| Manganese-oxidizing microbes widely occur in water phases and soil environments. They are able to oxidize Mn2+into Mn4+efficiently and therefore play an important role in the biogeochemical cycling of manganese. Many studies found that manganese oxides are a series of complex processes which include abiotic and biotic ways, and therefore microbial processes are considered to be primarily responsible for the formation of manganese oxides. However, the mechanism of bacterial oxidation has not been clear. So it is very crucial to investigate the mechanism of the microbes forming manganese oxides. This study focuses on the researches of manganese-oxidizing bacterial isolation and its mechanism of the manganese oxide.We collected several soil samples throughout the country and 54 manganese-oxidizing bacteria were separated including 25 higher oxidation strains. Five isolates were identified through 16S rDNA sequencing analysis, the results showed that all the manganese-oxidizing bacteria belong to Bacillus, including three B. cereus, one B. pumilus and one B. megaterium. In addition, we found that 7 strains from 18 B. thuringiensis had higher manganese oxidation rates than those of the isolates. Therefore, we selected a high manganese oxidation strain B. thuringiensis subsp. entomocidus HD-9 to investigate the oxidation mechanism.Through analyzing the HD-9 fermentation supernatant filtered with a 0.22μm sterile filter membrane, the manganese-oxidizing bands were visualized by native polyacrylamide gel electrophoresis in-gel ativity assays, subsequently the proteins in the bands were identified by tandem mass spectctrometry. By searching the database, a protein matched well with the catalase (CAT) from B. thuringiensis serovar konkukian str. 91-21. Otherwise, according to the literature reports we speculated that manganese oxide may associate with the MnSOD. So, the corresponding coding genes sodA and katB for MnSOD and CAT from B. thuringiensis 97-27 were amplified, expressed in the E. coli BL21(DE3) and purified by Ni-NTA resin. At the same time, we constructed the expression vector and expressed sodA and katB in B. thuringiensis BMB171, respectively.By measuring the manganese oxidation activity of the two enzymes in vitro, we found that CAT can directly oxide manganese, although manganese oxidizing rate is not high enough. Unfortunately, MnSOD has no ativity. When the two enzymes were added simultaneously, the Mn oxidation rate was even lower than that of separate enzyme. The manganese oxidizing rates of both the two recombinant strains were lower than that of original strain BMB171. When the HD-9 fermentation supernatant filtered with a 0.22 um sterile filter membrane was sterilized (121℃,20 min), its manganese oxidizing rate increased. On other hand, when MnSOD or CAT protein was added, the manganese oxidizing rate enhanced, while the manganese oxidizing rate decreased when both MnSOD and CAT were added. The result indicated that these two enzymes are playing important roles in the process of forming manganese oxide.In addition, we used microcalorimetric method for the first time to investigate the relationship between the expression of MnSOD and the formation of manganese oxides in E. coli. The thermokinetic parameters obtained from the metabolic power-time curves could act as a quantitative indicator of sensitive about the expression of MnSOD in manganese oxidation. We found that adding 2.0 mmol/L Mn2+ could make significant changes in heat release when sodA was expressed in E. coli BL21(DE3). It shows that MnSOD enzyme plays a certain role in the manganese oxidizing process, which provide an evidence for the mechanism of bacterial manganese oxidation, as well as a great significance in revealing the role of Mn oxidation enzymes. |
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