Part:BBa_K2346000

Nitric oxide reductase (NOR)

Cytochrome P450 55B1 from Chlamydomonas reinhardtii is reported to function as a nitric oxide reductase (NOR). We are still working on that. Since nitric oxide is nearly insoluable in water, it’s hard for us to create a aquatic reaction system for assaying the enzyme reactivity. We are still working on that. But currently we plan to create a reaction environment in a sealed container filled with solution, enzyme, and nitric oxide gas, By constant shake over, let’s say, two hours, we’ll then measure the change in the concentration of NO using the Gas-Phase Molecular Absorption Spectrometry to see if the presence of the enzyme made any difference on the concentration.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1
  Illegal XhoI site found at 1216
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1200
  
• 1000
  COMPATIBLE WITH RFC[1000]

Background

<img class="framed img" style="height: 30rem; width: auto;" src="/img/enzyme/nir1.png"></img>

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

<img class="framed img" style="height: 30rem; width: auto;" src="/img/enzyme/nir2.png"></img>

Nitrite reductase's tertiary structure

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>

Standard curve between Absorbance-540nm and Sodium Nitrite concentration

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.

<img class="framed img" src="/img/enzyme/nir4.png"></img>

NiR reactivity assay in vitro

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

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	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:

<tbody> </tbody>

1	In 500mL beaker <tbody> </tbody> 50mL Phosphoric Acid (d=1.70g/m) 20.0g p-aminobenzenesulfonamide (C6H8N2O2S) 1g n-(1-naphthyl)-ethylenediamine dihydrochloride 250mL ddH2O
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

<img class="framed img" src="/img/enzyme/nir5.png"></img>

Nitrite conc. against reaction time

<tbody> </tbody>

	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.