Screening, Identification and Degradation Characteristics of Nicotine-degrading Bacteria in Lasioderma serricorne

doi:10.13560/j.cnki.biotech.bull.1985.2022-1198

Abstract

Abstract:

The nicotine-degrading bacteria were screened and identified from Lasioderma serricorne, and their degradation characteristics were explored. The nicotine-degrading bacteria were primarily isolated and screened from L. serricorne by gradient dilution method, and re-screening was carried out using the four kinds of medium containing tobacco leaves. The strains were identified based on 16S rRNA gene sequencing combined with physiological and biochemical analysis. The whole genome sequence of the bacteria with degradation capabilities was analyzed to explore the degradation characteristics. Then, the potential degradation genes were explored through molecular docking and RT-qRCR. One bacterial strain J1 was isolated from L. serricorne, which significantly degraded nicotine. The strain J1 was identified as Mixta by 16S rRNA gene homology combined with physiological and biochemical characteristics. The results on the enzyme activities analysis showed that J1 did not produce β-glucosidase, fiber Enzymes, proteases, and amylases. The metabolism pathway and the key genes or enzymes involved in degrading nicotine in J1 were explored through functional annotation in the database. The nicotine dehydrogenase（NDH）possessed by J1 in the pyridine pathway was selected for molecular docking and functional verification. It was verified by RT-qPCR that the relative expression of ndh gene was up-regulated by 15.978 times using 1 g/L nicotine induction for 24 h compared with the control group. Additionally, the relative expression of ndh gene was up-regulated by 1.117 times when J1 was induced by 0.8% tobacco leaf extract for 24 h. The high-efficiency nicotine-degrading bacteria J1 was isolated and screened in L. serricorne. The degradation-related genes were verified. The results provided a new idea for the screening of nicotine-degrading bacteria and simultaneously enriched the nicotine-degrading microbial resources.

Key words: endophytic bacteria of Lasioderma serricorne, genus Mixta, nicotine tolerance, nicotine degradation

WANG Yu, YIN Ming-shen, YIN Xiao-yan, XI Jia-qin, YANG Jian-wei, NIU Qiu-hong. Screening, Identification and Degradation Characteristics of Nicotine-degrading Bacteria in Lasioderma serricorne[J]. Biotechnology Bulletin, 2023, 39(6): 308-315.

Figures/Tables 12

Fig. 1 Isolation and screening results of nicotine-degrading bacteria A: The tobacco plate spread with Lasioderma serricorne abrasive solution. B: The tobacco plate spread with ddH2O rinse solution after aseptic treatment of Lasioderma serricorne

Table 1 Morphological characteristics of high nicotine-degrading bacteria

菌株编号No. of strain	形态特征Morphological characteristics
J1	菌落小，圆形，表面光滑，白色
J2	菌落小，圆形，表面光滑，白色
YC1	菌落小，圆形，表面光滑，浅黄色
YC2	菌落大，圆形，表面黏稠，黄色
YC3	菌落小，圆形，表面光滑，乳白色
YC4	菌落大，圆形，表面褶皱，白色
YC5	菌落小，圆形，表面光滑，白色不透明
YC6	菌落小，圆形，表面黏稠，黄色
YC7	菌落大，圆形，表面光滑，乳白色
YC8	菌落小，圆形，表面黏稠，乳白色
YC9	菌落小，圆形，表面褶皱，浅黄色
YC10	菌落大，圆形，表面褶皱，浅黄色

Fig. 2 Rescreening results of nicotine-degrading bacteria on different tobacco medium plates

Fig. 3 Detection of enzyme activities in J1

Fig. 4 Phylogenetic tree of J1 based on 16S rRNA gene sequence

Fig. 5 Standard curve of nicotine UV absorption

Fig. 6 Changes of nicotine degradation rates of tobacco leaves treated with J1 bacterial solutions for 5 d *P<0.05, the same below

Fig. 7 Results of SDS-PAGE electrophoresis for J1 protein samples M is marker; lane 1-5 are ammonium sulfate saturation: 100%, 80%, 50%, 30%, and 0%

Fig. 8 Standard curve of protein concentration detected by BCA method

Table 2 Results of crude enzyme solution concentration detected by BCA method

样品Sample	蛋白浓度Protein concentration/（μg·μL^-1）
J1（0%）	2.317
J1（50%）	3.250
J1（80%）	4.360
J1（100%）	2.978

Fig. 9 Changes of nicotine degradation rates of tobacco leaves treated with crude enzyme solutions of J1 for 2 d

Fig. 10 Docking results of nicotine dehydrogenase（NDH）of J1 with nicotine

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