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: 8119144
• 负责人: Chenghong Lei
• 金额: $ 31.65万
• 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/8119144
• 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; Enzyme Stability; 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）：一个关键的基本科学问题是分离的酶如何在溶液或固定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。具体假设是：蛋白质的酶活性和稳定性可以在适当设计的开放纳米孔载体中显着增强，其功能是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）中捕获了三种不同的酶。介孔二氧化硅是一种典型的开孔纳米载体，孔径可达数十纳米。结果表明，与溶液中游离的酶相比，这三种酶在FMS中均表现出增强的活性；其次，通过改变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特异性活性；第三，我们发现FMS和混沌剂可以协同作用，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是一个密闭的空间，（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，我们提出具体的目标是：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，了解FMS约束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引导酶构象动力学增强活性的机制；4. 评价FMS中高活性、稳定的有机磷水解酶对大鼠体内有机磷神经毒性的解毒作用，展示一种集蛋白质（酶）药物储存、释放和传递于一体的一体化装置。