外源漆酶在污染土壤中的活性异变规律及稳定性调控/传质模式构建机制研究

Exogenous microbial enzyme remediation method is an efficient path to solve soil contamination. It is a green, in-situ and joint remediation technology. Exogenous enzyme remediation method the same as the other remediation methods are easy to lose activity, unstable and low efficient mass transfer likely in the contaminated soil. Laccase is a typical exogenous microbial enzyme. This project will test the relationships with laccase spatial structure and enzyme activity, contaminated soil and build stability of laccase, mass transfer model and catalytic oxidation behavior. These relationships are key problems aimed at resolving basic theoretical questions including the change law of laccase activity, stability system building and control principles of laccase and building on mass transfer model in soil environment. Specific studies include: 1. The influence rules of internal and external factors for laccase activity are studied to reveal the reason and mechanism laccase activity change. The relationship with the change ways of laccase spatial structure and enzyme activity is found out; 2. The methods for improving laccase stability in soil are researched to set up system and regulating mechanism of laccase stability based on protecting laccase catalytic activity; 3. Based on the theory of mass transfer in porous media, mass transfer models of laccase and pollutants in soil are established to determine mass transfer law and the major factors of limitation on mass-transfer efficiency, which will provide the theoretical basis for establishing efficient mass transfer patterns of laccase and pollutants. Based on the above, complete theoretical systems about laccase remediation method of soil contamination will be formed to provide theoretical support for high effective application of microbial enzyme remediation method.

外源微生物酶修复方法是缓解土壤污染的有效途径，是可靠的绿色、原位、可联合修复技术，它同其他修复技术相同，在土壤环境中存在酶易失活、稳定性波动大、传质效率低等问题。本项目从典型外源微生物酶-漆酶空间结构与活性关系、污染土壤与漆酶稳定性构建关系、传质模式与催化氧化行为关系等科学问题出发，旨在解决漆酶在污染土壤中活性异变规律、稳定性体系构建及调控原理、传质模式构建机制等基础理论问题，需重点研究：1.土壤内部和外部因子对漆酶酶活的影响规律，揭示漆酶活性异变原因和机理，弄清漆酶空间结构改变与活性的关系；2.通过增强漆酶在土壤中稳定性方法的研究，以保护漆酶催化活力为宗旨建立漆酶稳定性体系和调控机制；3.建立土壤中漆酶和污染物传质模型，弄清传质规律和限制传质效率的主要因素，为漆酶与污染物高效传质模式的构建提供理论基础。在此基础上建立完整的漆酶修复污染土壤理论体系，为微生物酶修复技术高效应用提供理论支撑。

外源微生物酶修复方法是解决土壤污染的有效途径。为了解决土壤环境中微生物酶失活，稳定性高波动和传质效率低的问题。漆酶作为一种典型的外源微生物酶，被选为该项目的研究对象，重点解决污染土壤中漆酶活性变化、稳定体系的构建及其调控原理、传质方式的构建机理等关键问题。在本项目中，首先研究了环境因素和外场力超声对漆酶活性和催化活性的影响机理，并确定了影响漆酶活性变化的主要因素；其次，研究了在不同底物体系下漆酶的失活动力学，结合多羟基化合物作用下漆酶的空间结构变化，确定了漆酶活性变化的原因和机理；之后，结合反胶束体系建立原理，研究了该体系与EK联用技术对蒽污染土壤的修复。最后，研究了在EK体系作用下菲在土壤中迁移和转化的物理化学过程，构建了菲迁移/转化模型。主要结果如下：1.不同的离子环境会导致漆酶的表面电荷发生变化，在高pH值和高温下会降低漆酶的稳定性，而适当的超声条件可使漆酶保持较高的活性。2.漆酶失活符合不同底物系统中的一阶动力学模型；多羟基化合物可与水分子形成氢键，增加溶液的粘度，维持漆酶肽链的紧密结构，并有助于提高漆酶的稳定性。3.表面活性剂通过疏水作用调节漆酶的电荷，使漆酶的疏水氨基酸残基暴露于非极性（或极性较小）的环境中，以提高漆酶的稳定性；Triton X-100构筑的反胶束体系可以提高漆酶的稳定性和催化活性，在表面活性剂和ABTS存在下，蒽的最大迁移转化率为49.3％。4.构建的数学模型适用于被菲污染的土壤，菲迁移/转化的主要影响因子是EK电压、土壤孔隙度和漆酶浓度。该项目不仅建立了污染土壤漆酶修复的完整理论体系，为微生物酶修复技术的有效应用提供了理论支持，而且促进了电动修复与漆酶结合技术的发展。该项目已发表（含录用）SCI论文9篇，中文核心论文6篇，申请了6项中国发明专利，培养了9名硕士研究生。