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