干酪乳杆菌LCR6013中NiR和电子供体的分离纯化及其协同降解亚硝酸盐

干酪乳杆菌LCR 6013经10.00 mg/L的亚硝酸钠诱导和溶菌酶破壁,粗酶溶液分别经过30%和60%饱和硫酸铵溶液分级沉淀,沉淀被溶解和透析后分别得蛋白液Ⅰ和蛋白液Ⅱ,通过阴离子DEAE Sepharose Fast Flow和葡聚糖凝胶G-100层析柱分离纯化。蛋白液Ⅱ纯化得亚硝酸盐还原酶蛋白(NiR),在加入细胞色素C才能降解亚硝酸盐,LCR6013的每升发酵液得到0.54 mg活性酶蛋白,其NiR比酶活为1851.20 U/mg,得率为2.08%,纯化后其NiR比活力提高16倍,经SDS-PAGE电泳确定LCR6013 NiR的单体分子质量约为45 ku。同时,由蛋白液Ⅰ纯化的蛋白加入LCR6013的NiR中,表现降解亚硝酸盐的活力,经鉴定为电子供体蛋白(El D),经SDS-PAGE电泳确定LCR6013中ElD的单体分子质量约为13 ku,与细胞色素C的单体分子质量相同。LCR6013的ElD、细胞色素C、FeSO_4和Na_2SO_3分别协同NiR能在48 h内将75.00 mg/L的亚硝酸钠完全降解,而LCR6013的ElD和细胞色素C降解效果最好。

Isolation and Purification of Nitrite Reductase and Electron Donor from Lactobacillus casei LCR 6013 and Their Synergetic Degradation of Nitrites

After Lactobacillus casei LCR 6013 was induced with sodium nitrite solution (10.00 mg/L) and treated with lysozyme, the crude enzyme solution was precipitated using 30% and 60% saturated ammonium sulfate solution, respectively. The precipitates were dissolved and dialyzed to obtain protein solution I and protein solution II, respectively, which were purified by anion DEAE Sepharose Fast Flow column chromatography and SephadexG-100 gel filtration. The nitrite reductase (NiR) purified from protein solution II could degrade nitrite only after adding cytochrome C. A total of 0.54 mg of active enzyme protein, with an activity of 1851.20 U/mg, was obtained from 1.00 L of LCR 6013 fermentation liquid, and the specific NiR activity of the purified enzyme increased by 16-fold with an yield of 2.08%. The monomer molecular weight of NiR was about 45.00 ku based on SDS-PAGE patterns. Moreover, when the protein purified from protein solution I was added to NiR from LCR 6013, it showed the ability to degrade nitrites. The protein was identified as an electron donor protein (ElD). The monomer molecular weight of the ElD, as confirmed using SDS-PAGE, was about 13.00 ku, which is the same as that of cytochrome C. It was also found that when combined with NiR from LCR 6013, the ElD from LCR 6013, cytochrome C, ferrous sulfate, and sodium sulfite could degrade 75.00 mg/L of sodium nitrite completely within 48 h, but ElD from LCR 6013 and cytochrome C were the most effective for nitrite degradation.