【作者】 朱艳华；

【导师】 冯雄汉；

【作者基本信息】 华中农业大学 ， 土壤学， 2017， 硕士

【摘要】 锰氧化物是普遍存在于深海、土壤和沉积物中的高反应活性矿物,锰平均氧化度的准确测定是了解其结构和特性,环境行为和潜在应用价值的重要前提。传统方法测定锰平均氧化度存在操作繁琐、分析周期长、对微量样品不太适用等缺点,而且每种方法都有一定的局限性。细菌氧化锰的反应是一个极其复杂的过程,目前认为多铜氧化酶的作用是催化形成锰氧化物的主要原因,但其催化氧化的机理并不是很清楚,锰氧化基因克隆表达纯化得到的重组蛋白,体外氧化Mn(Ⅱ)/Mn(Ⅲ)的特性更是急需探究。该机制的发现,将有助于我们更好的理解多铜氧化酶在生物成矿过程中的催化作用,对调控其氧化行为、影响重金属、有机污染物的迁移转化和地球化学循环过程具有重要的意义!本研究中,以草酸还原-高锰酸钾返滴定法和X射线吸收近边结构光谱(XANES)作为对照方法,利用新颖两步比色法对水羟锰矿、酸性水钠锰矿和黑锰矿进行锰平均氧化度的测定。接着探究将E.coli K-12 MG1655中cueO基因克隆外源表达、纯化出的重组蛋白CueO体外氧化Mn(Ⅱ)/Mn(Ⅲ)的特性及电子传递问题。在低锰离子、低铜离子浓度的体系中,借助DFOB捕获Mn(Ⅲ),分别定时监测以Mn(Ⅱ)和Mn(Ⅲ)为反应物时,反应体系中Mn(Ⅲ)-DFOB及高价锰的含量变化。取得的主要结果如下:1.测定高价锰含量的LBB比色法和总锰含量的甲醛肟比色法结合得到的新颖两步比色法,测定水羟锰矿、黑锰矿和酸性水钠锰矿的锰平均氧化度分别是4.00±0.02、2.67±0.01和4.00±0.01,其准确度和精确度明显高于草酸–还原高锰酸钾返滴定法和XANES光谱法的测定结果,而且该方法对微量样品(0.005 g)十分适用。2.重组蛋白CueO在低锰低铜(2 mM MnCl2、0.4 mM CuCl2)体系下依然能够生成高价锰氧化物,排除了化学氧化的影响,进一步证实CueO蛋白在体外的锰氧化活性。同时,Na4P2O7溶液对CueO蛋白的锰氧化活性具有明显的抑制作用,且抑制作用随着浓度增加而加强,当加入高浓度Na4P2O7(5 mM)溶液能够完全抑制CueO蛋白的锰氧化活性。3.在重组蛋白CueO体外氧化Mn(Ⅱ)的特性探究中,定时监测Mn(Ⅱ)为反应物时体系中Mn(Ⅲ)-DFOB和高价锰的含量变化。结果表明,CueO蛋白可以氧化Mn(Ⅱ)生成Mn(Ⅲ),氧化效率高达50%,且DFOB的加入可以增强CueO蛋白对Mn(Ⅱ)的氧化。同时定时取样加入LBB后颜色无变化,表明体系中无Mn(Ⅳ)生成。4.在重组蛋白CueO体外氧化Mn(Ⅲ)的特性探究中,定时监测乙酸锰(C6H9MnO6)为反应物时体系中Mn(Ⅲ)-DFOB和高价锰的含量变化。结果表明,CueO蛋白的加入能够抑制Mn(Ⅲ)的还原消失。同时定时取样加入LBB后颜色无变化,表明CueO蛋白不能氧化Mn(Ⅲ)生成Mn(Ⅳ),因此CueO蛋白不能独自催化完成两步电子传递的锰氧化过程。更多还原

【Abstract】 Manganese(Mn)oxide is a kind of highly reactive mineral,which is widely found in the deep sea,soil and sediment environments.Accurate determination of Mn AOS is a key step to understand the structures,properties,environment behaviors and potential applications of Mn oxides.The traditional methods for determining Mn AOS have the disadvantages of complicated operation,long analysis period and little applicability to the micro samples,and each method has some limitations.The bacterial Mn(Ⅱ)oxidation reaction is an extremely complex process.The catalytic oxidation of multicopper oxidases is considered to be the main reason for the formation of manganese oxides.However,the catalytic oxidation mechanism is not very clear,and it is urgent to explore characteristics of Mn(Ⅱ)/Mn(Ⅲ)oxidation in vitro by the recombinant protein CueO,of which manganese oxide gene were cloned and purified.The discovery of this mechanism,can help us to better understand the biological mineralization process for multicopper oxidases catalyzing.It plays an important role in the regulation of its oxidation behavior,the migration process of heavy metals and organic pollutants and the process of geochemical cycle!In this study,a novel two step colorimetric method was developed to determine the Mn AOS of vernadite,acid birnessite and hausmannite,and the oxalic acid-permanganate back titration method,and X-ray absorption near-edge spectroscopy(XANES)were adopted to determine the Mn AOS of the three Mn oxides for comparison.Moreover,we investigated the characteristics and the electron transfer of Mn(Ⅱ)/Mn(Ⅲ)oxidation in vitro by the recombinant protein CueO,which was cloned from E.coli K-12 MG1655 and purified.In the system of low concentration manganese and copper ions,with the help of DFOB to capture Mn(Ⅲ),the content of Mn(Ⅲ)-DFOB and high manganese with the reactant of Mn(Ⅱ)/Mn(Ⅲ)were monitored regularly.The main results are as follows:1.The novel two step colorimetric method,was combined with leucoberbelin blue I(LBB)and formaldoxime colorimetric method which were employed to obtain the content of high-valent Mn and total Mn,respectively.This novel method was used determine the Mn AOS of vernadite,acid birnessite and hausmannite,which was 4.00 ± 0.02、2.67 ± 0.01 and 4.00 ± 0.01,respectively.The result obtained by this method is more accurate than that obtained by the oxalic acid-permanganate back titration method and the XANES method.In addition,this novel method is also appropriate for trace amount of samples(0.005 g).2.The recombinant protein CueO can oxidize Mn(Ⅱ)to manganese oxides in the system with low concentration of manganese and copper ions(2 mM MnCl2,0.4 mM CuCl2),to eliminate the effect of chemical oxidation,to further confirm the manganese oxidizing activity of CueO protein in vitro.At the same time,the manganese oxidation activity of CueO protein was significantly inhibited with Na4P2O7 solution,and the inhibition was increased with the concentration increasing.When adding high concentration of Na4P2O7(5 mM),the manganese oxidation activity of CueO protein could be completely inhibited.3.In the study of the characteristics of Mn(Ⅱ)oxidation by recombinant protein CueO in vitro,the content of Mn(Ⅲ)-DFOB and high manganese with the reactant of Mn(Ⅱ)were monitored regularly in the system.The experimental results show that CueO protein can oxidize Mn(Ⅱ)to Mn(Ⅲ),with the oxidation efficiency reaching 50%,and the addition of DFOB could enhance Mn(Ⅱ)oxidation by CueO protein.At the same time,the color of the solution was not changed after the regular sampling mixed with LBB,which indicates that this experiment could not create Mn(Ⅳ).4.In the study of the characteristics of Mn(Ⅲ)oxidation by recombinant protein CueO in vitro,the content of Mn(Ⅲ)-DFOB and high manganese with the reactant of Mn(Ⅲ)acetate dihydrate(C6H9MnO6)were monitored regularly in the system.The results showed that the addition of CueO could inhibit the reduction of Mn(Ⅲ).At the same time,the color was not changed after the sample mixed with LBB,indicating that the CueO protein could not oxidize Mn(Ⅲ)to Mn(Ⅳ),so the CueO protein could not catalyze the two-step electron transfer of the manganese oxidation process alone.更多还原