CotA, a Multicopper Oxidase from Bacillus pumilus WH4, Exhibits Manganese-Oxidase ActivityReportar como inadecuado

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Multicopper oxidases MCOs are a family of enzymes that use copper ions as cofactors to oxidize various substrates. Previous research has demonstrated that several MCOs such as MnxG, MofA and MoxA can act as putative MnII oxidases. Meanwhile, the endospore coat protein CotA from Bacillus species has been confirmed as a typical MCO. To study the relationship between CotA and the MnII oxidation, the cotA gene from a highly active MnII-oxidizing strain Bacillus pumilus WH4 was cloned and overexpressed in Escherichia coli strain M15. The purified CotA contained approximately four copper atoms per molecule and showed spectroscopic properties typical of blue copper oxidases. Importantly, apart from the laccase activities, the CotA also displayed substantial MnII-oxidase activities both in liquid culture system and native polyacrylamide gel electrophoresis. The optimum MnII oxidase activity was obtained at 53°C in HEPES buffer pH 8.0 supplemented with 0.8 mM CuCl2. Besides, the addition of o-phenanthroline and EDTA both led to a complete suppression of MnII-oxidizing activity. The specific activity of purified CotA towards MnII was 0.27 U-mg. The Km, Vmax and kcat values towards MnII were 14.85±1.17 mM, 3.01×10−6±0.21 M·min−1 and 0.32±0.02 s−1, respectively. Moreover, the MnII-oxidizing activity of the recombinant E. coli strain M15-pQE-cotA was significantly increased when cultured both in Mn-containing K liquid medium and on agar plates. After 7-day liquid cultivation, M15-pQE-cotA resulted in 18.2% removal of MnII from the medium. Furthermore, the biogenic Mn oxides were clearly observed on the cell surfaces of M15-pQE-cotA by scanning electron microscopy. To our knowledge, this is the first report that provides the direct observation of MnII oxidation with the heterologously expressed protein CotA, Therefore, this novel finding not only establishes the foundation for in-depth study of MnII oxidation mechanisms, but also offers a potential biocatalyst for MnII removal.

Autor: Jianmei Su, Peng Bao, Tenglong Bai, Lin Deng, Hui Wu, Fan Liu, Jin He



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