Environmental Engineering, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Republic of Korea.
Environmental Engineering, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Republic of Korea.
Bioresour Technol. 2017 Dec;245(Pt B):1800-1807. doi: 10.1016/j.biortech.2017.05.006. Epub 2017 May 4.
This study aimed to clarify the initial 4-chlorophenol (4-CP) biodegradation pathway promoted by a two-component flavin-diffusible monooxygenase (TC-FDM) consisting of CphC-I and CphB contained in Arthrobacter chlorophenolicus A6 and the decomposition function of CphC-I. The TC-FDM genes were cloned from A. chlorophenolicus A6, and the corresponding enzymes were overexpressed. Since CphB was expressed in an insoluble form, Fre, a flavin reductase obtained from Escherichia coli, was used. These enzymes were purified using Ni-NTA resin. It was confirmed that TC-FDM catalyzes the oxidation of 4-CP and the sequential conversion of 4-CP to benzoquinone (BQN)→hydroquinone (HQN)→HQL. This indicated that CphC-I exhibits substrate specificity for 4-CP, BQN, and HQN. The activity of CphC-I for 4-CP was 63.22U/mg-protein, and the Michaelis-Menten kinetic parameters were v=0.21mM/min, K=0.19mM, and k/K=0.04mMmin. These results would be useful for the development of a novel biochemical treatment technology for 4-CP and phenolic hydrocarbons.
本研究旨在阐明由节杆菌（Arthrobacter chlorophenolicus）A6 中包含的两个组成部分黄素可扩散单加氧酶（TC-FDM）——CphC-I 和 CphB 促进的初始 4-氯苯酚（4-CP）生物降解途径，以及 CphC-I 的分解功能。从 A. chlorophenolicus A6 中克隆了 TC-FDM 基因，并对相应的酶进行了过表达。由于 CphB 以不溶形式表达，因此使用了从大肠杆菌中获得的黄素还原酶 Fre。这些酶使用 Ni-NTA 树脂进行了纯化。证实 TC-FDM 催化 4-CP 的氧化以及 4-CP 到苯醌（BQN）→对苯二酚（HQN）→对苯二酚（HQL）的连续转化。这表明 CphC-I 对 4-CP、BQN 和 HQN 具有底物特异性。CphC-I 对 4-CP 的活性为 63.22U/mg-蛋白，米氏动力学参数为 v=0.21mM/min、K=0.19mM 和 k/K=0.04mMmin。这些结果将有助于开发用于处理 4-CP 和酚类碳氢化合物的新型生化处理技术。
