Part:BBa_K2346001
Nitrite reductase (NiR):Reduction of nitrite ion(NO2-) to Nitrogen Monoxide (NO)
Nitrite reductase, is the product from the expression of the nirS gene amplified from Alcaligenes eutrophus DSM 530, a denitrifying soil bacteria. The nitrite reductase catalyzes the reduction of nitrite to nitric oxide, as the first step of the denitrificaton process. We expressed the nitrite reductase with a His-tag in E.coli using a pET28a vector. The proteins are then extracted from the cells and purified using a HIS-trap column. To assay the reactivity of the protein. We put the enzyme, sodium nitrite and pbs buffer together to establish a reaction system. We recorded the nitrite concentration over 24 hours, under temperature of 37 degree Celcius. The nitrite concentration was measured using a color-showing reagent consisting of p-aminobenzenesulfonamide and N-(1-Naphthyl)ethylenediamine dihydrochloride. The reagent reacts with nitrite to form the redish pink substance(diazonium salt) in the solution. The formed substance has a maximum absorbance at 540nm. A standard curve was first made by measuring the absorbance at 540nm of the reaction system under various nitrite concentration. According to our results, the absorbance dropped from 0.633 to 0.008 over 24 hours, which indicates a drop of nitrogen concentration from 0.196 to roughly zero.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1
Illegal XhoI site found at 1096 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 562
Illegal AgeI site found at 373 - 1000COMPATIBLE WITH RFC[1000]
Background
File:/wiki/images/8/8d/T--HFLS H2Z Hangzhou--img enzyme nir1.png
The first enzyme NiR is the product from expression of nirS gene amplified from Alcaligenes eutrophus DSM 530. The NiR catalyzes the reduction of nitrite (NO2-) to nitrogen monoxide(NO), as the first step of our substrate channeling system.
Biology
File:/wiki/images/b/b5/T--HFLS H2Z Hangzhou--img enzyme nir2.png
We expressed the nitrite reductase with a His-tag in E.coli using a pET28a vector. The proteins are then extracted from the cells and purified using a HIS-trap column. After further purification, we ran electrophoresis to check the correct expression of NiR. The result of protein electrophoresis is shown below.
Assay method
We used N-(1-naphthyl)ethylene diamine dihydrochloride spectrophotometric method, a common approach in waste water treatment, to measure the concentration of nitrite in the solution to track the reaction process. The nitrite concentration was measured using a color-showing reagent consisting of p-aminobenzenesulfonamide and 1g n-(1-naphthyl)-ethylenediamine dihydrochloride.
The reagent reacts with nitrite to form the redish pink diazo salt in the solution, which has a maximum absorbance around 540nm. A standard curve was first made by measuring the absorbance at 540nm of the reaction system under various nitrite concentration.
Stand curve between Absorbance-540nm and Sodium Nitrite concentration is shown below.
<img class="framed img" src="/img/enzyme/nir3.png"></img>
Reactivity assay in vitro
To assay the reactivity of the protein. We put the enzyme, sodium nitrite and pbs buffer together to establish a reaction system. We recorded the nitrite concentration over 24 hours, under temperature of 37 degree Celcius. A standard curve was first made(as seen above) by measuring the absorbance at 540nm of the reaction system under various nitrite concentration.
According to our results, the absorbance dropped from 0.633 to 0.008 over 24 hours, which indicates a drop of nitrite concentration from 0.196 mg/L to roughly zero. Currently we are still testing samples with shorter and various reaction time-length to plot the closest curve of the reactivity.
File:/wiki/images/f/f6/T--HFLS H2Z Hangzhou--img enzyme nir3.png
Since the application of these enzymes will be in vivo, we are satisfied with NiR’s reactivity was verified. We are much more interested in NiR’s ability to react in vivo with much greater concentration of nitrite.
Reactivity assay in vivo
Protocols
| Add IPTG into prepared cultures of E.coli (IPTG concentration: 0.1mg/L) | |
| Induce at 16 °C, 150 rpm overnight | |
| Add sodium nitrite solution at various concentration (Target concentration: ~65mg/L) | |
| Culture the bacteria at 37 °C,150 rpm | |
| Take samples (500uL) of the cultures at various time points | |
| Measure optical density(600nm) of the samples | |
| Centrifugate at 6,000 rpm for 60s | |
| Take 100uL samples from supernatant and dilute with 900uL ddH2O | |
| Take 100uL samples from the diluted solution and dilute again with 900 uL ddH2O | |
| Add 20uL of chromogenic agent to the solution | |
| Stand the solution for 20 mins and wait for the color to develop. | |
| Measure the absorbance(540nm) of the solutions | |
| Record data in Excel forms |
*Prepare the chromogenic agent as follows:
1 |
In 500mL beaker
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2 |
Transfer the solution to a 500mL volumetric flask and dilute with ddH2O to standard volume. |
NOTICE: We diluted the solution x100, so you have to time 100 the corresponding nitrite concentration using standard curve.
Results
File:/wiki/images/b/be/T--HFLS H2Z Hangzhou--img enzyme nir5.png
| According to our results, the absorbance at 540nm dropped from 0.410 to 90.105 over 45.5 hours, which indicates a drop of NaNO2 concentration from 62.0(mg/L) to 14.9(mg/L). | |
| According to the graph, the reactivity of NiR is the highest during 20-30hours. About 76% of total nitrite is degraded at the end of assay. Also, there is no obvious reduction in nitrite concentration in control group. |
| None |