Purification Nitrite Reductase And Electron Donor From Lactobacillus Casei LCR6013 And Their Synergetic Degradation Of Nitrites

Nitrites are potentially strong carcinogens and can cause methemoglobin under excessiveintake of nitrites. It is very important to control the concentration of nitrite in food. Our previous results showed that the strain Lactobacillus casei subsp. rhamnosus 6013 can degrade nitrites perfectly. But the characteristics of its nitrite reductase(LCR6013NiR), a critical limit-enzyme, and the electron donor(LCR6013ElD) and the mechanism of their synergetic degradation of nitrites has not yet been reported.After the Lactobacillus casei LCR 6013 was induced by sodium nitrite solution of 10.00 mg/L and treated with lysozyme, the crude enzyme solution was precipitated by saturated solution of(NH4)2SO4 with 30 %、60 %, respectively. The precipitates were dissolved and dialyzed, respectively, whereupon there was gotten protein solution I and protein solutionII, which were was respectively purified through anion DEAE Sepharose Fast Flow column chromatography and Sephadex G-100 gel filtration. The protein purified from protein solution II could degrade nitrite only after adding cytochrome C. 0.54 mg of active enzyme protein with an activity of 1851.20 U/mg can be obtained from 1.00 L of the fermentation liquid, and that the specific LCR6013 NiR activity of the purified enzyme increases by 16 folds with a recovery of 2. 08 %.The monomer molecular weight of NiR was 45.00 ku by means of SDS-PAGE. Molecular weight of LCR6013 NiR was 125 ku by means of GPC, So, LCR6013 NiR was a kind of enzyme of trimer, which may be classfied as CuNiR.Moreover, the protein purified from protein solution I was added to LCR6013 NiR, in which showed the ability of degrading nitrites. The protein was identified as electron donor protein(LCR6013ElD). The molecular weight of LCR6013 ElD was 13.00 ku which was same as cytochrome C by means of SDS-PAGE. It was also found that LCR6013 ElD, cytochrome C, ferrous sulfate, sodium sulfite could degrade 75.00 mg/L of sodium nitrite completely within 48 h together with LCR6013 NiR, but LCR6013 ElD and cytochrome C have the best effects on degrading nitrites.The properties of the LCR6013 NiR of nitrite degradation were investigated by adding LCR6013 ElD and cytochrome C into the reaction system. The optimum temperature of LCR6013 NiR for degrading nitrite was 37 °C; the optimal pH was 7.0; the optimal substrate concentration was 100 mg/mL; the optimal donor concentration was 20 mg/m L; LCR6013 NiR had low temperature stability, after the LCR6013 NiR was incubated at 55 °C for 30 min, the ratio of nitrite degradation dropped to 16.5 %; After the LCR6013 NiR was incubated under pH 2.0, 3.0, 4.0, 10.0, 11.0, 12.0 for 30 min, respectively, the ratio of nitrite degradation reached almost zero; The metal ions Ca2+and Mg2+ can promote the reaction of nitrite degradation. But, the metal ions Ag+and Zn2+ inhibit the reaction of nitrite degradation. Fe2+ has little effect on the reaction of nitrite degradation. Whether electron donor is purified ElD or cytochrome C, enzymatic properties of LCR6013 NiR were similar.