1. Background

  2. References


Nitrates play a significant role in food preservation by inhibiting bacteria growth and preserving color. However, excessive nitrates can pose serious health risks including acute poisoning and carcinogenic effects. The use of nitrate reductase enzyme offers a simple, efficient, and non-toxic method to degrade nitrates. This study focuses on constructing a recombinant expression vector to express the nitrate reductase enzyme from Bacillus cereus in E. coli. By using molecular dynamics simulation and rational design, the enzyme is modified to achieve high activity and stability for industrial production. Additionally, an enzymatic bioelectrode is designed for the enzyme, aiming at its application in nitrate detection and degradation in food.

Nitrates are compounds widely present in nature and food. They act as preservatives and color enhancers. However, overconsumption can lead to acute poisoning, carcinogenic effects, and malformations. Nitrate contamination in food and water is a pressing health and environmental issue, necessitating effective treatments. Traditional nitrate treatments have drawbacks like high energy consumption, low efficiency, and side-products. Hence, developing a safe, efficient, and eco-friendly nitrate degradation method is vital.

Nitrate reductase is an oxidoreductase enzyme capable of catalyzing nitrate reduction, found extensively in microorganisms and plants. It is pivotal in the natural nitrogen cycle, degrading nitrates to NO or NH3. This reduces nitrate accumulation in the environment, mitigating the toxic effects on living organisms. Based on cofactors and reaction products, nitrate reductase is categorized into: copper-type, cytochrome cd1 type, polycyclic heme c type, and iron oxidoreductase-dependent.

Current research on nitrate reductase primarily includes: isolation, purification, expression, and characterization from various microorganisms; understanding catalytic mechanisms and electron pathways; and developing biosensors or bioelectrodes for quick nitrate detection in food and water. However, due to its variety, complex structure, and unstable activity, studies are limited.

This project innovatively chooses to express the nitrate reductase enzyme from Bacillus cereus, known for its stability and efficiency over a wide pH range. By constructing a recombinant expression system and designing enzymatic bioelectrodes, the aim is industrial-scale preparation for food safety, especially in fermented foods like pickles.

The research primarily focuses on utilizing nitrate reductase to degrade nitrates in fermented foods, enhancing food safety and quality. Key areas include:

1. Constructing a recombinant expression vector for nitrate reductase and expressing it in E. coli BL21(DH5α).
2. Investigating the catalytic properties of nitrate reductase.
3. Designing enzymatic bioelectrodes and employing them for nitrate detection and degradation in food.


  1. [1] 张庆芳,李美玉,王晓辉,胡善松,于爽,迟乃玉.微生物亚硝酸盐还原酶的研究进展[J].微生物学通报,2019,46(11):3148-3157
  2. [2] Sam K A, Fairhurst S A, Thorneley R N F, et al. Pseudoazurin dramatically enhances the reaction profile of nitrite reduction by Paracoccus pantotrophus cytochrome cd1 and facilitates release of product nitric oxide[J]. Journal of Biological Chemistry, 2008, 283(18): 12555-12563.
  3. [3] Allen J W A, Higham C W, Zajicek R S, et al. A novel, kinetically stable, catalytically active, all-ferric, nitrite-bound complex of Paracoccus pantotrophus cytochrome cd1[J]. Biochemical Journal, 2002, 366(3): 883-888.
  4. [4] Klünemann T, Blankenfeldt W. Structure of heme d1-free cd1 nitrite reductase NirS[J]. Acta Crystallographica Section F: Structural Biology Communications, 2020, 76(6): 250-256.
  5. [5] A.S. Serra,S.R. Jorge,C.M. Silveira,J.J.G. Moura,E. Jubete,E. Ochoteco,G. Cabañero,H. Grande,M.G. Almeida. Cooperative use of cytochrome cd 1 nitrite reductase and its redox partner cytochrome c552 to improve the selectivity of nitrite biosensing[J]. Analytica Chimica Acta,2011,693(1).
  6. [6] Zhang X, Yang Q, Zhang Q, et al. Rapid detection of cytochrome cd1-containing nitrite reductase encoding gene nirS of denitrifying bacteria with loop-mediated isothermal amplification assay[J]. Scientific Reports, 2020, 10: 16484.
  7. [7] Silveira C M, Quintas P O, Moura I, et al. SERR Spectroelectrochemical Study of Cytochrome cd1 Nitrite Reductase Co-Immobilized with Physiological Redox Partner Cytochrome c552 on Biocompatible Metal Electrodes[J]. PLoS ONE, 2015, 10(6): e0129940.
  8. [8] Strehlitz B, Gründig B, Schumacher W, et al. A Nitrite Sensor Based on a Highly Sensitive Nitrite Reductase Mediator-Coupled Amperometric Detection[J]. Analytical Chemistry, 1996, 68(5): 807-816.
  9. [9] Niels Peter Revsbech,Michael Nielsen,Deby Fapyane. Ion Selective Amperometric Biosensors for Environmental Analysis of Nitrate, Nitrite and Sulfate[J]. Sensors,2020,20(15).
  10. [10] Fülöp V, Moir J W B, Ferguson S J, et al. The Anatomy of a Bifunctional Enzyme: Structural Basis for Reduction of Oxygen to Water and Synthesis of Nitric Oxide by Cytochrome cd1[J]. Cell, 1995, 81(3): 369-377.
  11. [11] 刘萍,罗岩,张莹,杨艳,孙君社,倪元颖. 亚硝酸还原酶的分离纯化及其性质研究[J]. 中国酿造,2011,30(9): 22-27.