【作者】 陈浩；

【导师】 刘冬梅；

【作者基本信息】 华南理工大学 ， 食品质量与安全， 2016， 硕士

【摘要】 亚硝酸盐是一种潜在的致癌物质,且过量摄入亚硝酸盐可诱发高铁血红蛋白症,因此严格控制食品中亚硝酸盐的含量非常重要。笔者研究组前期研究发现干酪乳杆菌鼠李糖亚种LCR 6013(Lactobacillus casei subsp.rhamnosus)能很好地降解亚硝酸盐。但其中关键限速酶亚硝酸盐还原酶(LCR6013NiR)及其电子供体(LCR6013ElD)的性质和协同降解亚硝酸盐的机理尚未见报道。干酪乳杆菌LCR 6013经10.00 mg/L的亚硝酸钠诱导,培养液经溶菌酶破壁,粗酶溶液分别经过30%、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,再通过阴离子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LCR6013NiR,仅在加入电子供体之一的细胞色素C才能降解亚硝酸盐,每升的LCR 6013发酵液可得到0.54 mg活性酶蛋白,其LCR 6013Ni R比酶活为1851.20 U/mg,得率为2.08%,纯化后LCR6013NiR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。经过GPC确定蛋白分子质量为125 ku,LCR 6013Ni R为三聚体,可能是CuNiR。同时,由蛋白液I纯化的蛋白加入LCR6013NiR中,表现降解亚硝酸盐的活力,经鉴定为电子供体蛋白LCR6013ElD,经SDS-PAGE电泳确定LCR6013 El D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。LCR6013ElD、细胞色素C、FeSO4和Na2SO3分别协同LCR6013Ni R能在48 h内将75.00 mg/L的亚硝酸钠完全降解,而LCR 6013ElD和细胞色素C降解效果最好。在含有LCR6013NiR的反应体系中,研究了LCR6013ElD和细胞色素C两种电子供体对LCR6013NiR降解亚硝酸盐的影响。LCR6013NiR降解亚硝酸盐的最适温度为37℃;最适pH为7.0;最适底物浓度为100 mg/m L;最适电子供体浓度为20 mg/mL;LCR6013NiR温度稳定性低,在55℃保持30 min后,亚硝酸盐降解率降到16.5%;LCR6013NiR在pH 2.0、3.0、4.0、10.0、11.0、12.0的环境中保持30 min后,LCR6013NiR亚硝酸盐的降解率都接近0;金属离子Ca2+、Mg2+能促进亚硝酸盐的降解,Ag+、Zn2+对降解亚硝酸盐有抑制作用,Fe2+对降解率影响不大。因此,电子供体分别为LCR6013ElD或细胞色素C对LCR6013NiR的酶学影响相似。更多还原

【Abstract】 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.更多还原