中文摘要：

【目的】研究亚硝酸盐胁迫下植物乳杆菌（Lactobacillus plantarum）WU14亚硝酸盐还原酶（nitritereductase,NirS）的作用机制,为发酵食品中应用乳酸菌纯种培养技术降解亚硝酸盐奠定基础。【方法】在37℃培养条件下,测定含0.02%—0.16%NaNO2的MRS培养液中L.plantarum WU14 24 h的生长密度、pH及NaNO2降解量。通过PCR扩增和TA克隆得到NirS,并克隆到乳酸乳球菌食品级细胞内高效诱导表达载体pRNA48中,获得重组菌L.lactis NZ9000/pRNA48-NirS。重组菌经30 ng.mL^-1 nisin诱导后,经SDS-PAGE和盐酸萘乙二胺法分析目的蛋白的表达情况及NirS酶活。通过生物信息学软件预测分析NirS编码的蛋白质二三级结构、跨膜结构及疏水性。【结果】L.plantarum WU14能够在NaNO2浓度小于0.12%的MRS培养基中生长并降解一部分NaNO2,在0.10%NaNO2培养液中发酵24 h后降解量达到最大,为56.34μg.mL^-1,NirS酶活达2 347.5 U.mL^-1。NirS编码的蛋白质是一种以α-螺旋和无规则卷曲为主,不存在信号肽和跨膜结构的亲水蛋白。NirS可在L.lactis NZ9000中高效表达,该重组菌能够在NaNO2浓度低于0.10%的GM17培养基中生长并降解一部分NaNO2,在含0.04%NaNO2的培养液中亚硝酸盐降解量达到最大,为22.21 mg.mL^-1,NirS酶活为925.41 U.mL^-1。【结论】L.plantarum WU14的NirS能够降解高浓度的亚硝酸盐,并且经食品级异源表达的NirS具有较高的酶活力。本研究为探索研究亚硝酸盐降解的机理,建立发酵食品中亚硝酸盐降解的可控发酵体系提供了参考。

英文摘要：

[Objective]The study aimed to explore the mechanism of nitrite reductase from Lactobacillus plantarum WU14 under nitrite stress so that lay a foundation for pure culture technology of lactic acid bacteria in fermented food. [Method] Growth density, pH and nitrite degradation quantity of L. plantarum WU14 were determined when the liquid medium contained sodium nitrite ranged from 0.02% to 0.16% under the condition of 37℃. The recombination strain Lactococcus lactic NZ9000/pRNA48-NirS was constructed followed the putative nitrite reductase gene from L. plantarum WU14 was amplified by PCR and then cloned into the food-grade cytoplasmic inducible expression vector pRNA48 of L. lactic NZ9000. After induced with 30 ng·mL^-1 nisin, the expressed target protein and the enzyme activity of nitrite reductase of the recombinant strains were analyzed by SDS-PAGE and Naphthyl ethylenediamine dihydrochloride spectrophotometric method. Using the bioinformatics software, the high level protein structure, membrane structure and hydrophobicity of nitrite reductase gene were predicted and analyzed. [Result]L. plantarum WU14 could routinely grow in MRS medium containing less than 0.12% nitrite, along with degradation of nitrite. After the strain L. plantarum WU14 was cultured for 24 hours in the liquid medium containing 0.10% sodium nitrite, the nitrite reductase activity of L. plantarum WU14 was 2 347.5 U·mL^-1, and the degradation quantity was 56.34 μg·mL^-1 according to the analysis of its nitrite degradation ability. the NirS gene could express in the recombinant strain. Nitrite reductase gene encodes a kind of hydrophilic protein containing alpha helix and random coil, no signal peptide and transmembrane structure. The recombination strain could routinely grow in GM17 medium containing less than 0.10% nitrite, meanwhile, its enzyme activity reached 925.41 U·mL^-1 and the degradation quantity reached 22.21 mg·mL^-1 after 24 h fermentation in the 0.04% nitrite concentration medium. [Conclusion]Nitrite reductase from L. pla