Isolating, purifying and determining of biochemical properties of catechol 1, 2 dioxygenase from microbial flora in petroleum-contaminated soils

Rabbani, Faeze; Jafarian, Vahab; Asoodeh, Ahmad

Isolating, purifying and determining of biochemical properties of catechol 1, 2 dioxygenase from microbial flora in petroleum-contaminated soils

Document Type : Original Research

Authors

Faeze Rabbani ¹

vahab jafarian ²

ahmad asoodeh ³

¹ Department of Biology, Faculty of Science, University of Zanjan

² Department of Biology, Faculty of Science, University of Zanjan, Zanjan, Iran

³ Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran

Abstract

The present study was accomplished to purify and biochemically characterize the phenol-degrading enzyme from the bacteria existed in petroleum-contaminated soils. The catechol 1, 2 dioxygenase was extracted from Aneurinibacillus migulanus Isolate ZNU05 and purified using Q-Sepharose ion exchange chromatography column. The enzyme activity was examined under different pHs (ranged from 4 to 9), at different temperatures (ranged from 20 to 70˚C), in the presence of various metal ions chloride salts (Ca²⁺, K⁺, Mn²⁺, Co²⁺, Zn²⁺, Mg²⁺, Cu²⁺ and Na⁺), and with various solvents (ethanol, ethyl acetate, petroleum ether, acetonitrile, N-amyl alcohol, N-hexane, and toluene). In addition, the enzyme activity was investigated using different substrates such as phenol, catechol, benzoic acid, pyrogallol and α-naphtol. SDS-PAGE analysis indicated that there was a single-band protein with a molecular weight of approximately 40 kDa. The catechol 1, 2 dioxygenase had a maximum activity at temperature 30˚C at pH 8.5. Moreover, the catalytic activity of the enzyme was increased in the presence of cobalt and zinc ions as well as organic solvent of amyl alcohol, while it was decreased or inhibited in the presence of the other metal ions and organic solvents used. Among different substrates on enzyme activity, catechol was the most favorable for the enzyme, so that, the V_max and K_m were 8.959 U/mg and 4.992 µg/mL for the substrate, respectively.

Keywords

petroleum pollutant

Phenol degradation

Protein purification

Catechol 1

2 dioxygenase

20.1001.1.23222115.1399.11.2.9.0

Subjects

Microbial biotechnology

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