College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310032, China.
Technology Center, China Tobacco Zhejiang Industrial Co., Ltd., Hangzhou, 310009, China.
Protein Expr Purif. 2021 Feb;178:105767. doi: 10.1016/j.pep.2020.105767. Epub 2020 Sep 26.
Nicotine contamination in tobacco waste effluent (TWE) from tobacco industry is a serious threat to public health and environment. Microbial degradation is an impending approach to remove nicotine and transform it into some other high value chemicals. Pseudomonas sp. JY-Q exhibits high efficiency of degradation, which can degrade 5 g/L of nicotine within 24 h. In strain JY-Q, we found the co-occurrence of two homologous key enzymes NicA2 and Nox, which catalyze nicotine to N-methylmyosmine, and then to pseudooxylnicotine via simultaneous hydrolysis. In this study, recombinant NicA2 and Nox were expressed in E. coli BL21(DE3) and purified. In vitro, the activity of recombinant NicA2 and Nox was accelerated by adding co-factor NAD, suggesting that they worked as dehydrogenases. The optimal reaction conditions, substrate affinity, catabolism efficiency, pH-stability and thermal-stability were determined. Nox showed lower efficiency, but at a higher stability level than NicA2. Nox exhibited wider pH range and higher temperature as optimal conditions for the enzymatic reaction. In addition, The Nox showed higher thermo-stability and acid-stability than that of NicA2. The study on enzymatic reaction kinetics showed that Nox had a lower Km and higher substrate affinity than NicA2. These results suggest that Nox plays more significant role than NicA2 in nicotine degradation in TWE, which usually is processed at low pH (4-5) and high temperature (above 40 °C). Genetic engineering is required to enhance the affinity and suitability of NicA2 for an increased additive effect on homologous NicA2 and Nox in strain JY-Q.
烟草工业废水中的尼古丁污染是对公共健康和环境的严重威胁。微生物降解是去除尼古丁并将其转化为其他高价值化学品的一种有前途的方法。假单胞菌 JY-Q 表现出高效的降解能力，可在 24 小时内降解 5 g/L 的尼古丁。在菌株 JY-Q 中，我们发现了两种同源关键酶 NicA2 和 Nox 的共存，它们催化尼古丁生成 N-甲基肌氨酸，然后通过同时水解生成伪氧尼古丁。在这项研究中，重组 NicA2 和 Nox 在大肠杆菌 BL21(DE3)中表达并纯化。在体外，添加辅因子 NAD 可加速重组 NicA2 和 Nox 的活性，表明它们作为脱氢酶起作用。确定了最佳反应条件、底物亲和力、分解代谢效率、pH 稳定性和热稳定性。Nox 的效率较低，但稳定性水平较高。Nox 表现出更低的 pH 值范围和更高的温度，作为酶反应的最佳条件。此外，Nox 表现出比 NicA2 更高的热稳定性和酸稳定性。酶反应动力学研究表明，Nox 的 Km 值较低，对底物的亲和力高于 NicA2。这些结果表明，在 TWE 中尼古丁降解中，Nox 比 NicA2 发挥更重要的作用，因为 TWE 通常在低 pH（4-5）和高温（40°C 以上）下进行处理。需要进行基因工程来增强 NicA2 的亲和力和适用性，以增加对同源 NicA2 和 Nox 的加性效应。
