固氮菌(Ensifer meliloti 1021)降解苯甲腈及其代谢酶的基因克隆和表达

doi:10.19741/j.issn.1673-4831.2018.0035

生态与农村环境学报 ›› 2019, Vol. 35 ›› Issue (2): 248-254.doi: 10.19741/j.issn.1673-4831.2018.0035

固氮菌(Ensifer meliloti 1021)降解苯甲腈及其代谢酶的基因克隆和表达

郭静静¹, 郭磊磊¹, 赵云岫¹, 葛峰², 戴亦军¹

1. 南京师范大学生命科学学院/江苏省微生物与功能基因组学重点实验室/江苏省微生物资源产业化工程技术研究中心, 江苏南京 210023;
2. 生态环境部南京环境科学研究所, 江苏南京 210042

收稿日期:2018-01-24 出版日期:2019-02-25 发布日期:2019-03-25
通讯作者: 葛峰, 戴亦军 E-mail:gefeng@nies.org;daiyijun@njnu.edu.cn
作者简介:郭静静(1990-),女,山东菏泽人,硕士,主要从事污染物生物修复研究。E-mail:guojjhz@163.com
基金资助:
国家自然科学基金（31570104）

Degradation of Benzonitrile by the Nitrogen-Fixing Bacterium Ensifer meliloti 1021 and the Cloning and Over-Expression of the Genes Encoding the Corresponding Metabolic Enzymes

GUO Jing-jing¹, GUO Lei-lei¹, ZHAO Yun-xiu¹, GE Feng², DAI Yi-jun¹

1. Jiangsu Key Laboratory for Microbes and Functional Genomics/Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources/College of Life Science, Nanjing Normal University, Nanjing 210023, China;
2. Nanjing Institute of Environmental Sciences, Ministry of Ecology and Environment, Nanjing 210042, China

Received:2018-01-24 Online:2019-02-25 Published:2019-03-25

摘要/Abstract

摘要：

对固氮菌（Ensifer meliloti 1021）降解环境污染物苯甲腈途径及其相关酶进行了基因克隆和表达研究。高效液相色谱法（HPLC）分析显示En.meliloti 1021静息细胞可将苯甲腈降解为苯甲酰胺和苯甲酸。En.meliloti 1021全基因组中没有腈水解酶（nitrilase）基因，但有1个腈水合酶（nitrile hydratase）基因和12个酰胺酶（amidase）基因，因而其降解苯甲腈生成苯甲酸是经腈水合酶/酰胺酶途径。PCR扩增En.meliloti 1021的腈水合酶基因并在Escherichia coli Rosetta（DE3）中成功表达。腈水合酶过表达的E.coli可在5 min内降解90%的浓度为97 mmol·L^-1的苯甲腈，10 min基本检测不到苯甲腈，产生66.9 mmol·L^-1苯甲酰胺。对12个酰胺酶进行了系统发育树分析，发现有4个酰胺酶与Genbank数据库中的苯甲酰胺水解酶（benzamide amidohydrolase）有较高的同源性。对这4个酰胺酶基因进行克隆表达以及酶活检测，发现只有登录号为CAC47672.1的酰胺酶有苯甲酰胺酶活性，可将苯甲酰胺水解为苯甲酸。该酰胺酶由434个氨基酸组成，分子量为47 kDa，等电点5.37。

关键词: 固氮菌, 腈水合酶, 酰胺酶, 苯甲腈, 微生物降解

Abstract:

The degradation pathway of the environmental pollutant benzonitrile by the nitrogen-fixing bacterium Ensifer meliloti 1021 was investigated. And the genes encoding the relevant metabolic enzymes were cloned and over-expressed. High performance liquid chromatography (HPLC) analysis revealed that En. meliloti 1021 resting cells degraded benzonitrile to benzamide and benzoic acid. A whole-genome analysis indicated that En. meliloti 1021 lacks nitrilase gene, but contains one nitrile hydratase (NHase) gene and 12 amidase genes. Therefore, the benzonitrile degradation pathway appears to be mediated by the NHase-amidase system. The NHase gene was amplified by PCR and over-expressed in Escherichia coli Rosetta (DE3)cells.The resulting cells degraded 97 mmol·L^-1 benzonitrile in 5 min (90% degradation rate). At 10 min, benzonitrile was undetectable, while 66.9 mmol·L^-1 benzamide was present. A phylogenetic tree including the En.meliloti 1021 amidase genes and three benzamide amidohydrolase genes indicated that four amidase genes clustered with the three benzamide amidohydrolase genes. Gene cloning and over-expression experiments proved that an amidase (Genbank accession number CAC47672.1)exhibits the benzamidase activity responsible for converting benzamide to benzoic acid. Benzamidase comprises 434 amino acids, with a molecular weight of 47 kDa and an isoelectric point of 5.37. The results of this study have a certain theoretical value and may be useful for eliminating benzonitrile from the environment.

Key words: Ensifer meliloti, nitrilehydratase, amidase, benzonitrile, microbialdegradation

中图分类号:

Q814

郭静静, 郭磊磊, 赵云岫, 葛峰, 戴亦军. 固氮菌(Ensifer meliloti 1021)降解苯甲腈及其代谢酶的基因克隆和表达[J]. 生态与农村环境学报, 2019, 35(2): 248-254.

GUO Jing-jing, GUO Lei-lei, ZHAO Yun-xiu, GE Feng, DAI Yi-jun. Degradation of Benzonitrile by the Nitrogen-Fixing Bacterium Ensifer meliloti 1021 and the Cloning and Over-Expression of the Genes Encoding the Corresponding Metabolic Enzymes[J]. Journal of Ecology and Rural Environment, 2019, 35(2): 248-254.

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固氮菌(Ensifer meliloti 1021)降解苯甲腈及其代谢酶的基因克隆和表达

Degradation of Benzonitrile by the Nitrogen-Fixing Bacterium Ensifer meliloti 1021 and the Cloning and Over-Expression of the Genes Encoding the Corresponding Metabolic Enzymes

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