Page 61 - 《精细化工》2022年第5期

第 39 卷第 5 期 精 细 化 工 Vol.39, No.5
2022 年 5 月 FINE CHEMICALS May 2022

生物工程
混合菌的构建及其对 3,4-二氯苯胺的生物转化

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倪金荧，赵 佳，张 跃，卿 青，朱 劼，王利群
（常州大学 药学院，江苏 常州 213164）

摘要：从染料废水中分离出以 3,4-二氯苯胺（3,4-DCA）为唯一碳源和能源生长的革兰氏阴性菌，筛选其中两株
形态差异较大的菌株 P11-1 和 L13 构建混合菌群，用于 3,4-DCA 的转化实验以削减其毒性。通过优化两株菌配
比构建混合菌群，混合菌群对 3,4-DCA 转化率达 73.33%，明显高于单菌。经菌株形态观察和 16S rDNA 序列分
析，确定两株菌均为铜绿假单胞菌。以单因素实验考察混合菌群在 3 d 内对 100 mg/L 3,4-DCA 的转化能力，并
通过正交实验进一步优化转化条件，得出最佳条件组合为：接菌量 3%（体积分数），温度 30 ℃，pH=8.0，摇
床转速 190 r/min，在该条件下 3,4-DCA 转化率达 89.26%，其中 pH 对菌群转化能力影响最大。GC-MS 分析菌
群代谢产物主要为 3,4-二氯乙酰苯胺，酶活分析发现 N-乙酰基转移酶（NAT）活性较高，即菌群通过乙酰化对
3,4-DCA 进行解毒。
关键词：3,4-二氯苯胺；铜绿假单胞菌；转化；N-乙酰基转移酶；3,4-二氯乙酰苯胺；生物工程
中图分类号：X703 文献标识码：A 文章编号：1003-5214 (2022) 05-0915-06

Construction of mixed bacterial strain and its biotransformation of
3,4-dichloroaniline

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NI Jinying, ZHAO Jia, ZHANG Yue, QING Qing, ZHU Jie, WANG Liqun
（School of Pharmacy, Changzhou University, Changzhou 213164, Jiangsu, China）

Abstract: Gram-negative bacteria growing on 3,4-dichloroaniline (3,4-DCA) as the only carbon source and
energy were isolated from dye wastewater. Two strains with great morphological differences, P11-1 and
L13, were screened to construct mixed bacterial strain, which was used to the transformation of 3,4-DCA to
reduce the toxicity of 3,4-DCA. By optimizing the proportion of the two strains to construct mixed bacterial
strain, the conversion rate of 3,4-DCA reached 73.33%, obviously higher than that of single strain. Both
strains were identified as Pseudomonas aeruginosa by morphology observation and 16S rDNA sequence
analysis. The transformation ability of mixed strain to 100 mg/L 3,4-DCA in 3 d was evaluated by single
factor experiment. The transformation conditions were further optimized by orthogonal experiment. The
results showed that the best conditions was obtained as follows: inoculum amount 3% (volume fraction),
30 ℃, pH=8.0, shaking velocity 190 r/min. Under these conditions, the transformation rate was up to
89.26%. Especially, pH had the greatest effect on the transformation ability of mixed strain. GC-MS result
revealed that the main metabolite of mixed strain was 3,4-dichloroacetanilide. Enzyme activity analysis
indicated that the mixed strain had higher N-acetyltransferase (NAT) activity, that is, it detoxified 3,4-DCA
by acetylation.
Key words: 3,4-dichloroaniline; Pseudomonas aeruginosa; transformation; N-acetyltransferase; 3,4-
dichloroacetanilide; biological engineering

3,4-二氯苯胺（3,4-DCA）作为一种卤代芳香族 残留于生产废水中，常通过工业和城市废水进入环
化合物，是多种精细化工产品的主要中间体，广泛 境中累积 [1-4] 。3,4-DCA 为剧毒化学品，在土壤和水
用于生产染料、药物、杀虫剂等有机产品，因其易 中具有高持久性和低生物降解性 [5-6] ，通过食物链影

收稿日期：2021-11-21; 定用日期：2022-01-19; DOI: 10.13550/j.jxhg.20211186
作者简介：倪金荧（1995—），女，硕士生，E-mail：1824529633@qq.com。联系人：王利群（1972—），女，教授，硕士生导师，E-mail：
wlq@cczu.edu.cn。