Enzyme Activity Enhancement in Functionalized Nanoporous Support

项目摘要 关键的基本科学问题之一是分离的酶如何保持其 溶液或固定化基质中的天然活性构象。我们的长期目标是 为了阐明功能化的酶活性增强的机制， 纳米多孔载体，以开发高活性和稳定的解毒酶， 癌症治疗、生物传感、蛋白质药物释放和递送。具体 假设是：蛋白质的酶活性和稳定性可以显著地 在适当设计的开放纳米多孔载体中增强，其功能为 一个封闭的和相互作用的纳米环境， 构象变化这一假设基于以下观察：首先，我们 已经在功能化介孔二氧化硅（FMS）中包埋了三种不同的酶。 介孔氧化硅是一种典型的开孔纳米多孔载体，孔径可达数十 纳米级。我们证明了所有三种酶在体外都表现出增强的活性， FMS与溶液中游离酶的比较;第二，酶比活性 可以通过改变蛋白质装载密度在很大程度上增加或减少， 第三，我们发现FMS和离液剂可以协同作用， 提高酶的活性;第四，我们发现实验证据表明， 是FMS中发生的有利的蛋白质构象变化。我们认为，（一） 柔性制造系统是一个有限的空间，（ii）柔性制造系统提供了一个互动的环境， 有利的蛋白质构象变化，从而增强酶活性， 稳定因此，我们提出的具体目标是：1。调查的必要性 介孔结构及孔径对酶活性的影响 增强; 2.研究蛋白质与FMS的相互作用以了解FMS 对酶活性增强的限制和交互作用; 3.发展 分子模型，并采用分子对接和分子动力学模拟， 探索FMS引导酶构象动力学的机制， 增强活性; 4.高活性、稳定性有机磷的疗效评价 FMS中的水解酶提供对有机磷的体内解毒 大鼠神经毒性，以证明蛋白质的一体化装置 （酶）药物储存、释放和递送。项目说明 关键的基本科学问题之一是分离的酶如何保持其 溶液或固定化基质中的天然活性构象。我们的长期目标是 为了阐明酶活性增强的机制， 开发用于医学应用的高活性和稳定的酶的纳米多孔载体 包括癌症和其他疾病的诊断、解毒和治疗。作为 由于这一努力，我们将评估高活性和稳定的 有机磷水解酶的功能性纳米多孔载体，以提供体内 解毒对有机磷神经毒性的大鼠，以证明 蛋白质（酶）药物储存、释放和输送一体化装置。

Project summary One of the key fundamental scientific questions is how isolated enzymes maintain their native active conformations in solution or in immobilization matrix. Our long-term goal is to elucidate the mechanisms for enzyme activity enhancements in functionalized nanoporous support to exploit highly-active and stable enzymes for detoxification, cancer treatment, biosensing, protein drug release and delivery. The specific hypothesis is that: a protein's enzymatic activity and stability can be significantly enhanced in an appropriately engineered open nanoporous support, which functions as a confined and interactive nanoenvironment for promoting a favorable protein conformational change. This hypothesis is based on the observations: First, we have entrapped three different enzymes in functionalized mesoporous silica (FMS). Mesoporous silica is a typical open nanoporous support with pore sizes as large as tens of nanometers. We demonstrated that all the three enzymes exhibit enhanced activity in FMS in comparison with the enzymes free in solution; Second, enzyme-specific activity can be increased or decreased to a large extent by changing protein loading density in FMS; Third, we found that FMS and chaotropic agents can act synergistically to enhance enzyme activity; Fourth, we found experimental evidences indicating there were favorable protein conformational changes occurring in FMS. We believe that, (i) FMS is a confined space, and (ii) FMS provides an interactive environment promoting a favorable protein conformational change, thereby enhancing enzyme activity and stability. Therefore, we propose the specific aims to: 1. Investigate necessity of mesoporous structure and effects of mesopore sizes on the enzyme activity enhancement; 2. Investigate the interactions of proteins with FMS to understanding FMS confinement and interactive effects on enzyme activity enhancement; 3. Develop molecular models and employ molecular docking and molecular dynamics simulations to probe the mechanism by which FMS steers enzyme conformational dynamics towards enhanced activity; 4. Evaluate the efficacy of highly-active and stable organophosphorus hydrolase in FMS to provide the in vivo detoxification towards organophosphorus neurotoxicity in the rat, to demonstrate an integrated all-in-one device of protein (enzyme) drug storage, release, and delivery. Project narrative One of the key fundamental scientific questions is how isolated enzymes maintain their native active conformations in solution or in immobilization matrix. Our long-term goal is to elucidate the mechanisms for enzyme activity enhancements in engineered nanoporous support to exploit highly-active and stable enzymes for medical applications including diagnostics, detoxification, and treatment for cancer and other diseases. As a result of this effort, we will evaluate the efficacy of highly-active and stable organophosphorus hydrolase in the functional nanoporous support to provide the in vivo detoxification towards organophosphorus neurotoxicity in the rat, to demonstrate an integrated all-in-one device of protein (enzyme) drug storage, release, and delivery.