Abstract
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 Mn(II) 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 Mn(II) oxidation, the cotA gene from a highly active Mn(II)-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 Mn(II)-oxidase activities both in liquid culture system and native polyacrylamide gel electrophoresis. The optimum Mn(II) 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 Mn(II)-oxidizing activity. The specific activity of purified CotA towards Mn(II) was 0.27 U/mg. The Km, Vmax and kcat values towards Mn(II) 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 Mn(II)-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 Mn(II) 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 Mn(II) oxidation with the heterologously expressed protein CotA, Therefore, this novel finding not only establishes the foundation for in-depth study of Mn(II) oxidation mechanisms, but also offers a potential biocatalyst for Mn(II) removal.
Highlights
Manganese is the second most abundant transition in the Earth’s crust and the fifth most abundant element on the Earth’s surface
The identity in amino acid sequence is 98% with the B. pumilus ATCC 7061 (ZP_03054403.1) [21], which was recently demonstrated to be a laccase-like Multicopper oxidases (MCOs) with similar properties as CotA of B. subtilis [20]
The cotA gene was amplified by PCR using genomic DNA from B. pumilus WH4 as template and 1530 bp PCR product was obtained
Summary
Manganese is the second most abundant transition in the Earth’s crust and the fifth most abundant element on the Earth’s surface. Manganese has three environmentally relevant oxidation states, Mn(II), Mn(III) and Mn(IV) [1,2]. Mn(II) is thermodynamically favored at low pH and Eh, whereas Mn(III) and Mn(IV) oxides are stable at high pH and Eh [3]. The soluble form of Mn(II), serves as a crucial micronutrient for organisms, while the insoluble form of Mn(III/IV) oxide, is a highly reactive mineral phase that participates in a wide range of redox and adsorptive reactions, playing a significant role in the bioavailability and geochemical cycling of many essential or toxic elements [1]. The chemical oxidation of Mn(II) by O2 in the pH range of 6.0–8.5 is at a considerably low level while in the presence of Mn(II)-oxidizing microorganisms, including a variety of bacteria and fungi, the oxidation rate can be accelerated by as much as five orders of magnitude [4,5]. Many microorganisms capable of oxidizing Mn(II) have been isolated and belong to diverse phyla, the biochemical mechanism of Mn(II) oxidation is still enigmatic [3]
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