Estimation of Nitrogenase | Enzymes| Biotechnology

In this article we will discuss about the principle, requirements and procedure for estimation of nitrogenase.

Nitrogenase is possessed by a group of prokaryotes which breaks the triple bond of elemental nitrogen (N≡N) present in the at­mosphere and convert it into NH₃, an as­similable form for plants. This enzyme can also reduce acetylene to ethylene.

Principle:

Nitrogenase reduces acetylene to ethylene and this ethylene is measured in a gas liquid chro­matograph (GLC). The activity is expressed as ‘n’ mole ethylene produced for unit time per gram of dry nodules.

Requirements:

1. Root nodules—Phaseolus.

2. Gas chromatograph with flame ioniza­tion detector.

3. Acetylene and ethylene gases.

4. Air tight syringes.

5. 100 ml conical flasks with small mouth to fit serum caps.

6. Conditions for operating GLC.

7. Carrier gas N₂/helium/argon having i flow rate 30-45 ml.

8. Column-Poropak, NRT or silica gel:

9. Gas for detector-H₂ and air.

Procedure:

1. First inject pure acetylene and prepare the sample peak.

2. Remove Phaseolus from soil without dis­turbing the nodules.

3. Excise root system as such. Don’t sepa­rate nodules from roots.

4. Place the root system into a 100 ml coni­cal flask and seal the flask with serum cap.

5. Using an air tight syringe remove 10 ml of air from the flask and inject 10ml acetylene into the flask.

6. Incubate at room temperature for 30- 60 minutes.

7. With an air tight syringe remove 0.5 to 1.0 ml of gas mixture from the flask and inject it into a preconditioned GLC. Look for acetylene and ethylene peaks and measure the height of the ethylene peak.

8. Remove nodules from the roots and find their dry weight.

9. A similar control assay should be con­ducted by injecting 10µg (2) of pure ethylene into a 100 ml sealed conical flask.

10. Remove 0.5 to 1.0 ml of ethylene, inject into GLC and measure ethylene peak.

Calculation:

1. Standard amount of Ethylene (E) in µ mol = 0.446 X2µl /Peak height in mm X attenuation.

2. Amount of ethylene produced in µ mol in the sample = E X Peak height of sam­ple ethylene in mm X attenuation.

3. Activity of nitrogenase = n mol or µ mol ethylene per unit sample per unit time (unit sample = dry weight of nodules).