Identification Of Highly Efficient Nitrite Degrading Strain LJ01 And Its Nitrite Reductase Characterization

Nitrite is a potential carcinogen, which can easily accumulate in vegetable fermentation process and bring potential food safety issues, moreover excessive intake of nitrite induced methemoglobinemia. Thus, it is very important to strictly control the content of nitrite in food.In our previous study, a strain of Bacillus sp. LJ01 with high nitrite degradation ability was screened from soybean paste. In this study, we were identified the novel strain and the characterization of nitrite reductase from that strain.Firstly, we were identified Bacillus sp. LJ01 as a Bacillus cereus based on its morphological, physiological, and biochemical characteristics, along with its 16 S r DNA gene sequence. In the logarithmic phase, the rate of nitrite reduction of LJ01 was lower, and the degradation rate reached the maximum at the stable stage, then decreased. The maximum Na NO2 degradation concentration of LJ01 in 24 hours was 250 μg/m L and its degradation rate was 99.31%.The degradation of nitrite by LJ01 in LB system was studied by the method of ammonium nitrogen and ECD- gas chromatography. The percentage content of N2 O in the system was 440.45×10-6 and 65.98×10-6 respectively, which showed significant different to compare with the control group(p≤0.05). Ammonium nitrogen test showed that both the experimental group and the control group were not produced TAN. The result of enzyme activity of the intracellular and extracellular components showed that the degradation of nitrite was due to the action of Ni R in the intracellular groups. The crude enzyme solution of LJ01 was purified by DEAE Sepharose Fast Flow and Sephadex G-150 gel filtration,respectively. The fractions which had Ni R activity was collected and concentrated by polyethylene glycol 20000, the molecular weight of Ni R was about 30 ku measured by SDS-PAGE.The cloning and expression of technology was used to improve the quantity of Ni R,constructing the recombinant plasmid p ET-32a(+) and recombinant strain BL21, after cultured, the recombinant strain was induced by 1m M IPTG to product recombinant Ni R.The crude enzyme solution was seperated by the way of nickel affinity chromatography and DEAE Sepharose Fast Flow, the high purity of the recombinant protein Ni R was got, thecharacterization of Ni R was detected. The results shows that the molecular weight of recombinant Ni R is 67 ku; iron and copper ion were simultaneously present in the enzyme and the contents were 51.0 mg/Kg and 184.5 mg/Kg, respectively; results of circular dichroism shows that the helix structure has been occupied the largest proportion in the recombinant Ni R.