亚硝酸盐胁迫下植物乳杆菌WU14亚硝酸盐还原酶的食品级高效诱导表达及其酶学性质研究

doi:10.3864/j.issn.0578-1752.2015.07.16

摘要/Abstract

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（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 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56.34 μg·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。

关键词: 植物乳杆菌WU14, 亚硝酸盐胁迫, 亚硝酸盐还原酶, 食品级高效诱导表达

Abstract: 【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.

Key words: Lactobacillus plantarum WU14, nitrite stress, nitrite reductase, food-grade induced expression

应碧，昌晓宇，刘志文，周通，陈瑶，钟平安，徐波. 亚硝酸盐胁迫下植物乳杆菌WU14亚硝酸盐还原酶的食品级高效诱导表达及其酶学性质研究[J]. 中国农业科学, 2015, 48(7): 1415-1427.

YING Bi, CHANG Xiao-yu, LIU Zhi-wen, ZHOU Tong, CHEN Yao, ZHONG Ping-an, XU Bo. Food-Grade Induced Expression and Enzymatic Properties of Nitrite Reductase from Lactobacillus plantarum WU14 Under Nitrite Stress[J]. Scientia Agricultura Sinica, 2015, 48(7): 1415-1427.

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