 2 minutes; 94 ℃, 20 seconds; 55 ℃, 1 minute; 72 ℃, 2 minutes; 94 ℃, 20 seconds; 40 ℃, 1.5 minutes; 72 ℃, 2 minutes, 16 circulations.72 ℃ then, extended 10 minutes.
Taking turns the PCR product with second again is template, is that template is carried out third round PCR with N-3 and N, and condition is: 94 ℃, and 30 seconds; 55 ℃, 1 minute; 72 ℃, 2 minutes, 25 circulations.72 ℃ then, extended 10 minutes.
Third round PCR product is carried out 1% agarose gel electrophoresis, obtain the dna fragmentation about a 800bp.Unknown nucleotide sequence to known array C extreme direction carries out pcr amplification with same procedure.Dna fragmentation about 250bp appears in the result.
With same procedure in the present embodiment 3 these two dna fragmentations are checked order then.According to the sequence of this two bar segment, with they with 3 in obtain dna fragmentation and be spliced into a dna fragmentation.The open reading frame that the sequence of splicing dna fragmentation is wherein contained the coding Pancreatopeptidase E Pseudoalterin of a 1212bp through the blastX software analysis discovery of NCBI website.Initiator codon is positioned at 1bp, and terminator codon is positioned at 1210bp, 403 amino acid of encoding altogether.The complete genome sequence and the amino acid sequence coded thereof of Pancreatopeptidase E are as shown in Figure 7.
Embodiment 2: cold-adaptive microbe bacterium strain CF6-2 excretory is fitted the purifying of cold Pancreatopeptidase E
Cold-adaptive microbe bacterium strain CF6-2 is inoculated in fermention medium and (contains the 0.2wt% yeast powder, 0.3wt% elastin (available from sigma company), 0.5mMCaCl in the artificial seawater (according to the plain compound method preparation of the seawater of the salt Shui nationality Science and Technology Ltd. in sea, Qingdao)
2With 0.5mM Na
2HPO
4, pH 8.0), cultivate 48h at 15 ℃.Fermented liquid is at 5 ℃ of centrifugal 10min of 10000g.Supernatant with 50mM Tris-HCl damping fluid (pH 9.5) dialysed overnight after in 5 ℃ of centrifugal 15min of 10000g.Supernatant carries out gradient elution with 0～0.3M NaCl after passing DEAE-Sepharose Fast Flow chromatography column with 2.5min/ml speed.Proteolytic enzyme under the wash-out detects purity (result is as shown in Figure 3) with 12.5%SDS-PAGE.
Embodiment 3: the property testing of fitting cold Pancreatopeptidase E
1, fits the cracking ability of cold Pancreatopeptidase E to the different sorts bacterium
To test abandoning supernatant behind the centrifugal 1min of thalline 10000rpm, clean thalline with 50mM Tris-HCl damping fluid (pH 9.0).The thalline that has cleaned is used the resuspended 20min of boiling of 50mM Tris-HCl damping fluid (pH 9.0) again.Boiling the back sample uses identical damping fluid to be diluted to that light absorption value is 0.8 at the 595nm place; Getting 180 μ l bacterium liquid adding concentration is that 10 μ g/ml fit cold Pancreatopeptidase E enzyme liquid 20 μ l; Measure its 595nm place light absorption value after 25 ℃ of insulation half a hour behind the mixing and change, control sample is with Tris-HCl damping fluid replacement enzyme liquid.The light absorption value reduced rate is as phase counterincision bacterium active (result is as shown in table 1).
Table 1
2, join analysis with liquid matter and fit the restriction enzyme site of cold Pancreatopeptidase E on elastin
Take by weighing 10mg elastin adding 1ml and contain in 50mM Tris-HCl (pH 9.0) damping fluid of the right cold Pancreatopeptidase E of 5 micrograms, shake insulation 2h down at 25 ℃ behind the mixing.Enzymolysis sample is got supernatant 2 μ l and is carried out LC-MS technical Analysis (can explain referring to Agilent Ion Trap 6340 technological operations) behind the centrifugal 5min of 10000rpm.The result shows that right cold Pancreatopeptidase E can be from a plurality of sites degraded elastins (result is as shown in Figure 4).
3, the righttest enzyme temperature alive
The enzyme of under 0,5,10,15,20,25,30,35,40,45,50 ℃ of condition, measuring Pancreatopeptidase E is respectively lived, to confirm the optimal reactive temperature of enzyme.Measuring enzyme concrete grammar alive is:
Elastin verivate (available from sigma company) the adding 250 μ L concentration that take by weighing 5mg are the right cold Pancreatopeptidase E enzyme liquid of 10 μ g/ml, and reaction is 60 minutes under the differing temps, the centrifugal 10min termination reaction of 10000rpm.Get supernatant and measure light absorption value in the 595nm place.The result shows the righttest enzyme of this enzyme 25 ℃ of temperature (result is as shown in Figure 5) alive.
4, ph optimum
Under pH 6.5,7.0,7.5,8.0,8.5,9.0,9.5,10.0,10.5,11.0 conditions, measure the enzyme of fitting cold Pancreatopeptidase E respectively and live, to confirm the ph optimum of this enzyme.Buffer system (50mmol/L): pH 6.5-8.0, Na
2HPO
4-NaH
2PO
4Damping fluid; PH 7.5-9.5, Veronal sodium-HCl damping fluid; PH 9.5-11.0, NaHCO
3-NaOH damping fluid.
Concrete grammar is a damping fluid of preparing different pH with above-mentioned damping fluid, measures the enzyme of right cold Pancreatopeptidase E under condition of different pH according to the method in the present embodiment 3 down at 25 ℃ then and lives.The result shows that the ph optimum of this enzyme is 9.5 (result is as shown in Figure 6).
Claims (3)
1. a strain cold-adaptive microbe bacterium strain Pseudoalteromonas sp.CF6-2, this bacterial strain on July 27th, 2010 were preserved in Chinese typical culture collection center, address: Chinese Wuhan Wuhan University, its culture presevation is numbered CCTCC NO:M2010189.
2. right cold Pancreatopeptidase E that is extracted by the said cold-adaptive microbe bacterium strain Pseudoalteromonas of claim 1 sp.CF6-2 makes through following method:
Cold-adaptive microbe bacterium strain CF6-2 is inoculated in fermention medium; Cultivate 48h at 15 ℃; Fermented liquid is at 5 ℃ of centrifugal 10min of 10000g; Supernatant with the Tris-HCl damping fluid dialysed overnight of 50mM, pH 9.5 after in 5 ℃ of centrifugal 15min of 10000g, supernatant carries out gradient elution with 0～0.3M NaCl after passing DEAE-Sepharose Fast Flow chromatography column with 2.5min/ml speed;
Said fermention medium is to contain 0.2wt% yeast powder, 0.3wt% elastin, the 0.5mM CaCl available from sigma company in the artificial seawater
2With 0.5mM Na
2HPO
4, pH 8.0; Artificial seawater is according to the plain compound method preparation of the seawater of the salt Shui nationality Science and Technology Ltd. in sea, Qingdao.
3. the said deep-sea of claim 2 Pancreatopeptidase E application in sanitas, anti-inflammation and sterilization medicine and the daily cosmetics in preparation low temperature hypersaline environment.
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