Function Analysis of Chlorophenol Monooxygenase for Chlorophenol Degradation

Christina S. Kang; Han Seung Kim

doi:10.4028/www.scientific.net/AMR.1073-1076.700

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Function Analysis of Chlorophenol Monooxygenase for Chlorophenol Degradation

Abstract:

4-Chlorophenol is well-known as an extensively used antiseptic, and it may cause severe damage to the environment and human health. 4-Chlorophenol can be biologically degraded by Arthrobacter chlorophenolicus A6, which can be attributed to the cphC-I and cphB genes of the microorganism that encode for a two-component flavin-diffusible monooxygenase (TC-FDM), composed of oxygenase and reductase components. This study reports the cloning, overexpression, purification, and function analysis of the oxygenase and reductase components from the genes cphC-I and cphB. The genes were cloned into vector pET-24a, and 4 different strains of Escherichia coli were transformed with these recombinant genes. The optimization of expression conditions indicated that cphC-I is best overexpressed in E. coli BL21(DE3) incubated for 24 hours at 15°C with 0.5 mM of isopropyl-β-D-1-thiogalactopyranoside. However, cphB was not expressed into soluble form enzyme in any of the conditions, and therefore fre of E. coli was used instead to analyze the function of CphC-I. CphC-I was able to degrade approximately 13.86% of 4-chlorophenol, indicating that it is indeed a reduced flavin-dependent monooxygenase and utilizes 4-chlorophenol as a substrate. The results of this study are expected to establish the basic understanding of TC-FDM for its application to enzymatic bioremediation of phenol-contaminated environment.