Enzyme Activity Enhancement in Functionalized Nanoporous Support

功能化纳米孔载体中酶活性的增强

• 批准号: 7666881
• 负责人: Chenghong Lei
• 金额: $ 32.77万
• 依托单位: BATTELLE PACIFIC NORTHWEST LABORATORIES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7666881
• 关键词: Acute; Bacteria; Binding; Biological; Biosensing Techniques; Caliber; Cells; Circular Dichroism; Computer Simulation; Computing Methodologies; Confined Spaces; Coupled; Devices; Diagnostic; Diffuse; Disease; Disulfides; Docking; Drug Metabolic Detoxication; Drug Storage; Electrostatics; Engineering; Environment; Enzymes; Exhibits; Fluorescence; Foundations; Freeze Drying; Genetic Engineering; Goals; Hydrogen Bonding; Immobilization; Inclusion Bodies; Invaded; Investigation; Kinetics; Life; Link; Malignant Neoplasms; Medical; Methods; Modeling; Molecular; Molecular Conformation; Molecular Models; Nanotechnology; Peptides; Pharmaceutical Preparations; Process; Property; Protein Denaturation; Proteins; Rattus; Reaction; Recombinants; Relative (related person); Research; Silicon Dioxide; Solutions; Source; Spectroscopy, Fourier Transform Infrared; Structure; System; Temperature; Testing; Thermodynamics; Time; Transportation; Work; aryldialkylphosphatase; base; cancer therapy; density; design; electrostatic chemical interaction; enzyme activity; enzyme substrate; enzyme substrate complex; functional group; in vivo; molecular dynamics; molecular modeling; nano; nanocomposite; nanometer; nanoscience; nerve agent; neurotoxicity; new technology; prototype; public health relevance; response; theories

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.

描述（由申请人提供）：关键的基本科学问题之一是孤立的酶如何保持其在溶液或固定矩阵中的天然主动构象。我们的长期目标是阐明功能化纳米多孔支持中酶活性增强的机制，以利用高度活跃和稳定的酶进行排毒，癌症治疗，生物传感，蛋白质药物释放和递送。具体的假设是：在适当设计的开放式纳米孔支持中，蛋白质的酶活性和稳定性可以显着增强，该支持是促进有利的蛋白质构象变化的狭窄且相互作用的纳米环境。该假设基于观察结果：首先，我们在功能化介孔二氧化硅（FMS）中夹杂了三种不同的酶。介孔二氧化硅是一种典型的开放式纳米孔支撑，其孔径与数十纳米一样大。我们证明，与溶液中的无酶相比，所有三种酶在FMS中均表现出增强的活性。其次，酶特异性活性可以通过改变FMS中的蛋白质负荷密度在很大程度上增加或减少。第三，我们发现FMS和Chaotropic剂可以协同作用以增强酶活性。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是一个狭窄的空间，（ii）FMS提供了一个交互式环境，促进了有利的蛋白质构象变化，从而增强了酶活性和稳定性。因此，我们提出的具体目的是：1。研究中孔结构的必要性以及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限制和对酶活性增强的互动影响； 3。开发分子模型并采用分子对接和分子动力学模拟，以探测FMS驱动酶构象动力学向增强活性的机制； 4。评估FMS高度活跃和稳定的有机磷水解酶的功效，以提供大鼠中有机磷神经毒性的体内解毒，以证明一种综合的蛋白质（Emzyme）药物储存，释放，释放，释放和递送的综合综合设备。 公共卫生相关性：关键的基本科学问题之一是孤立的酶如何在解决方案或固定矩阵中保持原生活性构象。我们的长期目标是阐明工程纳米多孔支持中酶活性增强的机制，以利用高度活跃和稳定的酶来用于医疗应用，包括诊断，排毒和治疗癌症和其他疾病。由于这项工作，我们将评估高度活跃和稳定的有机磷酶水解酶在功能性纳米多孔支持中的疗效，以提供大鼠中有机磷神经毒性的体内解毒，以证明蛋白质（酶）储存的全合成的蛋白质（酶）药物储存，释放，释放，释放，释放，和递送。