Classic Pentachlorophenol Hydroxylating Phenylalanine 4-Monooxygenase from Indigenous Bacillus tropicus Strain AOA-CPS1: Cloning, Overexpression, Purification, Characterization and Structural Homology Modelling

Autor: Oladipupo A. Aregbesola, Mduduzi P. Mokoena, Ademola O. Olaniran, Ajit Kumar
Rok vydání: 2021
Předmět:
Zdroj: Applied Biochemistry and Biotechnology. 194:635-658
ISSN: 1559-0291
0273-2289
Popis: The metabolically promiscuous pentachlorophenol (PCP) hydroxylating Phe4MO (represented as CpsB) was detected, amplified (from the genome of Bacillus tropicus strain AOA-CPS1), cloned, overexpressed, purified and characterized here. The 1.755-kb gene cloned in the pET15b vector expressed a ≅ 64 kDa monomeric protein which was purified to homogeneity by single-step affinity chromatography, with a total yield of 82.1%. The optimum temperature and pH of the enzyme were found to be 30 °C and 7.0, respectively. CpsB showed functional stability between pH 6.0–7.5 and temperature 25–30 °C. The enzyme–substrate reaction kinetic studies showed the allosteric nature of the enzyme and followed pre-steady state using NADH as a co-substrate with apparent vmax, Km, kcat and kcat/Km values of 0.465 μM.s−1, 140 μM, 0.099 s−1 and 7.07 × 10−4 μM−1.s−1, respectively, for the substrate PCP. The in-gel trypsin digestion experiments and bioinformatic tools confirmed that the reported enzyme is a Phe4MO with multiple putative conserved domains and metal ion-binding site. Though Phe4MO has been reported to have a diverse catalytic function, here we report, for the first time, that it functions as a PCP dehalogenase or PCP-4-monooxygenase by hydroxylating PCP. Hence, the use of this enzyme may be further explored in the bioremediation of PCP and other related xenobiotics.
Databáze: OpenAIRE
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