一种检测2'-甲基-6'-乙基-2-氯乙酰苯胺水解酶活性的方法

doi:10.11934/j.issn.1673-4831.2016.04.026

生态与农村环境学报 ›› 2016, Vol. 32 ›› Issue (4): 682-686.doi: 10.11934/j.issn.1673-4831.2016.04.026

一种检测2'-甲基-6'-乙基-2-氯乙酰苯胺水解酶活性的方法

王飞^1,2, 李周坤², 董维亮², 崔中利²

1. 江西省农业微生物资源开发与利用工程实验室/江西农业大学生物科学与工程学院, 江西南昌 330045;
2. 南京农业大学生命科学学院/农业部农业环境微生物工程重点开放实验室, 江苏南京 210095

收稿日期:2015-08-30 出版日期:2016-07-25 发布日期:2016-07-26
通讯作者: 崔中利 E-mail:czl@njau.edu.cn E-mail:czl@njau.edu.cn
作者简介:王飞(1976-),男,湖北天门人,讲师,博士,主要从事环境微生物学研究。E-mail:wangfei179@163.com
基金资助:
国家自然科学基金（31560031，31570059）；江西省自然科学基金（20161BAB204178）

A New Method for Determining Activity of CMEPA Hydrolase

WANG Fei^1,2, LI Zhou-kun², DONG Wei-liang², CUI Zhong-li²

1. Jiangxi Engineering Laboratory for the Development and Utilization of Agricultural Microbial Resources/College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang 330045, China;
2. Key Laboratory of Microbiological Engineering Agricultural Environment, Ministry of Agriculture/College of Life Science, Nanjing Agricultural University, Nanjing 210095, China

Received:2015-08-30 Online:2016-07-25 Published:2016-07-26

摘要/Abstract

摘要：

建立了一种快速测定Delftia acidovorans T3-6菌株2'-甲基-6'-乙基-2-氯乙酰苯胺（CMEPA）水解酶活性的方法。在20 mmol·L^-1 Tris-HCl（pH 值为7.0）缓冲液中，10～50 μmol·L^-1的CMEPA水解产物2-甲基-6-乙基苯胺（MEA）在铁氰化钾催化下与4-氨基安替比林反应生成紫红色物质，该物质在535 nm处的吸光度（y）与MEA浓度（x）呈正相关（y=0.014 6x-0.005 4，R²=0.9988）。底物CMEPA、十二烷基硫酸钠（SDS）、甲醇等有机试剂以及1 mmol·L^-1金属离子对显色反应无显著影响，温度和pH值对显色反应影响显著。4-氨基安替比林显色法对菌株T3-6粗酶液酶活性的测定结果与二氯甲烷萃取-紫外分光光度法无显著差异。该方法也适用于其他苯胺苯酚类衍生物的测定。

关键词: 2-甲基-6-乙基苯胺(MEA), 4-氨基安替比林显色法, 分光光度法, Delftia acidovorans T3-6

Abstract:

A new method for rapid determination of the activity of 2'-methyl-6'-ethyl-2-chloroacetanilide(CMEPA) hydrolase in Delftia acidovorans T3-6 was established. CMEPA hydrolase can hydrolyze CMEPA into 2-methyl-6-ethyl-aniline (MEA), which, in turn, reacts with 4-aminoantipyrene in 20 mmol·L^-1 Tris-HCl (pH 7.0) buffer to produce a compound, purple in color, with absorbancy at 535 nm(y) positively related to the concentration of MEA(x) in the range of 10-50 μmol·L^-1. The linear equation goes as y=0.014 6x-0.005 4, R²=0.9988. The compounds of CMEPA, SDS, organic reagent such as methanol and 1 mmol·L^-1 metal ions have no significant effect on chromogenic reaction, whereas temperature and pH does. Compared with the dichloromethane extraction-UV spectrophotometry method, the 4-aminoantipyrene colorimetric method does not vary much in determining enzymatic activity of T3-6 crude enzyme. This method is also applicable to the determination of other derivatives of aniline and phenol.

Key words: 2-methyl-6-ethyl-aniline (MEA), 4-aminoantipyrinechromogenic method, spectrophotometry, Delftia acidovorans T3-6

中图分类号:

X592

王飞, 李周坤, 董维亮, 崔中利. 一种检测2'-甲基-6'-乙基-2-氯乙酰苯胺水解酶活性的方法[J]. 生态与农村环境学报, 2016, 32(4): 682-686.

WANG Fei, LI Zhou-kun, DONG Wei-liang, CUI Zhong-li. A New Method for Determining Activity of CMEPA Hydrolase[J]. Journal of Ecology and Rural Environment, 2016, 32(4): 682-686.

参考文献

[1] 徐军,邱星辉,曹宏,等.氯乙酰胺类除草剂微生物降解研究进展[J].应用与环境生物学报,2004,10(3):389-393.
[2] 原居林,胡大雁,杨卫明,等.印染废水对3种水生生物的毒性作用[J].生态与农村环境学报,2012,28(2):209-212.
[3] BOLLAG J M,BLATTMANN P,LAANIO T.Adsorption and Transformation of Four Substituted Anilines in Soil[J].Journal of Agricultural and Food Chemistry,1978,26(6):1302-1306.
[4] 梁建奎,张永祥,王然,等.Fe⁰体系降解2,4-二氯酚的影响因素及其反应机理[J].生态与农村环境学报,2011,27(6):64-67.
[5] 侯颖.芳氧苯氧丙酸类和氯乙酰胺类除草剂降解菌的分离鉴定、降解基因克隆表达及其代谢途径研究[D].南京:南京农业大学,2011.
[6] 沈仁富,王凌星,陈云忠,等.井水中苯胺的快速检测方法[J].中国卫生检验杂志,2005,14(6):723-723.
[7] GOTTLIEB S,MARSH P B.Quantitative Determination of Phenolic Fungicides[J].Industrial & Engineering Chemistry Analytical Edition,1946,18(1):16-19.
[8] El-DIB M A,ABDEL-RAHMAN M O,ALY O A.4-aminoantipyrine as a Chromogenic Agent for Aromatic Amine Determination in Natural Water[J].Water Research,1975,9(5):513-516.
[9] 侯颖,王飞,李静泉,等.Delftia sp. T3-6菌株及其粗酶液对2',6'-甲乙基-2-氯乙酰苯胺的降解[J].环境科学学报,2014,34(6):1396-1402.
[10] SHEN W,CHEN H,JIA K,et al.Cloning and Characterization of a Novel Amidase From Paracoccus sp.M-1,Showing Aryl Acylamidase and Acyl Transferase Activities[J].Applied Microbiology and Biotechnology,2012,94(4):1007-1018.
[11] LONG M C,WAN L,ZENG C,et al.Enhanced Visible Light Activity of BiVO₄ by Treating in HCl Aqueous Solution and Its Mechanism[J].Acta Physico:Chimica Sinica,2012,28(12):2917-2923.
[12] 李惠娟,李琼芳,刘永梅,等.Fe掺杂TiO₂ 催化剂的制备及光催化降解苯酚的研究[J].云南大学学报(自然科学版),2011(增刊2):402-407.
[13] 蔡裕丰,吴春妍.4-氨基安替比林直接光度法测定污水中高浓度挥发酚[J].环境科学导刊,2012,31(3):85-87.
[14] 吴芳,杨立群,刘松.4-氨基安替比林分光光度法测定水中微量苯胺类化合物的研究[J].化学工程与装备,2014(2):194-196.
[15] HONG J M,JUN J K,KIM H Y,et al.Colorimetric Signaling of Cu (Ⅱ) Ions by Oxidative Coupling of Anilines With 4-aminoantipyrine[J].Tetrahedron Letters,2015,56(40):5393-5396.

一种检测2'-甲基-6'-乙基-2-氯乙酰苯胺水解酶活性的方法

A New Method for Determining Activity of CMEPA Hydrolase

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