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Environ Sci Pollut Res Int. 2018 Sep 23. doi: 10.1007/s11356-018-3200-y. [Epub ahead of print]

Evaluation of aromatic hydrocarbon decomposition catalyzed by the dioxygenase system and substitution of ferredoxin and ferredoxin reductase.

Author information

1
Civil and Environmental Engineering, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 05029, South Korea.
2
Civil and Environmental Engineering, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 05029, South Korea. hankim@konkuk.ac.kr.

Abstract

In this study, the catalytic activity and kinetic characteristics of the aromatic hydrocarbon dioxygenase system and the possibility of substituting its ferredoxin and ferredoxin reductase components were evaluated. The genes encoding toluene dioxygenase and toluene dihydrodiol dehydrogenase were cloned from Pseudomonas putida F1, and the corresponding enzymes were overexpressed and purified to homogeneity. Oxidative hydroxylation of toluene to cis-toluene dihydrodiol was catalyzed by toluene dioxygenase, and its subsequent dehydrogenation to 3-methylcatechol was catalyzed by toluene dihydrodiol dehydrogenase. The specific activity of the dioxygenase was 2.82 U/mg-protein, which is highly remarkable compared with the values obtained in previous researches conducted with crude extracts or insoluble forms of enzymes. Kinetic parameters, as characterized by the Hill equation, were vmax = 497.2 μM/min, KM = 542.4 μM, and nH = 2.2, suggesting that toluene dioxygenase has at least three cooperative binding sites for toluene. In addition, the use of alternative ferredoxins and reductases was examined. Ferredoxin cloned from CYP153 could transfer electrons to the iron sulfur protein component of toluene dioxygenase. The ferredoxin could be reduced by ferredoxin, rubredoxin, and putidaredoxin reductases of CYP153, alkane-1 monooxygenase, and camphor 5-monooxygenase, respectively. The results provide useful information regarding the effective enzymatic biotreatment of hazardous aromatic hydrocarbon contaminants.

KEYWORDS:

Aromatic hydrocarbon contaminants; Catalytic activity; Dioxygenase system; Hill equation; Pseudomonas putida F1; Substitute enzyme components

PMID:
30244447
DOI:
10.1007/s11356-018-3200-y

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