新型生物炭工艺中高效锰氧化菌的除锰机理及其功能蛋白酶和基因研究

As is known to all, excessive levels of manganese in water could seriously endanger human health. Some problems greatly restrict the development of biological manganese removal technology such as that the present biological manganese removal technology is difficult to totally guarantee the safety of water and the mechanism of manganese removal is not deeply investigated. The project is to replace granular activated carbon with new type of activated carbon fiber (ACF) to develop high efficient biological activated carbon fiber process (BACF). The bioreactor is started with efficient manganese oxidizing bacteria already selected by us. Through study on water quality conditions, operating parameters, influence law of biomass on manganese removal and contribution to the biological removal of manganese, establish the kinetic model of manganese removal in bioreactor and elucidate the mechanism of manganese removal from the macroscopic view. It is discussed that structure and properties of oxidation products of Mn(Ⅱ) by crude enzyme catalysis at the different stages and the impact of environmental factors on enzyme catalysis and mineral catalysis, thus explaining biological oxidation process of Mn(Ⅱ) in microscopic scales. Functional protein is obtained by separating and purifying manganese-oxidizing bacteria and its enzymatic properties is studied to analyse structure of functional protease. The oxidation mechanism of manganese is revealed at the molecular level by extracting manganese-oxidizing bacterial genomes, adopting PCR sequencing or construction of genomic library and identifying encoding gene by using BLAST methods. The results of the project could provide technical support for water treatment of manganese contamination. It also has important theoretical significance and practical value on the development in mechanism of biological manganese removal and guarantee of water safety.

水中过量的锰严重危害人体健康，而目前生物除锰技术的机理研究不深入，难以充分保障用水安全，因而极大地制约了生物除锰技术的发展。本项目以新型活性炭纤维（ACF）代替颗粒炭，以项目组已有的高效锰氧化菌启动反应器，形成高效的生物活性炭新工艺（BACF）。研究水质条件、操作参数和生物量对除锰的影响规律及生物除锰的贡献，建立反应器除锰动力学模型，从宏观上解释除锰机理；分析粗酶催化Mn(Ⅱ)后各阶段产物的结构、性质，阐明环境因素对酶催化及矿物催化的影响，从微观上解释Mn(Ⅱ)的氧化历程；分离、纯化锰氧化菌的目的功能蛋白，研究其酶学特性，解析功能蛋白酶的结构。提取锰氧化菌基因组，采用PCR扩增测序和构建基因组文库方法，通过BLAST序列比对鉴定编码基因，从分子层面上揭示锰氧化机制。项目成果可为锰污染水处理提供技术支撑，对保障用水安全和发展生物除锰理论具有重要的理论意义和实用价值。

水中过量的锰严重危害人体健康，而目前生物除锰技术的机理研究不深入，极大地制约了生物除锰技术的发展。本项目以新型活性炭纤维（ACF）代替颗粒炭，以高效锰氧化菌启动反应器，创新性地开发了新型高效的BACF工艺。实现了BACF的快速启动，优化了工艺参数。系统考察了温度、pH和EBCT等影响因子，建立了除锰的综合动力学模型，分析了BACF对锰的去除途径，从宏观上解释了生物除锰机理。筛选、分离出两株新的高效锰氧化菌Lysinibacillus MK-1和Brevibacillus panacihumi MK-8，其除锰效率高达97%以上。揭示了粗酶催化Mn(Ⅱ)氧化的途径主要是胞外酶氧化，并经中间态Mn(III)最终氧化成Mn(IV)，阐明了金属离子对粗酶催化的影响，建立了粗酶催化Mn(Ⅱ)的动力学方程，确定了pH和锰等对粗酶锰氧化活性的影响。成功扩增出目的基因片段并测序，构建基因组文库，通过BLAST序列比对，确定了其分子量分别为70.85KDa和58.3 KDa，预测了蛋白的二级结构和组成，发现MokA和CopA蛋白和已知的锰氧化酶中均含有4个保守的铜离子结合位点，解析了功能蛋白酶的结构，从分子层面揭示了锰氧化机制。将扩增的MK-1、MK-8锰氧化基因导入到BL21(DE3)异源表达，成功构建了高效重组工程菌。工程菌的锰氧化率最高达到99.6%，高于原始野生菌。重组菌锰氧化的最适pH为7.0，在锰浓度较高的环境中氧化能力强。分离、纯化目的蛋白，发现MK-1锰氧化基因可能与其上下游的基因共同组成锰氧化酶的分泌和表达系统。纯化后的MokA重组蛋白在pH为7、Cu2+为0.4mM、37℃时锰氧化率最高，CopA重组蛋白在pH为8、Cu2+为0.4mM、37℃时锰氧化率最高。项目成果为锰污染水处理提供了技术支撑，对丰富生物除锰理论和保障用水安全具有重要的理论意义和实用价值。

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