《环境昆虫学报》

烟碱含量的高低是烟草质量控制的一个重要指标，如何降低这些烟叶中的烟碱含量是一个现实问题。微生物降解烟碱具有成本低和生态相容性好优点，但是目前烟碱降解菌应用于烟草生产中报道很少。基于此，本研究从湖南烟草种植地分离纯化一株菌，形态学结合扫描电镜表明该菌为杆状细菌，16S rRNA测序为产脲节杆菌Paenarthrobacter ureafaciens，命名为Pu17。酶活测定表明Pu17产蛋白酶，不产淀粉酶、纤维素酶和β-葡萄糖苷酶；ELISA分析了Pu17的降解特性，Pu17适宜生长烟碱浓度为2.0 g/L，适宜生长温度为30℃，适宜生长pH为6；利用Slogistic模型对Pu17在烟碱浓度为2 g/L的纯烟碱培养基和LB培养基中的生长分析，0.1%、1.0%和2.0%接种量对应的生长延滞期分别为27.91、12.16和11.68 h，在烟碱浓度为2.0 g/L的LB中1.0%和2.0%接种量对应的生长延滞期分别为2.11和0.36 h。高效液相色谱（HPLC）分析表明，Pu17能以烟碱为唯一碳氮源生长，在烟碱浓度为2.0 g/L培养基中培养48 h，Pu17降解烟碱达到92.75%；而在烟碱浓度为2.0 g/L的LB液体培养基中，60 h达到最大降解率为90.57%。大田喷施Pu17可以显著降解烟叶中的烟碱含量，Pu17在喷施后24 h时达到极显著差异水平（P＜0.0001）；60 h也达到差异显著水平（P = 0.0175）。本研究筛选获得了降解烟草烟碱的产脲节杆菌并明确了其降解特性，为烟草烟碱的降解提供了微生物资源，并为环境保护提供了新思路。

英文摘要:

The level of nicotine content is an important index of tobacco quality control, and how to reduce the content of nicotine in these tobacco leaves is a practical problem. Microbial degradation of nicotine has the advantages of low cost and good ecological compatibility, but there are few reports on the application of nicotine-degrading bacteria in tobacco production. Based on this, this study isolated and purified a strain from tobacco growing field in Hunan Province. Morphology combined with scanning electron microscopy showed that the strain was rod-shaped bacteria, 16S rRNA sequencing was Paenarthrobacter ureafaciens, named Pu17. Enzyme activity assay showed that Pu17 produced protease, but not amylase, cellulase and β-glucosidase. The degradation characteristics of Pu17 were analyzed by ELISA. The optimum growth rate of Pu17 was 2.0 g/L, the optimum growth temperature was 30℃, the optimum culture time was 48 h, and the optimum growth pH was 6. The Slogistic model was used to compare the growth of Pu17 in pure nicotinic medium and LB medium with a nicotine concentration of 2 g/L. The lag time in growth nicotinic medium with 0.1%, 1.0% and 2.0% inoculations was 27.91, 12.16 and 11.68 h, respectively. In LB with a nicotine concentration of 2.0 g/L, the growth delays corresponding to 1.0% and 2.0% inoculations were 2.11 and 0.36 h, respectively. High performance liquid chromatography (HPLC) analysis showed that Pu17 could grow with nicotine as the only nitrogen source, and cultured in the medium with nicotine concentration of 2.0 g/L for 48 h, Pu17 degraded nicotine up to 92.75%. In LB liquid medium with nicotine concentration of 2.0 g/L, the maximum degradation rate was 90.57% at 60 h. The nicotine content in tobacco leaves was significantly degraded by spraying Pu17 in the field, and Pu17 reached a significant difference level at 24 h after spraying (P < 0.0001). The difference was also significant at 60 h (P=0.0175). In this study, P. ureafaciens was screened to degrade tobacco nicotine and its degradation characteristics were defined, which provided microbial resources for the degradation of tobacco nicotine and provided a new idea for environmental protection.

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