CN106244603A - A kind of bacillus amyloliquefaciens nitrite reductase and gene and application - Google Patents
A kind of bacillus amyloliquefaciens nitrite reductase and gene and application Download PDFInfo
- Publication number
- CN106244603A CN106244603A CN201610628344.6A CN201610628344A CN106244603A CN 106244603 A CN106244603 A CN 106244603A CN 201610628344 A CN201610628344 A CN 201610628344A CN 106244603 A CN106244603 A CN 106244603A
- Authority
- CN
- China
- Prior art keywords
- bacillus amyloliquefaciens
- nitrite
- nitrite reductase
- gene
- coli
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—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)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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)
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a kind of bacillus amyloliquefaciens nitrite reductase and gene and application, belong to bioengineering field.The present invention is to utilize round pcr to carry out the nitrite reductase gene in Bacillus amyloliquefaciens H47 cloning and carrying out abduction delivering in escherichia coli, utilizes affinity chromatograph effect purification to obtain highly purified recombiant protein.For studying its zymologic property, probing into the approach of Bacillus amyloliquefaciens H47 degrading nitrite, improvement Bacillus amyloliquefaciens H47 nitrite degradation rate lays the foundation.The NiRBD contained in Bacillus amyloliquefaciens H47 is the one in nitrite reductase, it is ammonium by nitrate reductase, the degraded with ruminant greenfeed nitrite can be used it can also be used to the degraded of kitchen waste fermentation fertilizer nitrite.
Description
Technical field
The present invention relates to a kind of nitrite reductase gene, especially a kind of Bacillus amyloliquefaciens
The clonal expression of H47 nitrite reductase gene and application, belong to technical field of bioengineering.
Background technology
Nitrite can cause baby's methemoglobinemia, and baby is in congenital malformation, goiter and breaks down proteins
The reaction of product secondary amine generates strong carcinogen nitrosamine.Water body and nitrite in food finite quantity standard.Nitrite reductase
It is widely present in microorganism and plant, is the key enzyme during nature nitrogen cycle, can be by nitrite degradation
NO or ammonium, thus reduce the accumulation of nitrite nitrogen in environment, reduce the murder by poisoning to organism caused because of Nitrite accumulation
Effect.
Nitrite reductase can be divided three classes according to the difference of participation approach and product: (1) copper type nitrous acid is also
Protoenzyme (Cu-NiRs) and Cytochrome cd 1 type nitrite reductase (cd1NiRs) are participated in anti-nitre by nirK and NirS coding respectively
Change approach, catalysis nitrite degradation is NO.(2) NirBD and NrfAH participates in nitrite alienation approach, is catalyzed nitrite
It is degraded to ammonium.(3) NIT-6 and NirA participates in nitrite assimilatory pathway, and catalysis nitrite degradation is ammonium.In different microorganisms
Kind containing nitrite reductase and plant beyond count identical.This laboratory is isolated one solution starch spore from Mel
Bacillus degradable nitrite to measure its degrading nitrite product be ammonium salt.In denitrifying bacteria containing copper type nitrous acid also
Protoenzyme (Cu-NiRs) or Cytochrome cd 1 type nitrite reductase (cd1NiRs), be widely studied.But have no both at home and abroad
Kind and the report of character about the nitrite reductase contained by bacillus amyloliquefaciens.
Summary of the invention
Kind and bacillus amyloliquefaciens degraded nitrous acid for research bacillus amyloliquefaciens nitrite reductase
The mechanism of salt.The present invention is by encoding base to Bacillus amyloliquefaciens H47 nitrite reductase gene
Because containing kind and the degraded of nitrite reductase during this bacterium is studied in clonal expression and enzyme assay in escherichia coli
The approach of nitrite, and the nitrite reductase of higher degree is gone out by affinitive layer purification, for studying its zymologic property,
Improvement Bacillus amyloliquefaciens H47 nitrite degradation rate lays the foundation.
For achieving the above object, the present invention adopts the following technical scheme that
The gene of a kind of bacillus amyloliquefaciens nitrite reductase, it has the nucleotide shown in SEQ ID NO.1
Sequence, and express the recombiant protein of aminoacid sequence shown in SEQ ID NO.2.
A kind of bacillus amyloliquefaciens nitrite reductase, by the protein of gene code described in claim 1.
Containing the recombiant plasmid of above-mentioned bacillus amyloliquefaciens nitrite reductase gene, containing group ammonia in this recombiant plasmid
Acidity scale label.
The protein of described gene code and containing the transgenic cell line of described gene, genetic engineering bacterium, expression vector
Or cloning vehicle belongs to the scope of protection of the invention.
A kind of recombination bacillus coli producing nitrite reductase and structure, expression, comprise the steps:
(1) with bacillus amyloliquefaciens nitrite reductase encoding gene as template, the design of upstream and downstream primer is carried out,
Design restriction enzyme site is NdeI, BamHI；
(2) extract total gene DNA of bacillus amyloliquefaciens Bacillus amyloliquefaciens H47, with it be
Masterplate, obtains genetic fragment nirBD of the nitrite reductase of 2830bp by PCR amplification；
(3) utilize NdeI, BamHI toolenzyme amplified fragments and plasmid pCold II to be cut into respectively at 37 DEG C and have two
The fragment of individual sticky end；Use and reclaim test kit recovery two above fragment, and connect with T4DNA ligase, build restructuring
Expression vector pCold II-NiRBD, by recombinant plasmid transformed e. coli bl21 (DE3), by ammonia benzyl antibiotic plate screening
Positive transformants sequence verification；
(4) respectively by positive colony, add the BL21 group (negative control group) containing empty plasmid of IPTG induction, be not added with IPTG
The BL21 group (positive controls) containing recombiant plasmid of induction is inoculated in LB fluid medium (containing 0.15g/L NaNO2,
50mg/L ampicillin) cultivate to OD under the conditions of 37 DEG C600=0.4, then continue at 15 DEG C and cultivate and add final concentration of
The IPTG of 0.5mM, abduction delivering 24h, rotating speed 200rpm.4 DEG C, 8000rpm is centrifuged 10min and obtains fermented liquid supernatant, according to GB
Fermentation liquid nitrite is measured by method 2 spectrophotography described in 5009.33 2010 (slightly changing),
And collect thalline.
(5) method of described spectrophotometry fermentation liquid nitrite is: takes 3mL supernatant and adds deionization
Water, to 28.5mL, adds ferrous nitrilation potassium (106g/L) 0.75mL, acetic acid zinc solution (106g/L) 0.75mL, mixing reaction 30min.
8000r/min is centrifuged 10min.Taking 2mL supernatant, to add 2mL sodium sulfanilate solution (4g/L) in 25mL color comparison tube reverse all
Even reaction 3min adds 1mL hydrochloride naphthodiamide solution (2g/L) again, is settled to 25mL with deionized water, reverse uniform, reaction
15min, surveys absorbance at wavelength 538nm.
Described Bacillus amyloliquefaciens H47 filters out from Mel, is preserved in Chinese Typical Representative training
Supporting thing preservation center, Luo Jia Shan, wuchang, wuhan, preservation place Wuhan University, deposit number is CCTCC M 2013283, preservation
June 24 2013 date.
Described PCR primer is:
M1:5 '-GCCGCATATGATGGGAAAAAAACAGCTAGTC-3 ', (SEQ ID NO.3)
M2:5 '-GCCGGGATCCTCAATACAGGATGTATACATTCTC-3 ' (SEQ ID NO.4).
Described recombinant bacterium produces the step of nitrite reductase: utilize IPTG that recombinant bacterium is produced nitrite reductase
Carry out abduction delivering, after abduction delivering, ultrasonication thalline, utilize Ni+Post affinity chromatography purification obtains solubility esterase, miaow
Azoles eluting concentration is 500mM；Described abduction delivering condition is 37 by the e. coli bl21 (DE3) containing recombinant expression plasmid
DEG C, cultivate under the conditions of 200rpm to OD600=0.4, then continue at 15 DEG C and cultivate to the IPTG adding final concentration of 0.5mM, lure
Lead expression 24h, rotating speed 200rpm；
Enzyme activity determination method: with NaNO2For substrate, with methyl viologen as electron donor, 2mL reaction system comprises 50mM phosphorus
Acid buffer 1.6mL, methyl viologen (0.01%) 100 μ L, enzyme liquid 50 μ L, NaNO2(1mM) 50 μ L, NaHCO3And Hydros
Solution (0.8%) 200 μ L, 37 DEG C of reaction 30min.Acutely concussion terminates reaction, adds 1mL sodium sulfanilate solution (4g/L)
Reverse homogeneous reaction 3min adds 0.5mL hydrochloride naphthodiamide solution (2g/L) again, reverse uniform, reacts 15min, with the most enzyme-added liquid
Group zeroing, surveys absorbance at wavelength 538nm.
LB medium component is: tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, pH7.2.
Compared with other technologies, having the active effect that of this invention
(1) present invention separate from Bacillus amyloliquefaciens H47, purification nitrite reductase
Gene.This gene includes two adjacent open reading frame.By to big containing empty plasmid and recombiant plasmid induced through IPTG
Enterobacteria and be not added with IPTG induction the escherichia coli crude enzyme liquid containing recombiant plasmid carry out SDS-PAGE analysis and affinity chromatograph skill
Art verifies that engineering bacteria can be with abduction delivering NiRBD recombiant protein.
(2) present invention can obtain substantial amounts of intracellular NiRBD by IPTG abduction delivering, may apply to enter in industry
Row is a large amount of to be produced.
(3) present invention utilizes affinity chromatograph effect purification to obtain the NiRBD recombiant protein that purity is higher, solves from Xie Dian
Afnyloliquefaciens separates nitrite reductase and a difficult problem of the higher NiRBD of content.
(4) the recombiant protein electron donor to be added of the engineering bacterium expression purification that the present invention obtains could be degraded Asia effectively
Nitrate.
(5) present invention is mechanism and the regulation and control of research Bacillus amyloliquefaciens H47 degrading nitrite
Provide guarantee.
Accompanying drawing explanation
Fig. 1 is to utilize Bacillus amyloliquefaciens H47 genomic DNA to carry out for masterplate in embodiment 1
Pcr amplification product identifies figure, and wherein 1 is pcr amplification product, and M is DNA molecular quality standard.
Fig. 2 is recombiant plasmid pColdII-NiRBD digestion verification figure.
Fig. 3 is the Recombinant protein expression of Bacillus amyloliquefaciens H47 nitrite reductase.
Fig. 4 is the SDS-PAGE of NiRBD protein after purification.
Detailed description of the invention
The term used in the present invention, unless otherwise specified, typically has those of ordinary skill in the art usual
The implication understood.
Below in conjunction with concrete preparation embodiment and Application Example, and with reference to data, this is described in further detail
Bright.Should be understood that these embodiments present invention solely for the purpose of illustration, rather than limit the scope of the present invention by any way.
Below in an example, the various processes not described in detail and method are conventional methods as known in the art.
General explanation: the enzyme involved by embodiment is purchased from TaKaRa company, plasmid kit and agarose gel and returns
Receive test kit to carry out fully according to respective description purchased from Shanghai biological engineering company limited, operation.Primer synthesis is checked order with plasmid
By Shanghai, Sani company completes.
The clonal expression of embodiment 1:Bacillus amyloliquefaciens H47 nitrite reductase gene, step
Rapid as follows:
Application bacterial genomes DNA extraction agent box (TaKaRa company) extracts Bacillus according to its operation
Amyloliquefaciens H47 phage gene group STb gene, identifies its quality and purity, ultraviolet by agarose gel electrophoresis
Its concentration of spectrophotometric determination.According to full genome order-checking design upstream and downstream primer: m1 (SEQ ID NO:3,5 '-
GCCGCATATGATGGGAAAAAAACAGCTAGTC-3 ') and m2 (SEQ ID NO:4,5 '-
GCCGGGATCCTCAATACAGGATGTATACATTCTC-3 '), apply PCR method, with above-mentioned Bacillus
Amyloliquefaciens H47 genome is template, with above-mentioned m1 and m2 as specific primer, expands Bacillus
Amyloliquefaciens H47 nitrite reductase full length gene encoder block sequence.PCR reaction condition is: with genome
DNA is template, in 50 μ L reaction systems, adds 10 × ExTaqBuffer 5 μ L, 25mM MgCl2 2μL、2.5mM dNTP
Mixture 4 μ L, 20 μMs of each 1 μ L, Ex Taq enzyme (Takara company) the 1 μ L and DNA profiling 1 μ L of upstream and downstream primer, moisturizings to 50 μ L.
PCR cycle parameter is: 94 DEG C of denaturations 5min, then carries out 30 circulations (98 DEG C of degeneration 10s, 55 DEG C of annealing 30s, 72 DEG C of extensions
3min), finally 10min is extended in 72 DEG C.PCR primer with 1% agarose gel electrophoresis separate, result as it is shown in figure 1,
Having obvious DNA band between 2000 and 3000bp, size is about 2800.Reclaim test kit with rubber tapping and reclaim this DNA band, warp
Order-checking, result such as sequence.
The DNA sequence of the NiRBD gene that rubber tapping is reclaimed gained is carried out respectively with commercialization expression plasmid pColdII
NdeI, BamHI double digestion, reacts 1h in 37 DEG C, is then separately recovered sheet segment DNA and plasmid DNA.
Above-mentioned two fragment is attached, in coupled reaction system, at 16 DEG C, is attached 14～16h, i.e. obtains
Obtaining recombiant plasmid pCold II-NiRBD, described linked system is: reclaim enzyme action pColdII 2 μ L, reclaims genes of interest fragment
6 μ L, T4DNA ligase 1 μ L, T4DNA ligase buffer 2 μ L, moisturizing to 20 μ L；
20 μ L are connected product convert to 100 μ L e. coli bl21 (DE3) competent cells, then mix, ice bath
30min, then 42 DEG C of heat shock 90s, then ice bath 5min, adds the LB culture medium recovery for competent cell of 880 μ L.Cell
Culture fluid is placed on 37 DEG C, after cultivating 40min, takes 75 μ L and coats in Amp resistant panel in the shaking table of 200rpm, 37 DEG C of cultivations
After 8～12h, verifying the single bacterium colony grown, a step sequence verification gene order of going forward side by side is the most correct.
Plasmid pCold II-NiRBD single endonuclease digestion and double digestion method are identified, qualification result is as in figure 2 it is shown, swimming lane 1 is double
Digestion products, swimming lane 2 is double digestion product.From figure, clearly visible recombiant plasmid has a band, and molecule after single endonuclease digestion
Amount is coincide with recombiant plasmid size 7200bp in theory；Recombiant plasmid has two band after double digestion, and one is about
4300bp, one is about 2800bp, illustrates that the carrier built becomes two bar segment, recombiant plasmid through enzyme NdeI, BamHI double digestion
PCold II-NiRBD successfully constructs.
Implement the escherichia coli induction table of row 2:Bacillus amyloliquefaciens H47 nitrite reductase
Reach, specific as follows:
The positive colony bacterium of embodiment 1 gained is inoculated in 3mlLB fluid medium (containing 50mg/L ampicillin)
In 37 DEG C, incubated overnight under 200rpm, then it is inoculated in LB fluid medium (containing 0.15g/L NaNO with 2% inoculum concentration2And
50mg/L ampicillin) cultivate to OD under the conditions of 37 DEG C600=0.4, then continue at 15 DEG C and cultivate and add final concentration of
The IPTG of 0.5mM, abduction delivering 24h, rotating speed 200rpm.4 DEG C, 8000rpm is centrifuged 10min and obtains fermented liquid supernatant according to GB
Fermentation liquid nitrite is measured by method 2 spectrophotography described in 5009.33 2010 (slightly changing).
And collect thalline.Setting up matched group, one group is to add the BL21 group (negative control group) containing empty plasmid that IPTG induces, separately simultaneously
One group of BL21 group (positive controls) containing recombiant plasmid being to be not added with IPTG induction, is entered by the abduction delivering process of the present embodiment
Row abduction delivering, carries out the operation of the same terms to the thalline fermentation liquid of negative control group and positive controls, measures fermentation liquid
Content of nitrite result such as table 1.Can show that the BL21 group nitrite reductase gene containing recombiant plasmid successfully lures
Lead expression and there is the enzyme activity.
Table 1
The method of described spectrophotometry fermentation liquid nitrite is: takes 3mL supernatant and adds deionized water
To 28.5mL, add ferrous nitrilation potassium (106g/L) 0.75mL, acetic acid zinc solution (106g/L) 0.75mL, mixing reaction 30min.
8000r/min is centrifuged 10min.Taking 2mL supernatant, to add 2mL sodium sulfanilate solution (4g/L) in 25mL color comparison tube reverse all
Even reaction 3min adds 1mL hydrochloride naphthodiamide solution (2g/L) again, is settled to 25mL with deionized water, reverse uniform, reaction
15min, surveys absorbance at wavelength 538nm.
LB medium component is: tryptone (Tryptone) 10g/L, yeast extract (Yeast extract) 5g/L,
Sodium chloride (NaCl) 10g/L, pH 7.2.
Embodiment 3: the extraction of crude protein, purification and enzyme activity determination in recombinant bacterium
Restructuring thalline obtained in embodiment 2 and matched group thalline sterilized water are washed twice, cultivates by every 100ml
Liquid adds 10ml, and the broken 5min of phosphate buffer (pH7.0) ice bath excusing from death of the 0.02M of 4 DEG C of pre-coolings, in 4 DEG C of 8000rpm after crushing
Centrifugal 5min, taking its supernatant is NiR crude protein liquid, carries out SDS-PAGE detection under same concentrations, and result is as it is shown on figure 3, swim
Road 1 is the BL21 group crude enzyme liquid containing empty plasmid adding IPTG induction, and swimming lane 3 is the BL21 containing recombiant plasmid being not added with IPTG induction
Group crude enzyme liquid, swimming lane 5 is the BL21 group crude enzyme liquid containing recombiant plasmid adding IPTG induction, illustrates that genetic engineering bacterium has after induction
Article two, obvious specifically expressing band, and band molecular weight is consistent with expection sized molecules amount.
Utilizing Ni+ post affinity chromatography purification to obtain solubility nitrite reductase, imidazoles eluting concentration is 500mM.
Product after purification is with sodium nitrite as substrate, and methyl viologen is that electron donor surveys the work of its enzyme, and recombiant protein detects activity, and
Detecting its purity by SDS-PAGE, result is as shown in Figure 4.Swimming lane 6 is albumen after purification, and swimming lane 7 is for being not added with IPTG induction
Supernatant after BL21 group thalline breaking cellular wall containing recombiant plasmid, swimming lane 8 is the BL21 group containing recombiant plasmid adding IPTG induction
Supernatant after thalline breaking cellular wall.Band is recombiant protein two Asias of arrow indication in comparison swimming lane 6,7,8 band explanation swimming lane 6
Base.
Enzyme activity determination method: with NaNO2For substrate, with methyl viologen as electron donor, 2mL reaction system comprises 50mM phosphorus
Acid buffer 1.6mL, methyl viologen (0.01%) 100 μ L, enzyme liquid 50 μ L, NaNO2(1mM) 50 μ L, NaHCO3And Hydros
Solution (0.8%) 200 μ L, 37 DEG C of reaction 30min.Acutely concussion terminates reaction, adds 1mL sodium sulfanilate solution (4g/L)
Reverse homogeneous reaction 3min adds 0.5mL hydrochloride naphthodiamide solution (2g/L) again, reverse uniform, reacts 15min, with the most enzyme-added liquid
Group zeroing, surveys absorbance at wavelength 538nm.
Claims (10)
1. a bacillus amyloliquefaciens nitrite reductase gene, it is characterised in that there is the core shown in SEQ ID NO.1
Nucleotide sequence, and express the recombiant protein of aminoacid sequence shown in SEQ ID NO.2.
2. a bacillus amyloliquefaciens nitrite reductase, it is characterised in that the albumen of gene code described in claim 1
Matter.
3. contain the genetic engineering bacterium of gene described in claim 1.
4. contain the recombiant plasmid of bacillus amyloliquefaciens nitrite reductase gene described in claim 1.
Genetic engineering bacterium the most according to claim 3, it is characterised in that preferably recombination bacillus coli E.coli BL21
(DE3) it is that host expresses.
Recombiant plasmid the most according to claim 4, it is characterised in that containing histidine-tagged in this recombiant plasmid.
7. the construction method of recombination bacillus coli described in claim 5, it is characterised in that choose the cold-induced expression of escherichia coli
PCold II, builds recombinant expression carrier, is that host expresses with large intestine bar E.coli BL21 (DE3).
8. the method that escherichia coli described in claim 5 produce nitrite reductase, it is characterised in that comprise the steps:
(1) with bacillus amyloliquefaciens Bacillus amyloliquefaciens H47 nitrite reductase gene as mould
Plate, carries out the design of upper and lower primer, and the restriction enzyme site of design is NdeI, BamH I；
(2) DNA of bacillus amyloliquefaciens Bacillus amyloliquefaciens H47 is extracted, and as template, logical
Cross the genetic fragment of round pcr amplification bacillus amyloliquefaciens H47 nitrite reductase；Described bacillus amyloliquefaciens H47
Deposit number be CCTCC M 2013283, preservation date on June 24th, 2013.
(3) utilize NdeI, BamHI toolenzyme that amplified fragments and plasmid pCold II are carried out at 37 DEG C double digestion respectively, use
Reclaim two fragments after test kit reclaims double digestion, and connect with T4DNA ligase, then proceed in recombination bacillus coli, profit
Carry out screening with antibiotics ampicillin and obtain positive colony bacterium；
(4) utilize IPTG that recombinant bacterium is produced nitrite reductase enzyme and carry out cold-induced expression, after abduction delivering, ultrasonication bacterium
Body, utilizes Ni+Post affinity chromatography purification obtains solubility nitrite reductase, and imidazoles eluting concentration is 500mM；Described lure
Sliver part be by the e. coli bl21 (DE3) containing recombinant expression plasmid pCold II-NiRBD 37 DEG C, train under the conditions of 200rpm
Support to OD600=0.4, then continue at 15 DEG C and cultivate to the IPTG adding final concentration of 0.5mM, abduction delivering 24h, rotating speed
200rpm；Medium component is: tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, NaNO20.15g/L, pH
7.2。
9. the method described in claim 5, it is characterised in that have on described primer be 5 '-
GCCGCATATGATGGGAAAAAAACAGCTAGTC-3 ', downstream primer is 5 '-
GCCGGGATCCTCAATACAGGATGTATACATTCTC-3’。
10. during prepared in claim 8 nitrite reductase determination of activity, it is characterised in that electron donor first need to be added
Benzyl viologen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610628344.6A CN106244603A (en) | 2016-08-03 | 2016-08-03 | A kind of bacillus amyloliquefaciens nitrite reductase and gene and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610628344.6A CN106244603A (en) | 2016-08-03 | 2016-08-03 | A kind of bacillus amyloliquefaciens nitrite reductase and gene and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106244603A true CN106244603A (en) | 2016-12-21 |
Family
ID=57605878
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610628344.6A Pending CN106244603A (en) | 2016-08-03 | 2016-08-03 | A kind of bacillus amyloliquefaciens nitrite reductase and gene and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106244603A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103451128A (en) * | 2013-07-12 | 2013-12-18 | 江南大学 | Bacillus amyloliquefaciens and application thereof in preventing and controlling peach bleeding disease |
| CN105063066A (en) * | 2015-08-10 | 2015-11-18 | 华南理工大学 | Bacillus cereus NiR (nitrite reductase), gene and application |
-
2016
- 2016-08-03 CN CN201610628344.6A patent/CN106244603A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103451128A (en) * | 2013-07-12 | 2013-12-18 | 江南大学 | Bacillus amyloliquefaciens and application thereof in preventing and controlling peach bleeding disease |
| CN105063066A (en) * | 2015-08-10 | 2015-11-18 | 华南理工大学 | Bacillus cereus NiR (nitrite reductase), gene and application |
Non-Patent Citations (2)
| Title |
|---|
| NCBI: "nitrite reductase [Bacillus amyloliquefaciens]，NCBI Reference Sequence: WP_058906549.1", 《GENBANK》 * |
| 刘冬梅 等: "蜡样芽孢杆菌LJ01的鉴定及亚硝酸盐还原酶性质", 《华南理工大学学报（自然科学版）》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109825484B (en) | Zearalenone hydrolase ZHD101 mutant and method for hydrolyzing zearalenone by using mutant | |
| CN1678624B (en) | Benzoate- and anthranilate-inducible promoters | |
| Lin et al. | Cloning, sequencing and analysis of expression of a Butyrivibrio fibrisolvens gene encoding a β-glucosidase | |
| CN107267486A (en) | A kind of beta galactosidase combination mutant with high transglycosylation and its preparation method and application | |
| CN107164395B (en) | Molecular marker of algal toxin degrading bacteria, algal toxin degrading bacteria and preparation method of algal toxin degrading bacteria | |
| CN102965355B (en) | Carboxylesterase and application thereof in degradation of pesticides malathion and carbaryl | |
| CN102424811A (en) | Cadaverine-producing engineering strain | |
| CN104109659A (en) | Carboxylesterase as well as coding gene and application thereof | |
| CN108220219A (en) | A set of lactobacillus plantarum food-grade expression system and its application in heterologous protein expression | |
| CN108559708A (en) | The preparation method and obtained strains of the bacillus licheniformis of reinforcing YvbW expression and its application | |
| CN106434707B (en) | A kind of bacterial laccase gene, bacterial laccase and its application from hay bacillus ZNXH1 | |
| CN106434706B (en) | A kind of bacterial laccase gene, bacterial laccase and its application from hay bacillus ZXN4 | |
| CN107034199A (en) | The bacillus pumilus CotA Laccase mutants that a kind of Fixedpoint mutation modified stability and activity are improved | |
| CN105200020B (en) | A kind of high substrate specificity bacillus pumilus CotA laccase being transformed by compound point mutation | |
| CN104774829B (en) | A kind of specific enzyme activity and stability-enhanced pattern of fusion nitrile hydratase | |
| Suzuki et al. | Extracellular production of an RNA aptamer by ribonuclease-free marine bacteria harboring engineered plasmids: a proposal for industrial RNA drug production | |
| CN104946681A (en) | Microbiological method for detecting heavy-metal cadmium in water body | |
| CN106244603A (en) | A kind of bacillus amyloliquefaciens nitrite reductase and gene and application | |
| CN107034200A (en) | The bacillus pumilus CotA laccase complex mutation bodies that a kind of amount of soluble expression is improved | |
| CN115838713A (en) | Protease and application thereof in L-carnosine synthesis | |
| CN113735282B (en) | Old yellow enzyme OYE2 protein and application thereof in chromium pollution | |
| CN107227302A (en) | The bacillus pumilus CotA Laccase mutants that a kind of amount of soluble expression is improved | |
| CN113832117A (en) | Enzyme for degrading oxytetracycline, and coding gene and application thereof | |
| CN102776147B (en) | Gene recombinaton Pierre auspicious Russia series bacillus and its construction method and application | |
| Liu et al. | Construction of a hydrazine oxidoreductase gene recombinant bacterium for effective N2H4 removal from wastewater |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20161221 |