CN109609524A - Protein and the application of a kind of lactobacillus plantarum nitrite reductase gene and its coding - Google Patents
Protein and the application of a kind of lactobacillus plantarum nitrite reductase gene and its coding Download PDFInfo
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- CN109609524A CN109609524A CN201910116450.XA CN201910116450A CN109609524A CN 109609524 A CN109609524 A CN 109609524A CN 201910116450 A CN201910116450 A CN 201910116450A CN 109609524 A CN109609524 A CN 109609524A
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- protein
- nitrite reductase
- lactobacillus plantarum
- nitrite
- nir
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- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 150000001989 diazonium salts Chemical class 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 235000021121 fermented vegetables Nutrition 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- 108010062266 glycyl-glycyl-argininal Proteins 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 108010092114 histidylphenylalanine Proteins 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 201000002313 intestinal cancer Diseases 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 108010017391 lysylvaline Proteins 0.000 description 1
- 235000013622 meat product Nutrition 0.000 description 1
- 208000005135 methemoglobinemia Diseases 0.000 description 1
- 108010005942 methionylglycine Proteins 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N nitrate group Chemical group [N+](=O)([O-])[O-] NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 108010073025 phenylalanylphenylalanine Proteins 0.000 description 1
- 108010051242 phenylalanylserine Proteins 0.000 description 1
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229940016590 sarkosyl Drugs 0.000 description 1
- 108700004121 sarkosyl Proteins 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- KSAVQLQVUXSOCR-UHFFFAOYSA-M sodium lauroyl sarcosinate Chemical compound [Na+].CCCCCCCCCCCC(=O)N(C)CC([O-])=O KSAVQLQVUXSOCR-UHFFFAOYSA-M 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 108010031491 threonyl-lysyl-glutamic acid Proteins 0.000 description 1
- 108010044292 tryptophyltyrosine Proteins 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
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- 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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0044—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7)
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- 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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- C12Y—ENZYMES
- C12Y107/00—Oxidoreductases acting on other nitrogenous compounds as donors (1.7)
- C12Y107/01—Oxidoreductases acting on other nitrogenous compounds as donors (1.7) with NAD+ or NADP+ as acceptor (1.7.1)
- C12Y107/01004—Nitrite reductase [NAD(P)H] (1.7.1.4)
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Abstract
The invention discloses protein and the applications of a kind of lactobacillus plantarum nitrite reductase gene and its coding, belong to bioengineering field.The present invention is that the nitrite reductase gene in Lactobacillus plantarum is cloned to using round pcr and carried out in Escherichia coli inducing expression, obtains the recombinant protein of high-purity using affinity chromatography effect purifying.The protein of the high-purity purified using method of the invention can effectively be degraded the nitrite in food, and can be used for its structure and property Quality Research.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to a kind of lactobacillus plantarum nitrite reductase gene
Recombinant vector, the host with carrier conversion and utilization the transformant great expression nitrite reductase and nitrite
The high-performance affinity chromatography purification process of reductase.
Background technique
Nitrite is a kind of potential carcinogen, is easily accumulated during fermented vegetable, is brought to product potential
A product safety problem, and Excess free enthalpy nitrite can induce methemoglobinemia, therefore strict control food nitrite nitre
The content of hydrochlorate is extremely important.Many studies have shown that nitrite is the precursor of nitrosamine, nitrosamine is a kind of strong carcinogen,
A variety of cancerations in digestive system can be induced, such as gastric cancer, intestinal cancer and liver cancer.In view of food, there may be exceeded nitrite dirts
Potential food-safety problem containing nitrite in dye and meat products seeks the effectively method gesture of control or degrading nitrite
It must go, other than the lactic acid bacteria of edible safety is added, the life of nitrite is also carried out using nitrite reductase (NiR)
Object degradation.But to the nitrite reductase for how obtaining a large amount of edible safety, there are no good methods at present.Mainly
It is to separate skill with routine protein because the difficulty that isolates and purifies of zymoprotein causes production cost to be too high to realize industrialization
Art is compared, fixing metal ions affinity chromatography technology (Imobilized metal ion affinitychromatography,
Abbreviation IMAC) it is used for the separation of zymoprotein, selectivity is high, is able to achieve high-throughput, high absorption isolate and purify.Isolated principle master
Affinity interaction occurs using histidine, tryptophan, cysteine of protein surface etc. and fixing metal ions and realize enzyme egg
The separation of white matter.Therefore, designing polyhistidyl, tryptophan, cysteine, arginine etc. is affinity tag and purpose zymoprotein
Purified again with IMAC after fusion, can reach expected separating effect.
Nitrite reductase and great expression report are extracted from lactic acid bacteria both at home and abroad seldom, utilizes letter in the prior art
The method of single Mechanical Crushing and centrifugation collects thick enzyme, due to containing a large amount of foreign protein in crude enzyme liquid, so that subsequent separation
With purification work complexity, target product nitrite reductase can not be largely obtained.Application No. is in 201210037774.2
State's invention patent discloses " a kind of nitrite reductase gene of lactobacillus plantarum and its protein and the application of coding ",
Whether middle verifying nitrite reductase expresses successfully the holoprotein SDS-PAGE electrophoresis knot that main foundation is recombinant microorganism
Fruit does not go deep into the isolating and purifying of the recombinant protein, enzyme activity and application further, due to microbial engineering bacteria E.coli BL21,
E.coli DE 3 and JM105 can express nitrite reductase before conversion, the method by inventing above, can not be fine
Whether the expression for identifying nitrite reductase succeeds.
Summary of the invention
The deficiency of great expression, extraction and purifying for the existing nitrite reductase of solution etc., the present invention provide
- kind of nitrite reductase encoding gene recombination method, and provide a kind of nitrite recombinant protein great expression and
The method of purifying carries out nitrous acid mainly using the prokaryotic expression carrier pET-28a (+) for containing histidine tag (His label)
The recombination of salt reductase gene, then recombinant vector is transformed into host, and carry out great expression nitrite also using transformant
Protoenzyme carries out purifying nitrite albumen using affinity chromatography.
To achieve the goals above, present invention employs following technical solutions:
A kind of lactobacillus plantarum nitrite reductase gene, nucleotide sequence is as shown in SEQ ID NO.1.
Contain histidine tag in recombinant plasmid containing lactobacillus plantarum nitrite reductase gene.
A kind of protein of lactobacillus plantarum nitrite reductase gene coding, amino acid sequence such as SEQ ID
Shown in NO.2.The preparation method of the protein of above-mentioned reductase gene coding, includes the following steps:
(1) using lactobacillus plantarum nitrite reductase encoding gene in NCBI as template, upstream and downstream primer design is carried out,
The restriction enzyme site of design is NcoI, XhoI；
(2) bacterium solution after Lactobacillus plantarum cultivates 16h at 37 DEG C, extracts its DNA, and as mould
Plate expands the genetic fragment of lactobacillus plantarum nitrite reductase by round pcr (as shown in SEQ ID NO.1)；
(3) amplified fragments and plasmid pET-28a (+) are cut into respectively at 4 DEG C with two using NcoI, XhoI toolenzyme
The segment of a cohesive end；Two above segment is recycled using QIAquick Gel Extraction Kit, and is connected with T4DNA ligase, then be transferred to weight
Group Escherichia coli in, using antibiotic card receive mycin carry out screening can be obtained positive engineering bacteria；
(4) it by above-mentioned engineering bacteria through inducing expression, is purified using affinity chromatography to get recombination nitrate reductase is arrived
Enzyme.
Affiliated recombination bacillus coli is E.coli DH5 α or E.coli BL21.
Upstream primer described in step (1) is 5-CTCGAGAAAAGACCATGGCCAA-3 (NcoI),
Downstream primer is 5-CTTAAGAATCCTGTTTGGAC-3 (XhoI).
The inducing expression is that inducer IPTG is added to be induced, and the concentration of IPTG is 1mmol/L.
The affinity chromatography is Ni sepharose affinity chromatography.
When prepared nitrite reductase determination of activity, 15 μ L 0.1mol/L need to be added in 500 μ L reaction systems
Electron donor methyl viologen, NiR determination of activity use Na2S2O4- MV method.
The protein that the present invention is prepared, the nitrite in the food that can effectively degrade, and can be used for it
Structure and property Quality Research.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) present invention engineering bacteria is verified by affinity chromatography effect can be with inducing expression nitrite reductase (NiR)
Recombinant protein.
(2) present invention can obtain a large amount of nitrite reductase by IPTG inducing expression, can be applied to industry
Middle mass production nitrite reductase.
(3) present invention obtains the higher NiR recombinant protein of purity using affinity chromatography effect purifying, solves from plant cream
The problem of the higher nitrite reductase of purity is isolated in bacillus.
(4) recombinant protein of engineering bacterium expression that the present invention obtains can effective degrading nitrite, can be produced into
Enzyme preparation, the nitrite in the food that can effectively degrade；It is anti-for lactobacillus plantarum nitrite reductase to be alternatively arranged as antigen
The preparation of body, and can be used for the research to its structure and properties.
Detailed description of the invention
Fig. 1 be in embodiment 1 using Lactobacillus plantarum genomic DNA be template carry out PCR amplification production
Object qualification figure, wherein M is DNA molecular amount；1~5 is pcr amplification product.
Fig. 2 is that the recombinant plasmid constructed in embodiment 2 carries out double digestion qualification figure, and wherein M is DNA molecular amount；1 is
PET28a-nir is by NcoI, XhoI product；2 be plasmid pET28a-nir.
Fig. 3 is that the electrophoresis detection knot after the E.coli BL21 containing recombinant plasmid pET28a-nir is induced in embodiment 3
Fruit.M indicates protein marker；1 precipitates for pET28a；2 be pET28a supernatant；3 be not induce pET28a/BL21 (DE3) heavy
It forms sediment；4 be not induce pET28a/BL21 (DE3) supernatant；5 be induction pET28a-nir/BL21 (DE3) supernatant；6 be induction
PET28a-nir/BL21 (DE3) precipitating.
Fig. 4 is the SDS-PAGE electrophoresis of the protein of the NiR in embodiment 4 after purification, and wherein M is protein
marker；1-4 is Ni2 +80mmol/L, 100mmol/L, 150mmol/L, 300mmol/L imidazole solution eluent of column.
Fig. 5 is zymologic property measurement in embodiment 5, and pH is to the effect tendency figure of enzymatic activity, and wherein a is to recombinate NiR most
Suitable pH, b are the pH stability of enzyme.
Fig. 6 is zymologic property measurement in embodiment 5, and temperature is to the effect tendency figure of enzymatic activity, and wherein a is recombination NiR
Optimum temperature, b are the thermal stability of enzyme.
Specific embodiment
Below with reference to embodiment, invention is further described in detail, but the claimed scope of the invention is not limited to
In this.
Embodiment 1
The preparation of the genomic DNA of lactobacillus plantarum nitrite reductase, includes the following steps:
1, PCR primer designs
PCR primer is designed with the DNA of object lactobacillus nitrite reductase in Genbank:
Upstream primer (SEQ IDNO.3) is 5-CTCGAGAAAAGACCATGGCCAA-3 (NcoI),
Downstream primer (SEQ IDNO.4) be 5-CTTAAGAATCCTGTTTGGAC-3 (XhoI), and it is artificial synthesized this to upper
Downstream primer；
2, extraction (the bacterial genomes DNA small scale purification reagent of the genomic DNA of lactobacillus plantarum nitrite reductase
Box Takara Minibest Bacterial Genomic DNA Extraction Kit Ver.2.0 is Takara
The product of Biotechnology company is purchased from Guangzhou Rui Zhen Bioisystech Co., Ltd), wherein solution is in kit
Solution, specific steps are as follows:
(1) lactobacillus plantarum (Lactobacillus plantarum) 3~4mL of culture is centrifuged 1min in 12000rpm
Supernatant is abandoned, bacterial sediment is placed in the EP of 1.5mL, and adding 0.6mL concentration is 10% lysozyme soln, is mixed by inversion 5~10
37 DEG C of heat preservation at least 30min are put after secondary；
Lactobacillus plantarum (Lactobacillus plantarum) is preserved in China Microbiological bacterium on July 6th, 2014
Kind preservation administration committee common micro-organisms center, address: the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences is micro-
Biological study institute, postcode: 100101, biological deposits number are CGMCC NO.1.12935.
(2) 12000rpm room temperature is centrifuged 10min, carefully abandons supernatant.
(3) 150 μ L SP Buffer (Al containing RNase) sufficiently suspended bacterial precipitating is added.
(4) the Lysozyme solution of 20 μ L is added, is stored at room temperature 5min after evenly mixing.
(5) the EDTA Buffer of 30 μ L is added, is stored at room temperature 5min after evenly mixing.
(6) the Solution A of 200 μ L is added, acutely 65 DEG C of heat preservation 10min after concussion.
(7) the Solution B of 400 μ L is added, acutely shakes 15s.
(8) the Solution C of 650 μ L, uniform mixing of turning upside down is added.
(9) 12000rpm is centrifuged l min.
(10) upper organic phase is first discarded, then aqueous phase solution (colourless lower layer) is moved into and is previously placed in 1.5mL centrifuge tube
On Filter Cup in, 12000rpm be centrifuged l min.
(11) Filter Cup is abandoned, the DB Buffer of 450 μ L is added in filtrate, is uniformly mixed.
(12) the Spin Column in kit is placed on Collection Tube.
(13) mixed liquor of above-mentioned (11) is transferred in Spin Colum, 12000rpm is centrifuged 1min, abandons filtrate.
(14) the Rinse A of 500 μ L is added into SpinColumn, 12000rpm is centrifuged l min, abandons filtrate.
(15) the Rinse B of 700 μ L is added into Spin Colum, 12000rpm is centrifuged 1min, abandons filtrate.
(16) repetitive operation step (15).
(17) Spin Column is placed on Collection Tube, 12000rpm is centrifuged 1min.
(18) Spin Column is placed on the centrifuge tube of new 1.5mL, is added in the center of Spin column film
The sterile purified water of 60 μ L, is stored at room temperature 1minA.
(19) 12000rpm is centrifuged l min eluted dna.
3, the amplification of target gene
Using the lactobacillus plantarum genomic DNA extracted in step 2 as template, using PCR method amplifying target genes segment,
PCR reaction condition: 94 DEG C of denaturation 5min.Loop parameter are as follows: 94 DEG C of 30s, 65 DEG C of 45s (every circle reduces 2 DEG C when annealing), 72 DEG C
1min, 5 circulations.Loop parameter are as follows: 94 DEG C of 30s, 55 DEG C of 45s, 72 DEG C of 1min, 72 DEG C of 10s, 25 circulations, PCR reaction knot
Beam obtains the genomic DNA of the lactobacillus plantarum nitrite reductase of PCR extension, as shown in SEQ IDNO.1；It sets up simultaneously
Negative control carries out aforesaid operations using sterile water as template.
With 1% agarose electrophoresis testing goal gene PCR product；Electrophoresis detection result such as Fig. 1, there is one near 1300kb
The apparent DNA band of item, the DNA band are above-mentioned resulting lactobacillus plantarum NiR genomic DNA.
Embodiment 2
The structure of the recombinant expression carrier of the genomic DNA of lactobacillus plantarum NiR containing the resulting PCR of embodiment 1 extension
It builds, steps are as follows:
By the amplified fragments DNA and plasmid of the lactobacillus plantarum nitrite reductase of the resulting PCR of embodiment 1 extension
PET-28a (+) carries out NcoI, XhoI digestion respectively, and piece segment DNA is then separately recovered in 37 DEG C of overnight 14~16h in reaction condition
And Plasmid DNA.
Above-mentioned two segment is attached, in 4 DEG C of 14~16h of refrigerator cold-storage reaction overnight to get arrive connection product,
Middle coupled reaction system are as follows: recycling digestion pET28a (+) 19 μ L recycles 2.5 μ L, T4DNA connection of target gene fragment, 1 μ L,
2.5 μ L of T4DNA ligase buffer solution；
Above-mentioned connection product is converted to E.coli DH5 α cell, i.e. acquisition recombinant plasmid pET28a (+)-nir again；
Recombinant plasmid pET28a (+)-nir of acquisition is analyzed through NcoI and XhoI double digestion, 1% agarose gel electrophoresis,
It can be seen that the carrier segments of about 5.4kb and the target gene fragment (Fig. 2) of 1.3kb.To recombinant expression carrier pET28a (+)-nir into
Row DNA sequencing is inserted into gene order as the result is shown and implementation sequence is completely the same, successfully constructs recombinant expression carrier pET28a
(+)-nir。
Embodiment 3
Expression vector pET28a (+)-nir plasmid that embodiment 2 is built, is transferred to E.coli BL21 competent cell
In, it obtains recombination bacillus coli and carries out inducing expression, the specific steps of which are as follows:
By the expression strain inoculated built into LB/Kan fluid nutrient medium, then 37 DEG C of shaken overnight culture 14h divide
It is not inoculated in the triangular flask containing 100mL LB/Kan fluid nutrient medium by 1% bacterium solution amount, 37 DEG C of shaken cultivation 4h (bacterium solutions
OD600 reaches 0.4~0.6) when, IPTG, which is added, makes ultimate density 1mmol/L；It is compared simultaneously with empty carrier and not inducing.
Bacterium solution 8000r/min is centrifuged 10min, discards supernatant liquid, precipitating thallus is washed twice with PBS buffer solution (pH7.4), will be washed
Thallus afterwards is resuspended in the PBS buffer solution (pH7.4) of 10mL pre-cooling, and ultrasound cracking 10min, each 3s are spaced 5s, power
200W.Then 10000r/min, is centrifuged 20min by 4 DEG C, respectively supernatant, to be precipitated as protein sample solidifying through denaturing polyacrylamide
Gel electrophoresis (SDS-PAGE) detection, records and analyzes result.From the figure 3, it may be seen that swimming lane is unloaded, does not induce host strain and induction bacterium
The broken supernatant of strain is showed no destination protein, and occurs special item at the position broken precipitating swimming lane about 45kD of inducible strain
Band is consistent with the relative molecular mass (44.959kD) of expected expression, and amino acid sequence is as shown in SEQ ID NO.2, explanation
Destination protein NiR successful expression in host strain E.coliBL21 (DE3), but destination protein is only detected in precipitating, thus
It is found that destination protein NiR is in Escherichia coli with the insoluble expression of inclusion bodies.
Embodiment 4
It will be crushed the precipitating after being centrifuged in embodiment 3 and carry out renaturing inclusion bodies, protein purification and enzyme activity determination, specific steps
It is as follows:
By the sediment in embodiment 3 with 20ml inclusion body cleaning solution (2mol/L urea, 1 ‰ triton x-100s, 1 ‰ ten
Sarkosyl sodium, 1mmol/LDTT, 20mmol/L Tris-HCl (pH 8.0), 1mmol/L EDTA, 50mmol/L
NaCl it) is washed 3 times under the conditions of 4 DEG C, sets 8000r/min centrifugation 20min；Precipitating 10ml protein denaturation liquid after washing
Inclusion body is resuspended in (6mol/L urea, 1mmol/L DTT, 1% sarcosyl), and 8000r/min is centrifuged 30min, abandons
Fall precipitating；By above-mentioned deformed protein liquid renaturation solution (20mmol/L Tris-HCl, 50mmol/L NaCl, 1mmol/L
DTT, 0.1% triton x-100,20% glycerol) after 4 DEG C of stirring dialysis for 24 hours, gradually change to 10% glycerol of final concentration, 4 DEG C of stirrings
Dialysis is for 24 hours.Destination protein inclusion body in bag filter is crude enzyme liquid after denaturation dissolution.Crude protein is affine with Ni sepharose
Chromatography carries out affinitive layer purification, and 4 DEG C of protein liquid preservations of elution, protein sample is through denaturing polyacrylamide gel electrophoresis
(SDS-PAGE) it detects, records and analyzes result.
The measurement of NiR enzymatic activity: NiR determination of activity uses Na2S2O4- MV method.It is supplied using methyl viologen as artificial electron
Body makes NiR be catalyzed NO2 -It is reduced to NO or NH3.The consumption of nitrite reductase can pass through nitrite total in reaction solution
Amount subtracts remaining NO2 -It measures.NO2 -Content can be measured with hydrochloric acid-naphthalene-ethylenediamine method, i.e., in acid condition with p-aminophenyl
Diazo reaction occurs for sulfonic acid, and the diazonium compound of generation generates aubergine azo-compound with hydrochloride naphthodiamide again, can be
Chromogenic assay under 538nm.
Measure 500 μ L:0.1mol/L phosphate buffer (pH 6.5) of enzyme activity reaction system, 50 μ L, 0.1mol/L NaNO2
25 μ L, 0.1mol/L MV, 15 μ L, 0.1mol/L Na2S2O480 μ L, 300 μ L of enzyme solution.10min is reacted in 37 DEG C of water-baths, acutely
Oscillation terminates reaction (using phosphate buffer as blank).10 μ L are taken to measure nitrate residue with hydrochloric acid-naphthalene-ethylenediamine method.It is sub-
Vitality of nitrate reductase unit by 37 DEG C, restoring enzyme amount consumed by 1 μm of ol nitrite to indicate per minute.Than work
Power is indicated with the unit of activity number of enzyme in 1mg protein.
Under cryogenic, resulting inclusion body will be centrifuged first to be washed with buffer, and will remove inclusion body surface impurity, then right
Inclusion body is dissolved, and supernatant is collected.Through dialysis renaturation, high concentration urea is removed, recombinant protein space conformation is gradually recovered,
Albumen after detection renaturation has corresponding bioactivity.
Protein renaturation liquid passes through Ni sepharose affinity chromatography purifying recombination NiR (such as Fig. 4).Purify NiR total protein
Content is 10.3mg, Rate activity 226.48U/mg, total activity 2332.72U, overall recovery 63.00%, and purification is
7.32, as shown in table 1, it can be seen that recombinant protein can effective degrading nitrite.
The purifying of the recombination of table 1 NiR
Table 1Summary of NiRenzyme
Embodiment 5
The zymologic property of the nitrite reductase purified in measurement embodiment 4, the specific steps are as follows:
Recombinate the property analysis of NiR: optimal reactive temperature: under conditions of pH value is 6.5, in 4 DEG C, 10 DEG C, 20 DEG C, 25
DEG C, 30 DEG C, 35 DEG C, 37 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, measure enzyme activity at a temperature of 70 DEG C, be 100% to calculate phase with 37 DEG C of enzyme activity
To enzyme activity.
The thermal stability of enzyme: enzyme solution is respectively placed in different temperatures (4 DEG C, 10 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 37
DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C) under water-bath 40min, detect remnant enzyme activity at optimal reaction pH and optimal reactive temperature,
And enzyme activity when not keeping the temperature is the opposite enzyme activity of 100% calculating.
Optimal reaction pH value: under most suitable enzyme activity reaction temperature, pH be 2.0,3.0,4.0,4.5,5.0,5.5,6.0,
6.5, enzyme activity is measured under conditions of 7.0,7.5,8.0,9.0,10.0,11.0,12.0, be 100% to calculate phase with the enzyme activity of pH6.5
To enzyme activity, enzyme activity curve is drawn.
The pH stability of enzyme: by enzyme solution be respectively placed in different pH buffers (pH2.0,3.0,4.0,4.5,5.0,5.5,
6.0,6.5,7.0,7.5,8.0,9.0,10.0,11.0,12.0) in water-bath 40min, in optimal reaction pH and optimal reactive temperature
Lower detection remnant enzyme activity, and enzyme activity when not keeping the temperature is 100% to calculate relative activity, draws enzyme activity curve.
It is reduced afterwards by a in Fig. 5 it is found that recombinating NiR and first being increased within the scope of pH 2.0-12.0 with respect to enzyme activity, is 6.5 in pH
When reach maximum value, gradually decreased later with respect to enzyme activity, therefore, recombinate NiR optimum pH be 6.5.Under the conditions of 37 DEG C, point
Not Cai Yong different pH buffers be incubated for recombination NiR, the results showed that, stability of the enzyme between pH 6.5-8.0 is preferable, relatively
Enzyme activity is maintained at 60% or more.Wherein, when pH is 7.0 most stable (in Fig. 5 shown in b).
By a in Fig. 6 it is found that recombination NiR optimum temperature be 37 DEG C, when temperature is at 4-70 DEG C with 91% or more it is opposite
Enzyme activity.Under the conditions of 6.5 pH, it is incubated for recombination NiR at different temperatures respectively, the results showed that, the enzyme is between 4 DEG C -70 DEG C
Temperature stability is preferable, and opposite enzyme activity is maintained at 85% or more.Wherein, at 37 DEG C most stable (in Fig. 6 shown in b), have wide
General thermal adaptability.
Sequence table
<110>University Of Dalian
<120>protein and the application of a kind of lactobacillus plantarum nitrite reductase gene and its coding
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1257
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
atggcaaaaa ttattattgt cggggctgca catggcggcc gcgaaacggt caatggctta 60
ttagcagcta atacggataa tgagattcat tggtacgagc acgggcaatt cgcgacggct 120
ttggattggg ctccggcgga tgctgaaaaa gagcggttgg ccttgtccca gcaggtgact 180
ctattcgacc aaacgacggt cacaagaatt accccagcga cccacacgat tactgctcgt 240
aatcaacggg ggcaattaca gactgatcat tacgatcgat tggtattgag cgttggatcg 300
ttaccaatcc agttaccgat tcccggagcc gaattatctg gtgttcgatc gattcaaaac 360
cgtgccagca tcaatgagtt aaaattggcc gctaagtcag cagcaattaa aaacgtggtc 420
gtgattggag gcggctatat tgggatgaat tttgcagcct tatttaaaca aaccggcaag 480
caagtaactg ttattgatgt gaacgctcgg ccattcagtc acaatcttga ttcagaattt 540
acgcaaattt tagctgcagc gagtgttgag aatgggctgc aattaaagat ggaagagcgg 600
gtgacggcca tattaggttc aacacacgtg acagcggtac aaacgaatcg tggtcagtat 660
gctgccgact tagtccttgt cgcagtcggc aatcggccga atactgcatg gctacgggga 720
actttgacgc tagattctga gggattaatt gagacggatg attattttca aacaagtgtt 780
ccagatattt acgcgattgg cgatgcgact aaagttcggt ttacgcccac gggtactaaa 840
gagcggatca ctttaggcag cgcggccagt catgctggtc ggttattagc gcataacttg 900
ttaacggatc agcgaattgt gtttcccggc gttcaggcaa cgtccgcgct taatgcggcg 960
ggatattact ttgctgctac gggcctaaac acgcaactag ctgtccgtat gcaacaacca 1020
gtgttagcaa cttacatcgc ggttccccga ttggtggcat ccgcaccagc tcgattgaat 1080
gcgaccgttc attttaagtt gttttatgat aaaactcatc ggatactagg tgcacaaata 1140
atggctacag cagaattaac ggcggtcatc aataccgttt cgctcgcaat ccagatggga 1200
gcaacgctcg agcaattagc ctatggtgac tttttctttc aaccgggatt aagctag 1257
<210> 2
<211> 418
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Met Ala Lys Ile Ile Ile Val Gly Ala Ala His Gly Gly Arg Glu Thr
1 5 10 15
Val Asn Gly Leu Leu Ala Ala Asn Thr Asp Asn Glu Ile His Trp Tyr
20 25 30
Glu His Gly Gln Phe Ala Thr Ala Leu Asp Trp Ala Pro Ala Asp Ala
35 40 45
Glu Lys Glu Arg Leu Ala Leu Ser Gln Gln Val Thr Leu Phe Asp Gln
50 55 60
Thr Thr Val Thr Arg Ile Thr Pro Ala Thr His Thr Ile Thr Ala Arg
65 70 75 80
Asn Gln Arg Gly Gln Leu Gln Thr Asp His Tyr Asp Arg Leu Val Leu
85 90 95
Ser Val Gly Ser Leu Pro Ile Gln Leu Pro Ile Pro Gly Ala Glu Leu
100 105 110
Ser Gly Val Arg Ser Ile Gln Asn Arg Ala Ser Ile Asn Glu Leu Lys
115 120 125
Leu Ala Ala Lys Ser Ala Ala Ile Lys Asn Val Val Val Ile Gly Gly
130 135 140
Gly Tyr Ile Gly Met Asn Phe Ala Ala Leu Phe Lys Gln Thr Gly Lys
145 150 155 160
Gln Val Thr Val Ile Asp Val Asn Ala Arg Pro Phe Ser His Asn Leu
165 170 175
Asp Ser Glu Phe Thr Gln Ile Leu Ala Ala Ala Ser Val Glu Asn Gly
180 185 190
Leu Gln Leu Lys Met Glu Glu Arg Val Thr Ala Ile Leu Gly Ser Thr
195 200 205
His Val Thr Ala Val Gln Thr Asn Arg Gly Gln Tyr Ala Ala Asp Leu
210 215 220
Val Leu Val Ala Val Gly Asn Arg Pro Asn Thr Ala Trp Leu Arg Gly
225 230 235 240
Thr Leu Thr Leu Asp Ser Glu Gly Leu Ile Glu Thr Asp Asp Tyr Phe
245 250 255
Gln Thr Ser Val Pro Asp Ile Tyr Ala Ile Gly Asp Ala Thr Lys Val
260 265 270
Arg Phe Thr Pro Thr Gly Thr Lys Glu Arg Ile Thr Leu Gly Ser Ala
275 280 285
Ala Ser His Ala Gly Arg Leu Leu Ala His Asn Leu Leu Thr Asp Gln
290 295 300
Arg Ile Val Phe Pro Gly Val Gln Ala Thr Ser Ala Leu Asn Ala Ala
305 310 315 320
Gly Tyr Tyr Phe Ala Ala Thr Gly Leu Asn Thr Gln Leu Ala Val Arg
325 330 335
Met Gln Gln Pro Val Leu Ala Thr Tyr Ile Ala Val Pro Arg Leu Val
340 345 350
Ala Ser Ala Pro Ala Arg Leu Asn Ala Thr Val His Phe Lys Leu Phe
355 360 365
Tyr Asp Lys Thr His Arg Ile Leu Gly Ala Gln Ile Met Ala Thr Ala
370 375 380
Glu Leu Thr Ala Val Ile Asn Thr Val Ser Leu Ala Ile Gln Met Gly
385 390 395 400
Ala Thr Leu Glu Gln Leu Ala Tyr Gly Asp Phe Phe Phe Gln Pro Gly
405 410 415
Leu Ser
<210> 3
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ctcgagaaaa gaccatggcc aa 22
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
cttaagaatc ctgtttggac 20
Claims (10)
1. a kind of lactobacillus plantarum nitrite reductase gene, which is characterized in that its nucleotides sequence is classified as SEQ ID NO.1 institute
Show.
2. the protein of the coding of gene described in claim 1.
3. protein according to claim 2, which is characterized in that its amino acid sequence is as shown in SEQ ID NO.2.
4. the recombinant expression carrier containing gene described in claim 1.
5. recombinant expression carrier according to claim 4, which is characterized in that the expression vector is pET-28a (+).
6. the host cell containing gene described in claim 1, which is characterized in that the host cell is Bacillus coli cells.
7. a kind of lactobacillus plantarum nitrite reductase gene according to claim 1, which is characterized in that described goes back
The preparation method of the protein of nitroreductase gene coding, includes the following steps:
(1) preparation of the genomic DNA of lactobacillus plantarum nitrite reductase
1) PCR primer is designed with the DNA of lactobacillus plantarum nitrite reductase in Genbank:
Upstream primer (SEQ IDNO.3) is 5-CTCGAGAAAAGACCATGGCCAA-3 (NcoI),
Downstream primer (SEQ IDNO.4) be 5-CTTAAGAATCCTGTTTGGAC-3 (XhoI), and it is artificial synthesized this to upstream and downstream
Primer；
2) extraction of the genomic DNA of lactobacillus plantarum nitrite reductase；
3) amplification of target gene；
(2) building of recombinant expression carrier pET28a (+)-nir
(3) expression vector pET28a (+)-nir plasmid built, is transferred in E.coli BL21 competent cell, is recombinated
Escherichia coli carry out inducing expression
By the expression strain inoculated built into LB/Kan fluid nutrient medium, then 37 DEG C of shaken overnight culture 14h are pressed respectively
1% bacterium solution amount is inoculated in the triangular flask containing 100mL LB/Kan fluid nutrient medium, 37 DEG C of shaken cultivation 4h, and IPTG is added
Make ultimate density 1mmol/L；Bacterium solution 8000r/min is centrifuged 10min, discards supernatant liquid, precipitating thallus is slow with pH7.4 PBS
Fliud flushing washes twice, and the thallus after washing is resuspended in the pH7.4PBS buffer of 10mL pre-cooling, ultrasound cracking 10min, often
Secondary 3s is spaced 5s, power 200W；Then 10000r/min, 4 DEG C, be centrifuged 20min, respectively supernatant, be precipitated as protein sample warp
Denaturing polyacrylamide gel electrophoresis SDS-PAGE detection, records and analyzes as a result, swimming lane is unloaded, does not induce host strain and lures
The broken supernatant for leading bacterial strain is showed no destination protein, and occurs at the position broken precipitating swimming lane about 45kD of inducible strain special
Band is consistent with the relative molecular mass 44.959kD of expected expression, and illustration purpose albumen NiR is in host strain E.coliBL21
Successful expression, but destination protein is only detected in precipitating；
(4) renaturing inclusion bodies, protein purification to get to recombination nitrite reductase；
Sediment is washed 3 times under the conditions of 4 DEG C with 20ml inclusion body cleaning solution, sets 8000r/min centrifugation 20min；After washing
Inclusion body is resuspended in precipitating 10ml, and 8000r/min is centrifuged 30min, discards precipitating；By above-mentioned deformed protein liquid renaturation solution
After 4 DEG C of stirring dialysis for 24 hours, 10% glycerol of final concentration is gradually changed to, for 24 hours, the destination protein in bag filter is forgiven for 4 DEG C of stirring dialysis
Body is crude enzyme liquid after denaturation dissolution, as recombination nitrite reductase, crude enzyme liquid Ni sepharose affinity chromatography
Carry out affinitive layer purification, 4 DEG C of protein liquid preservations of elution.
8. the preparation method of protein according to claim 7, which is characterized in that the inclusion body cleaning solution be 2~
3mol/L urea, 1 ‰ triton x-100s, 1 ‰ sarcosyls, 1~2mmol/LDTT, 20mmol/L pH's 8.0
Tris-HCl, 1mmol/L EDTA, 50mmol/L NaCl；
The protein denaturation liquid is 5~6mol/L urea, 2~3mmol/L DTT, 1% sarcosyl.
9. the preparation method of protein according to claim 7, which is characterized in that the recombination nitrite reductase
It is insoluble in Escherichia coli, it is expressed with inclusion bodies.
10. the application of recombination nitrite reductase degrading nitrite according to claim 7.
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Cited By (3)
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| CN110133091A (en) * | 2019-04-29 | 2019-08-16 | 中国科学院武汉物理与数学研究所 | 05SAR-PAGE and its preparation method and application |
| CN113151333A (en) * | 2020-12-31 | 2021-07-23 | 安徽丰原发酵技术工程研究有限公司 | Method for producing recombinant nitrite reductase |
| CN113311053A (en) * | 2021-06-29 | 2021-08-27 | 中国科学院精密测量科学与技术创新研究院 | Gel for protein electrophoresis, marker, application and kit |
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| CN113151333A (en) * | 2020-12-31 | 2021-07-23 | 安徽丰原发酵技术工程研究有限公司 | Method for producing recombinant nitrite reductase |
| CN113311053A (en) * | 2021-06-29 | 2021-08-27 | 中国科学院精密测量科学与技术创新研究院 | Gel for protein electrophoresis, marker, application and kit |
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