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.

烟草业产生的烟草废液中的尼古丁污染严重威胁公共卫生和环境。微生物降解是一种去除尼古丁并将其转化为其他高价值化学品的迫在眉睫的方法。假单胞菌属。JY-Q的降解效率很高，可以在24小时内降解5 g / L的尼古丁。在菌株JY-Q中，我们发现了两个同源关键酶NicA2和Nox共同存在，它们将尼古丁催化到N-甲基肌胺，然后通过同时水解催化到假氧烟碱。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，通过加入辅因子NAD可以加速重组NicA2和Nox的活性⁺，表明它们起脱氢酶的作用。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，但稳定性高于NicA2。Nox表现出更宽的pH范围和更高的温度，这是酶促反应的最佳条件。另外，Nox显示出比NicA2更高的热稳定性和酸稳定性。酶促反应动力学研究表明，Nox的K值较低。m和比NicA2高的底物亲和力。这些结果表明，在TWE中尼古丁降解中，NOx的作用比NicA2更重要，后者通常在低pH（4-5）和高温（40°C以上）下加工。需要基因工程来增强NicA2的亲和力和适用性，以增加对JY-Q菌株中同源NicA2和Nox的加性作用。