巨大芽胞杆菌好氧发酵生产亚硝酸还原酶机制及该酶反应特性研究

项目名称： 巨大芽胞杆菌好氧发酵生产亚硝酸还原酶机制及该酶反应特性研究

项目编号： No.20876164

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 金属学与金属工艺

项目作者： 刘萍

作者单位： 中国农业大学

项目金额： 35万元

中文摘要： 由于环境中亚硝酸盐的大量富集及食品加工中添加化学合成的亚硝酸盐，使农产品原料、水源及加工食品中有大量亚硝酸盐残留。亚硝酸盐是一种对人体有害的物质，它不仅使人直接中毒、致死，而且是致癌物亚硝胺的前体。目前，生物方法降解亚硝酸盐残留是研究的重点和热点，主要是利用可降解亚硝酸盐的微生物或其产生的亚硝酸还原酶（nitrite reductase，NiR）来降低亚硝酸盐含量。 本项目研究在前期筛选菌种、NiR分离纯化及产酶条件初步优化的研究基础上，进一步探讨了利用添加外源电子供体及溶氧控制提高巨大芽孢杆菌B. megaterium MPF-906发酵产NiR酶活的方法，并探讨了各适宜发酵策略对发酵过程的影响；研究不同的初始碳源及氮源浓度对发酵过程的影响及发酵动力学；通过逐步优化培养基，获得了应用于养殖水中的适宜培养基组成，同时建立了巨大芽孢杆菌菌剂的制备工艺；通过正交试验确定了巨大芽孢杆菌产亚硝酸还原酶酶活力测定的适宜反应体系；将该酶加入到血红蛋白与亚硝酸盐为底物的反应体系中，在适宜的条件下即可生成无硝着色剂亚硝基血红蛋白，替代亚硝酸钠的发色作用，即可以降低肉制品中亚硝酸钠的残留量。

中文关键词： 巨大芽孢杆菌；亚硝酸盐还原酶；亚硝酸盐；发酵；反应特性

英文摘要： Because of enrichment of nitrite in environment and adding chemical synthesis of nitrite in food processing, there are a lot of nitrite residues in agricultural materials, water and processed foods. Nitrite is a kind of harmful substances, which makes people not only direct poisoning, and death, and is a precursor of nitrosamines carcinogens. At present, bio-degradation of residual nitrite is the focus of research. These problems could be resolved by using microorganisms or nitrite reductase (NiR) which was produced by microorganisms. Based on previous study of isolation of Bacillus megaterium MPF-906 and purification of NiR, in this work, we report the effects of adding artificial electron donors and dissolved oxygen on NiR activity, and effects of these methods on fermentation process were also investigated. The effect of different initial concentrations of sodium nitrite and glucose on fermentation process were also investigated. Micro ecology preparation of this strain was got.By orthogonal test, suitable reaction system for the determination of NiR activity is established. This enzyme is added to the reaction system of HbNO synthesis, in which nitrite and hemoglobin are added as the substrates which can generate non-nitrate stain nitroso hemoglobin in appropriate conditions.

英文关键词： acillus megaterium MPF-906； Nitrite Reductase; Nitrite； Fermentation; Reactivity