精噁唑禾草灵降解菌株Rhodococcus sp. DSB-1分离鉴定、降解机制及其应用研究  

Isolation, identification and application of a fenoxaprop ethyl-degrading strain Rhodococcus sp. DSB-1

作　　者：张浩 滑紫微 牛秋红[1] 惠丰立[1] 董维亮 周杰 陈兆进 李娜 Hao Zhang;Ziwei Hua;Qiuhong Niu;Fengli Hui;Weiliang Dong;Jie Zhou;Zhaojin Chen;Na Li(School of Life Science and Technology,Nanyang Normal University,Nanyang 473061,Henan Province,China;College of Biotechnology and Pharmaceutical Engineering,Nanjing Tech University,Nanjing 211816,Jiangsu Province,China;School of Agricultural Engineering,Nanyang Normal University,Nanyang 473061,Henan Province,China)

机构地区：[1]南阳师范学院生命科学与技术学院,河南南阳473061 [2]南京工业大学生物与制药工程学院,江苏南京211816 [3]南阳师范学院农业工程学院,河南南阳473061

出　　处：《微生物学报》2020年第10期2226-2241,共16页Acta Microbiologica Sinica

基　　金：河南省能源微生物资源可持续利用创新型科技团队;河南省高等学校重点科研项目(20A180020);南阳师范学院高层次人才科研启动费资助项目(2019ZX015,2019ZX013)。

摘　　要：【目的】分离并鉴定精噁唑禾草灵高效降解菌株,为开发高效降解菌剂,强化精噁唑禾草灵原位修复,保证黄瓜产品安全提供菌株资源和理论依据。【方法】利用富集培养的方法分离降解菌株,并通过形态学、生理生化特征和16S rRNA基因进化分析进行鉴定;HPLC/MS鉴定菌株降解精噁唑禾草灵的中间产物,采用鸟枪法建库克隆降解过程中关键的水解酶基因,并进行异源表达,利用Michaelis-Menten双倒数曲线图测定酶动力学参数;通过正交试验确定菌株液态发酵参数,并通过对黄瓜灌根接种的方式,研究降解菌株对黄瓜根际土壤中精噁唑禾草灵的降解以及甘露醇对降解效率的强化作用。【结果】Rhodococcus sp.DSB-1在24 h内能将100 mg/L精噁唑禾草灵完全转化为精噁唑禾草灵酸,降解最适温度和pH分别为30℃和8.0。克隆得到一个精噁唑禾草灵水解酶基因,命名为pepE。水解酶PepE对精噁唑禾草灵的Km为28.2μmol/L,kcat/Km为11.0 L/(μmol·s)。在发酵温度30℃、通气量1:0.4、搅拌速度200 r/min、培养时间48 h条件下,液态发酵所得菌剂对精噁唑禾草灵的降解效率最高。投加至黄瓜根际的菌株DSB-1可以在黄瓜根系定殖,12d内完全降解黄瓜根际环境中10mg/kg的精噁唑禾草灵。此外还发现添加甘露醇可强化菌株的修复能力,降解效率相对于未添加的处理提高14.8%。【结论】菌株DSB-1具有原位修复精噁唑禾草灵污染土壤的潜力。[Objective] The purpose of this study is to isolate and identify the high-efficient degradation strains of fenoxaprop ethyl, providing strain resources and theoretical basis for the development of degradation agents, enhancing the in-situ bioremediation of fenoxaprop ethyl residue contaminated soil, and ensuring the safety of cucumber products. [Methods] The degradation strain was isolated by enrichment culture method and identified by morphology, physiological, biochemical characteristics and 16S rRNA gene evolution analysis. Intermediate products during fenoxaprop ethyl degradation were analyzed by HPLC/MS. The key hydrolase gene was cloned by shotgun method, and heterologously expressed. Michaelis-Menten double reciprocal curve and orthogonal tests were used to determine the enzyme kinetic and liquid fermentation parameters of the degradation strain. Through the irrigation of degradation strain, the fenoxaprop ethyl degradation in cucumber rhizosphere soil and the enhancement effect of mannitol on degradation efficiency were studied. [Results] Rhodococcus sp. DSB-1 was able to transform 100 mg/L fenoxaprop ethyl to fenoxaprop acid as sole carbon source within 24 h. The optimum degradation temperature and pH were 30 ℃ and 8.0, respectively. A fenoxaprop ethyl hydrolase gene named pepE was cloned from the genome of strain DSB-1 by shotgun method. The Km and kcat/Km of the hydrolase PepE towards fenoxaprop ethyl were 28.2 μmol/L and 11.0 L/(μmol·s). The degradation agent acquired possessed the highest degradation efficiency towards fenoxaprop ethyl under the fermentation conditions: temperature of 30 ℃, ventilation rate of 1:0.4, stirring speed of 200 r/min and culture time of 48 h. Strain DSB-1 could colonize on the cucumber root surface, and completely degraded 10 mg/kg fenoxaprop ethyl residue in cucumber rhizosphere soil within 12 d. Addition of mannitol could improve the degradation efficiency by 14.8% compared with the non-added treatment. [Conclusion] Strain DSB-1 could be potentially applied in bio

关 键 词：精噁唑禾草灵 微生物降解 水解酶基因pepE 定殖 强化修复 

分 类 号：X172[环境科学与工程—环境科学] X592