| Mn oxide is one of important component in soils and sediments, widely participating in the tranformation of many heavy metal and organic substances, which palys an predominant role in the biogeochemical cycles. Microorganisms, primarily bacteria but also fungi, are known to catalyze the oxidation of Mn(Ⅱ) and the formation of Mn(Ⅲ,Ⅳ) oxide minerals. Biological Mn(Ⅱ) oxidation is generally fast relative to abiotic Mn(Ⅱ) oxidation processes (faster up to 5 orders of magnitude). So the biological processes are believed to be the primary factor responsible for formation of naturally occurring environmental Mn oxides. It is significant of studying on the processes and mechesims of biological Mn(Ⅱ) oxidation in soil for understanding the evolution of ecosystem and cycles of substances.In this study, we isolated 47 Mn(Ⅱ)-oxidizing bacteria strains from different soil profile throughout the country abound with iron-manganese nodules. Our research focused on the biochemical characterictics, molecular biological mechanism and enzymology of four Mn(Ⅱ)-oxidizing strains:Bacillus sp. J4142, Bacillus sp. T1122, Bacillus sp. WH4 isolated from Fe-Mn nodules of Que yu, Shandong Province, Taian, Shandong Province and Wuhan, Hubei Province, respectively,as well as the standard strain B. thuringiensis serovar konkukian str.97-27 preserved in our laberatery.We discussed the mechanisms of Mn(Ⅱ) oxidation by Bacillus strains in various aspects. First, we extracted spores from Bacillus sp. J4142 and Bacillus sp. T1122, and dectected their Mn(Ⅱ) oxidation activities. Unfortunately, the purified spores showed no apparent oxidation activity against Mn(Ⅱ). Second, the filtrates of Bacillus sp. J4142 and Bacillus sp. T1122 appeared to be able to oxidize Mn(Ⅱ) into Mn(Ⅲ)/Mn(Ⅳ) oxides to some extent. Furthermore, the Mn(Ⅱ) oxidation activities did not decrease with the heat and inhibitors treatments, which demonstrated that no enzyme was involved in the processes. On the other hand, the molecular weight of the Mn(Ⅱ) oxidation activities in the ultrafiltrates were lower than 10 kDa, hinted at the possibility of involvement of extracellular reactive oxygen species (ROS) generated by the microbes during growth. Third, we cloned four gene, encoding proteins with homology to multicopper oxidase (MCO), and successfully expressed in Escherichia coli. The recombinant MCO protein of Bacillus sp. WH4 was purified and it had high Mn(Ⅱ) oxidation activity and laccase-like activity. The phenomenon was firstly reported in this study. Last, B. thurigensis BMB171 gained the capability of Mn(Ⅱ) oxidation when the putative MCO protein from Bacillus sp. WH4 was expressed in it, which proved that the Mn(Ⅱ) oxidation was directly catalyzed by this enzyme. Vitreoscilla hemoglobin (VHb) is an oxygen-binding protein of prokaryotic origin, and various studies showed that it was able to enhance the dissolved oxygen intake of cells We expressed vgb gene in B. thurigensis BMB171 in order to improve the capability of Mn(Ⅱ) oxidation. The purpose of this part is to provide a new way of lowering the production cost for using Bacillus sp. WH4 to Mn removal of the groundwater. VHb expression in B. thurigensis BMB171 improved the cell growth, but no obvious increase on the Mn(Ⅱ) oxidation activity was observed. Accordingly, further research remained to do in the next step. |
PDF Full Text Request