Enzyme Activity Enhancement in Functionalized Nanoporous Support

描述（由申请人提供）：关键的基本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活性构象。我们的长期目标是阐明功能化纳米多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：蛋白质的酶活性和稳定性可以在适当工程化的开放式纳米多孔支持物中显著增强，该支持物用作用于促进有利的蛋白质构象变化的受限和相互作用的纳米环境。这一假设是基于以下观察：首先，我们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，孔径可达几十纳米。结果表明：与游离酶相比，三种酶在FMS中均表现出较高的活性;第二，改变FMS中蛋白质的装载密度，可以在很大程度上提高或降低酶的比活性;第三，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，有有利的蛋白质构象变化发生在FMS。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，促进有利的蛋白质构象变化，从而提高酶的活性和稳定性。因此，我们提出的具体目标是：1。探讨介孔结构的必要性及介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;开发分子模型，并采用分子对接和分子动力学模拟来探索FMS引导酶构象动力学增强活性的机制; 4.评价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，以证明蛋白（酶）药物储存、释放和递送的一体化装置。 公共卫生相关性：分离的酶如何在溶液或固定化基质中保持其天然活性构象是一个关键的基础科学问题。我们的长期目标是阐明工程化纳米多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学应用，包括诊断，解毒和治疗癌症和其他疾病。作为这项工作的结果，我们将评估高活性和稳定的有机磷水解酶在功能性纳米多孔支持物中的功效，以提供对大鼠有机磷神经毒性的体内解毒，以证明蛋白质（酶）药物储存、释放和递送的一体化装置。

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.