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클로로페놀의 생화학적 분해를 위한 분해효소(chlorophenol monooxygenase) 소단위체의 대량발현조건 최적화
이소라 ( Christina S. Kang ),류송정 ( Song Jung Ryu ),김한승 ( Han S. Kim ) 한국폐기물자원순환학회 2014 젊은 연구자 학술연구발표회 Vol.13 No.0
In this study, the feasibility of enzymatic bioremediation of 4-chlorophenol was examined using the TC-FDM of Arthrobacter chloropheno1ieus A6. Here we report the cloning, overexpress ion, and purification of the reductase component from the gene cphB (AAN08757) of A. chlorophenolicus A6, which is a putative chlorophenol monooxygenase small subunit. The gene cphB was cloned into vector pET-24a with or without GB1 and GST solubility/affinity tags, and 3 different strains of Escherichia coli (BL21 (DE3), BL21-CodonPlus® (DE3)-RIL, and Tuner (DE3)) were transformed with these recombinant genes. The expression conditions for soluble form enzymes were optimized by varying incubation time, temperature, and concentration of inducer (isopropyl β-D-1-thiogalactopyranoside). In addition, the soluble enzymes were purified by GST affinity chromatography, and the protein yield was analyzed with the Bradford protein assay using bovine serum albumin as a standard. The results of this study are expected to establish the basis of the use of TC-FDM in enzymatic bioremediation of phenol-contaminated geoenvironment.
류송정,이소라,김한승,Ryu, Song-Jung,Kang, Christina S.,Kim, Han S. 한국지하수토양환경학회 2014 지하수토양환경 Vol.19 No.3
Arthrobacter chlorophenolicus A6 possesses several monooxygenases (CphC-I, CphC-II, and CphB) that can catalyze the transformation of 4-chlorophenol (4-CP) to hydroxylated intermediates in the initial steps of substrate metabolism. The corresponding genes of the monooxygenases were cloned, and the competent cells were transformed with these recombinant plasmids. Although CphC-II and CphB were expressed as insoluble forms, CphC-I was successfully expressed as a soluble form and isolated by purification. The specific activity of the purified CphC-I was analyzed by using 4-CP, 4-chlorocatechol (4-CC), and catechol (CAT) as substrates. The specific activities for 4-CP, 4-CC, and CAT were determined to be 0.312 U/mg, 0.462 U/mg, 0.246 U/mg, respectively. The results of this study indicated that CphC-I is able to catalyze the degradation of 4-CC and CAT in addition to 4-CP, which is a primary substrate. This research is expected to provide the fundamental information for the development of an eco-friendly biochemical degradation of aromatic hydrocarbons.