| Mn oxides formed after Mn(II) oxidation are mineral composition with highly reactive activity, determining the shapes, migration and transformation of many substances in the environment. Microbial Mn(II) oxidation is considered to be the main driving force of nature manganese oxide. Mn(II)-oxidizing bacteria in the aquatic environment had been intensively investigated. However, little information is available on their distribution and biogeochemical significance of those in the terrestrial soil environments. In this research, a soil-borne Escherichia coli MB266 strain with high Mn(II)-oxidizing activity were isolated. Using the methods of random transposon mutagenesis library screening, gene disruption and complementary, cell surface display analysis and in vitro test of active ingredient et al., the molecular mechanism of Mn(II) oxidation actuated by multi-copper oxidase at the cell surface in this bacteria were research. Further, the biodegradation of endocrine disruptors by Mn(II)-oxidizing bacteria mix Mn oxides and the constructing of biosensor for phenols detection were research. The main contents are as follows:1. Six high Mn(II)-oxidizing activity bacteria isolated from different soil layers of Brown were continuous cultured 7 d in K medium contain Mn(II). Result showns the concentration of Mn oxide produced by these isolates with incubation time increases and reached a peak after 5 days,The Mn(II) oxidation of 6 isolates were increased during the time course at p H<8.0 and reached a peak after 5 days. Therefore, Mn oxidation apparently occurred through biotic transformation process. Interestingly, SEM revealed that 5 isolates formed regular microspherical aggregates contains culture and Mn oxides in the three-week culture process, and other 1 isolates not formed regular microspherical aggregates. EDX analysis confirmed that the main elements of these aggregates was C, O, and Mn. XRD analysis suggested a possible link between bacteria formed Mn oxides and Mn oxide mineral components present in the soil.A Escherichia coli MB266 isolated from soil with highest Mn(II)-oxidizing activity in all isolated strains, formed an Mn(III)/Mn(IV) oxide black deposit layer and aggregates in rich Mn(II) culture. The Tn5 transposon mutants of a multi-copper oxidase(Multicopper oxidase, MCO) gene result the reduced of Mn(II)-oxidizing activity in the Tn5 transposon mutant libraries of MB266. Real time-q PCR analysis demonstrated MCO gene of MB266 was the only expression increased oxidoreductases elevated gene in the detected 30 possible Mn(II) oxidation-related genes. These results suggested that multi-copper enzymes and biological Mn(II) oxidation of MB266 strain were linked. Purified MCO266 protein was obtained by expressing the heterologous mco266 genes in JM109. In vitro tests proved that the purified MCO266, the recipient strain E. coli JM109 and recombinant bacteria expressing MCO266 did not show Mn(II)-oxidizing activity.To further confirming the Mn(II) oxidation of MB266 is occured on its cell surface, N-terminal of ice nucleation protein Ina Q(Ina Q-N) from Pseudomonas syringae was used as a carrier protein, We engineered MCO266 onto the cell surfaces of the activity-negative recipient E. coli JM109. The surface displayed engineering bacteria was named MB253 which was verified with higher Mn(II) oxidation activity. The Mn(II)-oxidizing activity of purified MCO266 was demonstrable when combined with cell outer membrane component(COMC) fractions, and could be suppressed by proteinase K. These results shown Mn(II) oxidation of MCO266 occurs at the cell surface, and requires the participation of the extracellular membrane protein. By mixed MCO266 and purified Ccm F, G and H components of cytochrome c maturation enzymes Ccm, as well as co-expression intracellular MCO266 and Ccm FGH, Mn(II) oxidation activity was not detected in the mixture or co-expression recombinant bacteria. Surface displayed recombinant bacteria can formed aggregates when continuous cultured in rich Mn(II) media. XPS experiments confirmed the aggregates were Mn oxides mainly and the proportions of Mn oxides were different in aggregates formed by different bacterial. XRD experiments confirmed the Mn oxides formed by bacteria were bixbyite. FT-IR confirmed that some small molecules are also involved in the Mn(II) oxidation reaction. The study also confirmed that the initial Mn(II) concentration in the medium and nutritional status of the cells not only affected the Mn(II) oxidation activity of wild-type strain and cell surface display recombinant bacteria, but also influence the formed of aggregates. On the basis of these studies, a possible molecular mechanism of Mn(II) oxidation in E. coli cell surface actuated by the multi-copper oxidase was proposed.2. In this study, we report a new method for the complete degradation of EDCs by a biodegradation-active composite of biogenic manganese oxides and multicopper oxidase Cot A. Using the characteristics of engineered E.coli cells surface-expressing the multicopper oxidase Cot A were capable of forming manganese oxide, a new EDCs degradation system by constructed. This system were consisted by engineered E.coli MB500 live cells surface-expressing Cot A and Mn oxides aggregates formed by MB500, and had dual-oxidation activity of multicopper oxide and manganese oxide. The oxidative reaction of the biocomposite proceeded at acidic p H and room temperature and did not require cofactors. Using stable isotope C13-labeled BPA and NP as a substrate, 7 kinds of BPA intermediate degradation products and 10 kinds of NP intermediate degradation products were identified. The final degradation product 13CO2 was detected in the final degradation system. Bioassays using Caenorhabditis elegans as an indicator organism demonstrated that all three EDCs were completely degraded by the biocomposite and that estrogenic activity was eliminated. Moreover, a consecutive three-round treatment process verified that comparable degradation by the biocomposite occurred in repeated reactions and that activity could be recovered easily.3.In this study, an engineered E.coli cells MB275 surface-immobilizing a bacterial laccase were constructed. The expression and surface localization of laccases on target cells were confirmed by Western blot analysis, immunofluorescence microscopy, and flow cytometry. Increased tandem-aligned anchors with three repeats of the N-terminal domain of an ice nucleation protein, as well as low-level expression induction, were used to obtain a highly active E. coli whole cell laccase-based catalytic system with an activity of 32.7 U/m L cells. A novel biosensor was developed by directly absorbing live MB275 cells onto glassy-carbon electrodes. The electrochemical response of the biosensor under optimized p H conditions was linear within the concentration range of 5.0 μmol/L to 500.0 μmol/L for several phenols(catechol, caffeic acid, dopamine, gallic acid). The proposed biosensor’s detection limit of 1.0 μmol/L to 5.0 μmol/L was comparable to those of other biosensors based on purified chemically-modified laccases. The developed system exhibited high stability and reproducibility. The biosensor offered a considerable level of accuracy for the determination of the phenols contents of red wine, pharmaceutical injection, and wastewater samples. The proposed biosensor has distinctive features, including a structurally nonglycosylated microenvironment, highly enzymatic reactivity, and a simple but regenerable electrode preparation. Thus, this novel biosensor has much potential for the facile, accurate, and cost-effective detection of phenols compounds. |
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