| Manganese-oxidizing bacteria, which are widely found in soil, fresh water, sludge and other sediments, and marine environments, can strongly catalyze the oxidation of Mn(II) to Mn(IV) and plays an important role in the biogeochemical cycling of manganese. The biological oxidation process catalyzed by Mn(II)-oxidizing bacteria is much faster than non-biological oxidation(as much as five orders of magnitude), so the bio-oxidation process is considered to be the main reason for the environment manganese oxide. The bacterial Mn(II) oxidation reaction is an extremely complex process, where the catalytic oxidation mechanism is not very clear, and the identification and characterization of biological manganese oxides is still lacking. Biological manganese oxides have great significance in sewage treatment and the transformation of heavy metals and organic pollutants due to their strong adsorption, oxidation and catalytic characteristics.Three bacteria were focused in this study: Bacillus pumilus WH4, which is a Mn(II)-oxidizing bacterium isolated from Fe-Mn nodules in soil, Escherichia coli K-12 MG1655 and Bacillus thuringiensis 97-27(Bt 97-27). Firstly, the potential Mn(II) oxidases were cloned respectively from these three strains, then the recombinant proteins were overexpressed and purified in E. coli strains. Furthermore, the mechanisms of Mn(II) oxidation in vitro and in vivo were further investigated, and the generated manganese oxides were characterized and analyzed. In the reaction system containing 5 mM Mn(II), the abilities of Mn(II) oxidation and Mn(II) removal were determined in the purified recombinase and recombinant strain, respectively. Additionally, the laccase and dye decolorization activities of recombinase were also investigated. The main results are as follows:1. The spore coat protein coding gene cotA was cloned from B. pumilus WH4 and heterologously expressed in E. coli M15. The purified recombinant protein CotA was proved to be a typical multicopper oxidase(MCO) by spectrometry analysis and copper ion content determination. CotA also possessed the typical laccase activity, which could oxidize 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)(ABTS), syringaldazine(SGZ) and 2,6-dimethoxyphenol(2,6-DMP), respectively. Moreover, CotA could remove 75% of Congo red(initial concentration of 500 mg/L) after static cultivation at 37°C for 24 h. Importantly, CotA displayed Mn(II)-oxidase activities both in the Native-PAGE and in the liquid culture system, which catalyzed the Mn(II) oxidation to generate Mn(IV) oxides. The optimum Mn(II) oxidase activity of CotA was obtained at 53°C in HEPES buffer(pH 8.0) supplemented with 0.8 mM CuCl2. The addition of o-phenanthroline and EDTA both led to a complete suppression of the Mn(II)-oxidase activities. 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 and 0.32±0.02 s-1, respectively. Additionally, the Mn(II)-oxidizing activity of the recombinant E. coli strain M15-pQE-cotA was significantly increased compared with the mother strain when cultured both in Mn(II)-containing K liquid medium and on agar plates in vivo. After seven-day liquid cultivation with 5 mM Mn(II), M15-pQE-cotA resulted in 18.2% removal of Mn(II) from the K medium. Furthermore, many biogenic Mn oxides were clearly observed on the cell surfaces of M15-pQE-cotA by scanning electron microscopy(SEM). To our knowledge, CotA is the first MCO that purified through the heterologous expression technique and meanwhile possesses an active Mn(II)-oxidizing ability, which provides the direct evidence of Mn(II) oxidation catalyzed by the recombinant protein CotA.2. A multicopper oxidase gene cueO was cloned from E. coli MG1655, and expressed in E. coli BL21(DE3). The enzyme activity of purified holo CueO was significantly higher than apo-CueO, indicating CueO was strongly depended on the copper ions. CueO also exhibited typical laccase characteristics, which could oxidize ABTS and 2,6-DMP. The recombinant protein CueO showed a strong Mn(II) oxidase activity both in the Native-PAGE and in the liquid reaction system. The optimum Mn(II) oxidase activity of CueO was obtained at 55°C in HEPES buffer(pH 8.0) with 1.0 mM CuCl2. The kinetic constant kcat /Km for oxidizing Mn(II) and specific activity of CueO were 5.6 fold and 5.4 fold higher than those of CotA. The addition of EDTA and SDS could almost completely inhibit the activity of CueO in Mn(II) oxidation, while o-phenanthroline and DTT also played a certain degree of inhibition. Due to the high Mn(II) oxidase activity of CueO, we established an in vitro catalytic system mediated by CueO to prepare biological manganese oxide(BioMnOx), which was confirmed to be very similar with hausmannite γ-Mn3O4 by X-ray diffraction(XRD) analysis. The BioMnOx catalyzed and formed by CueO was found to contain 53.6% of Mn(II), 18.4% Mn(III) and 28.0% Mn(IV) by X-ray photoelectron spectroscopy(XPS) analysis. BioMnOx nanoparticles showed an obvious polyhedron structure with a diameter of 150-350 nm by transmission electron microscopy(TEM) observation, which were much bigger than the chemically synthesized Mn3O4. Importantly, CueO could remove 35.7% of Mn(II) after a seven-day reaction in vitro. Moreover, the cueO-overexpressing E. coli strain(ECueO) had a significantly higher Mn(II)-oxidizing activity than the mother strain. ECueO cultivated with Mn(II) and Cu(II) could also oxidize 58.1% of dissolved Mn(II), and simultaneously remove 97.7% of Mn(II). The surface of ECueO was covered with a large number of manganese oxides observed by SEM.3. The catalase gene katB was cloned from Bt 97-27 and expressed in E. coli BL21(DE3). The purified CatB protein exhibited as an polymer on Native-PAGE, it not only had an active catalase activity that catalyzed the degradation of H2O2, but also had a peroxidase activity that catalyzed the oxidation of ABTS, indicating that CatB might be a biofunctional enzyme belong to a type of CAT-POD. The present study is the first time to confirm that CatB can oxidize Mn(II) in the liquid reaction system, but requires the addition of H2O2 to induce the Mn(II) oxidation in the Native-PAGE gel. The optimal Mn(II) oxidizing activity of Cat B was at 76°C in HEPES buffer(pH 8.0). The addition of EDTA and SDS could completely inhibited the Mn(II)-oxidase acivity of CatB. In the present study, we established an in vitro catalytic system mediated by CatB to prepare biological manganese oxide(BioMnOx), which was further confirmed to be similar with birnessite syn-MnO2 by XRD analysis. BioMnOx was shown to have the obvious sheet strip and launch-like structure by SEM and TEM. Besides, we also found an increase of Mn(II)-oxidizing ability in E. coli recombinant strain BL21-pET-katB because of the heterologous expression of CatB, whereas no obvious induction of Mn(II) oxidation was observed in Bt BMB171-pHT1K-katB strain.Therefore, this study proposes three new Mn(II) oxidases, which not only enrich the Mn(II) oxidase category, but also further clarify the direct relationship between the MCO and Mn(II) oxidation process. The present study identifies the composition and characteristics of the biological manganese oxide, which has a guiding significance for the future in-depth studies on the biological Mn(II) oxidation reaction process and regulation mechanisms. Meanwhile, the Mn(II) oxidases and produced manganese oxides in the present study have great potential applications in Mn(II) wastewater treatment, heavy metal pollution restoration and decolorization technology and other aspects. |
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