Enzyme Activity Enhancement in Functionalized Nanoporous Support

描述(由申请人提供)：一个关键的基本科学问题是分离的酶如何在溶液或固定化基质中保持其天然的活性构象。我们的长期目标是阐明功能化纳米孔载体中酶活性增强的机制，以开发高活性和稳定的酶用于解毒、癌症治疗、生物传感、蛋白质药物释放和递送。具体的假设是：在适当设计的开放纳米孔载体中，蛋白质的酶活性和稳定性可以显著提高，其功能是作为一个受限的和交互的纳米环境来促进有利的蛋白质构象变化。这一假设是基于以下观察结果：首先，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孔型纳米载体，其孔径可达几十纳米。我们发现这三种酶在FMS中都表现出比溶液中无酶的更高的活性；其次，通过改变FMS中的蛋白质负载密度可以在很大程度上提高或降低酶的比活性；第三，我们发现FMS和杂化试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FMS中发生了有利的蛋白质构象变化。我们认为，(I)FMS是一个有限的空间，(Ii)FMS提供了一个相互作用的环境，促进了有利的蛋白质构象变化，从而提高了酶的活性和稳定性。因此，我们提出的具体目标是：1.研究介孔结构的必要性和中孔大小对酶活性增强的影响；2.研究蛋白质与FMS的相互作用，以了解FMS的限制和相互作用对酶活性增强的影响；3.建立分子模型，利用分子对接和分子动力学模拟来探索FMS引导酶构象动力学向增强活性的机制；4.评估FMS中高活性和稳定的有机磷水解酶对大鼠有机磷神经毒性的体内解毒作用，以展示一种蛋白质(酶)药物储存、释放和传递一体化装置。 与公共卫生相关：关键的基础科学问题之一是分离的酶如何在溶液或固定化基质中保持其天然的活性构象。我们的长期目标是阐明工程纳米孔载体中酶活性增强的机制，以开发高活性和稳定的酶用于医疗应用，包括诊断、解毒和治疗癌症和其他疾病。因此，我们将评估高活性和稳定的有机磷水解酶在功能性纳米孔载体中的有效性，以提供对有机磷神经毒性的体内解毒，以展示一种集蛋白质(酶)药物储存、释放和释放为一体的装置。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.