 IVITR Nitrogen It is a major component of, biomolecules is one of the most abundant elements as

nitrogen gas (N2 atmosphere), plants can only utilize reduced forms of this element. ICBP Plants
biological nitrogen fixation
 (BNF), is carried out by diazotrophic organisms These organisms contains the enzyme nitrogenase
to catalyze the reduction of atmospheric nitrogen (N2) to ammonia (NH3) which is composed of two
components, the FeMo protein and the Fe protein.
 nitrogenzase structure of the bacterium Azotobacter Vinelandi, has been reported Figure 1b [2]. For
the case of the Nases Ga. diazotrophicus until now there are not information about their structure and
reaction mechanism.
 The research is focused isolation and characterization of nitrogenase enzyme from diazotrophic bacteria of
sugarcane. Once
 The first part of the methodology consisted of the extraction specifically the roots from sugarcane
plants (Figure 2a), from crops of sugar mill of the province of Imbabura. For the isolation of the
nitrogen-fixing bacterial strains, the root of the sugarcane plant was sterilized with ethanol solution for
distilled water to remove excess soil present in the roots Figure 2b. Later, nodules from the roots were
inoculated in Petri dishes in culture media of Luria Bertani (LB).
 The second part of the methodology was focused on the use of the Pal-5 strain of Ga. diazotrophicus,
to growth of strain in a solid medium rich in sugars (Agar-SYP) (Figure 3). Subsequently a pre-
inoculum is performed in a liquid medium (SYP). Finally, the pre-inoculum is inoculated in an
appropriate medium (NFB) to overexpress Nase
 From the strain inoculated from roots of sug . After 24 h, we observed the growth of some different
colonies of bacteria. In the next step single colony of the different bacteria will be cultured in NFB
selective media for nitrogen fixing bacteria in order to identify de fixing microorganisms.
 From the study of the growth of Ga. diazotrophicus in NFB media, it was determined that the optimal
incubation time is 4 days (Figure 6). Additionally, in order to evaluate the influence of the
concentration of dioxygen on the nitrogenase activity, tests were carried out on the growth of the
bacteria at different O2 concentrations and the nitrogenase activity was measured by reduction of
ethylene using gas chromatography. The result obtained from the optimal growth at 5% of [O2]
(Figure 7) under our growing conditions differs from that reported by Newton [4].
 Finally, it is expected to isolate and purify the Fe-Mo protein and Fe from the Ga. Diazotrophicus
bacteria of sugarcane to perform characterization studies such as electronic paramagnetic resonance
(EPR) to analyze and compare with the results obtained by Newton [4]
 A protocol for the growth of the Ga. diazotrophicus PAL-5 under conditions of Nase overexpression
was established.
 In the case of diazotrophicus bacteria from sugar cane we are working on the develop of a method of
isolation. Shortly, purification of Nase from the commercial strain and from the natural strain will be
performed. Once we obtained pure Nase, epr studies of the enzyme will be carried out and its 3-D
structure will be determined, which would be only the second crystallographic structure
determined until now. The above will be a great contribution to the elucidation of the nitrogen
reduction mechanism by nitrogenase