Enzyme Activity Enhancement in Functionalized Nanoporous Support

项目总结 关键的基本科学问题之一是分离的酶如何维持其 溶液或固定化基质中的天然活性构象。我们的长期目标是 阐明功能化后酶活性增强的机制 纳米多孔载体用于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。具体的 假设是：一种蛋白质的酶活性和稳定性可以显著 在适当设计的开放纳米孔支撑体中增强，其功能如下 用于促进有利蛋白质的受限和交互的纳米环境 构象变化。这一假设是基于观察到的：第一，我们 在官能化介孔二氧化硅(FMS)中包埋了三种不同的酶。 介孔二氧化硅是一种典型的开放式纳米孔载体，其孔径可达数十 纳米级的。我们证明了这三种酶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的负载密度在很大程度上增加或减少 FMS；第三，我们发现FMS和促进剂可以协同作用 提高酶活性；第四，我们发现实验证据表明 在FMS中发生了有利的蛋白质构象变化。我们相信，(一) FMS是一个有限的空间，(Ii)FMS提供了一个互动环境，促进了 有利蛋白质构象变化，从而增强酶活性和 稳定性。因此，我们提出的具体目标是：1.调查必要性 中孔结构及中孔尺寸对酶活性的影响 增强；2.研究蛋白质与FMS的相互作用以了解FMS 限制和交互作用对酶活性提高的影响；3.发展 分子模型，并使用分子对接和分子动力学模拟 探讨FMS引导酶构象动力学的机制 活性增强；4.评价高活性稳定有机磷的药效 FMS中的水解酶对有机磷的体内解毒作用 对大鼠的神经毒性，以展示一种集成的蛋白质一体机 (酶)药物的储存、释放和输送。项目叙事 关键的基本科学问题之一是分离的酶如何维持其 溶液或固定化基质中的天然活性构象。我们的长期目标是 阐明工程菌中酶活性增强的机制 纳米多孔载体用于开发高活性和稳定的医用酶 包括癌症和其他疾病的诊断、解毒和治疗。作为一名 通过这一努力，我们将评估高活性和稳定性的疗效 有机磷水解酶在功能性纳米孔中的载体提供体内 对大鼠有机磷神经毒性的解毒作用 蛋白质(酶)药物储存、释放、输送一体机。

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.