CAJ | 학술논문

【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（nitrite reductase，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 WU1424 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2347.5 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。
【목적】연구아초산염협박하식물유간균（Lactobacillus plantarum）WU14아초산염환원매（nitrite reductase，NirS）적작용궤제，위발효식품중응용유산균순충배양기술강해아초산염전정기출。【방법】재37℃배양조건하，측정함0.02%—0.16%NaNO2적 MRS 배양액중 L. plantarum WU1424 h 적생장밀도、pH 급 NaNO2강해량。통과 PCR 확증화 TA 극륭득도 NirS，병극륭도유산유구균식품급세포내고효유도표체재체 pRNA48중，획득중조균L. lactis NZ9000/pRNA48-NirS。중조균경30 ng·mL-1 nisin 유도후，경 SDS-PAGE 화염산내을이알법분석목적단백적표체정황급 NirS 매활。통과생물신식학연건예측분석 NirS 편마적단백질이삼급결구、과막결구급소수성。【결과】L. plantarum WU14능구재 NaNO2농도소우0.12%적 MRS 배양기중생장병강해일부분 NaNO2，재0.10% NaNO2배양액중발효24 h 후강해량체도최대，위56.34μg·mL-1，NirS 매활체2347.5 U·mL-1。NirS 편마적단백질시일충이α-라선화무규칙권곡위주，불존재신호태화과막결구적친수단백。NirS 가재 L. lactis NZ9000중고효표체，해중조균능구재 NaNO2농도저우0.10%적 GM17배양기중생장병강해일부분 NaNO2，재함0.04%NaNO2적배양액중아초산염강해량체도최대，위22.21 mg·mL-1，NirS 매활위925.41 U·mL-1。【결론】L. plantarum WU14적 NirS 능구강해고농도적아초산염，병차경식품급이원표체적 NirS 구유교고적매활력。본연구위탐색연구아초산염강해적궤리，건립발효식품중아초산염강해적가공발효체계제공료삼고。
[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. plantarum WU14 could degrade the high concentration nitrite, and NirS of food-grade induced expression possessed higher enzyme activity. The study laid a foundation for research of the mechanism of nitrite degradation and established a controllable system of nitrite degradation of fermented foods.