Multi-enzyme immobilization in metal-organic materials for rapid and sustainable degradation of biomass

当代生活和社会在很大程度上依赖于能源和材料来源。某些资源是不可持续的，这意味着它们一旦消耗就不能迅速补充，而其他资源是可持续的，可以再生。不幸的是，不可持续的资源正在迅速枯竭；因此，改善对可持续资源的利用可以使国家在环境和经济上都受益。植物是有前途的可持续资源，它们提供燃料、材料和营养。然而，这些有价值的成分不能轻易地从植物中提取出来，因为它们被困在植物的表皮中。典型的植物表皮是由复杂的物种组成的，例如：1)由众多交联的大分子组成的密集网络，称为纤维素；2)一堆被称为脂质的小分子，它们排列并组装成植物细胞膜；3)糖蛋白，由糖样分子与蛋白质连接形成的分子。这些物种为植物建立了坚硬的保护层，不易被破坏。该项目将开发特殊的生物材料来打破这种保护层。一系列酶，加速纤维素、脂质和糖蛋白分解的特殊蛋白质，将被放置在被称为Ca-MOM的固体晶体的支架和缝隙中。这些酶将协同降解植物的保护层，而不会产生不利的副产物。Ca-MOM晶体将稳定并帮助重新收集酶，使它们可重复使用。同时，酶在晶体支架中的位置也将被探索，以了解酶在所开发的生物材料中的性能。这些努力不仅将产生有效和可持续地分解植物表皮的生物材料，以便提取有价值的能源资源和材料，而且还将了解酶在这些生物材料中的作用。所展示的策略还将促进科学的进步，因为它可以适用于分解含有有价值成分并需要多种酶合作的其他天然来源。研究工作将与一项教育计划相结合，通过让当地代表性不足的群体参与绿色化学研究，为他们带来研究机会。该首席研究员还将开发一个名为“绿色化学-绿色星球”的教育项目，以便通过诸如北达科他州（ND） 4-H和ND军事儿童行动项目等已建立的项目向当地小学生介绍绿色化学概念。技术摘要/摘要植物是可持续的能源和材料来源，但有价值的成分是由植物细胞壁保护的。分解这些细胞壁的挑战是难以降解密集的纤维素网络，这是细胞壁硬度的主要原因，以及其他成分（如脂质和糖蛋白）的复杂性，这些成分也会增强黏度和硬度。利用纤维素酶和辅助酶降解纤维素和其他组分是植物生物质降解的绿色选择，因为它们具有特异性和生物相容性，但挑战在于需要多种酶，包括蛋白酶，这些酶会破坏伙伴酶。该项目将通过酶-MOM共结晶将三种纤维素酶、一种脂肪酶和一种蛋白酶固定在金属有机材料（MOM）上，从而克服这一挑战。在合成的共晶中，每个酶部分暴露在反应介质中以与底物接触，而部分埋在MOM表面下以保护酶以减少蛋白水解损伤。开发的生物催化剂将在模式植物生物量的降解上进行演示，然后使用电子顺磁共振波谱技术探索合成的多酶/MOM生物催化剂在MOM配体上的结构-性质关系。假设是：1)同时固定化5种酶提高了植物生物量降解的成本效率，并加速了植物生物量降解，减少了蛋白质水解损伤；2)所开发的生物催化剂的生物催化性能取决于酶暴露在MOM表面和MOM配体上的结构基础。这些假设将通过三个目标进行验证：1)开发用于快速生物降解植物生物量的5合1/Ca-BDC生物催化剂，2)确定5合1/Ca-BDC生物催化剂的结构基础，以及3)建立5合1/ ca - mom的结构-性能关系以及所开发生物催化剂的生物催化性能。该项目的教育计划将平均每年为1名美国原住民学生、2名本科生和1名当地高中生提供参与绿色化学研究的机会。此外，还将开发一项名为“绿色化学-绿色星球”的教育计划，通过北达科他州（ND）的4-H系统和ND的军事儿童行动计划，提高众多K-12学生的科学知识。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Non-technical summary/abstractContemporary life and society depend heavily on energy and materials sources. Certain sources are non-sustainable, meaning that they cannot be replenished quickly once consumed, while others are sustainable and can be regenerated. Unfortunately, non-sustainable sources are running out rapidly; thus, the nation could benefit both environmentally and economically from an improved use of sustainable sources. Plants are promising sustainable sources, which offer fuels, materials, and nutrients. Yet, these valuable components cannot be easily taken out from plants, because they are trapped in plant skins. Typical plant skins are composed of complex species, such as i) an intense network of numerous, cross-linked large molecules, known as cellulose, ii) a bunch of small molecules known as lipids which line up and assemble into plant cell membranes, and iii) glycoproteins, molecules formed by sugar-like molecules connected with proteins. These species build up a hard protection layer to plants, which cannot be broken down easily. This project will develop special biomaterials to break down such a protection layer. A series of enzymes, special proteins which speed up the breaking-down of cellulose, lipids, and glycoproteins, will be placed in the scaffolds and gaps of a solid crystal, known as Ca-MOM. These enzymes will cooperate to degrade the protection layer of plants without generating adverse by-products. The Ca-MOM crystal will stabilize and help re-collect the enzymes, making them reusable. Meanwhile, the positions of the enzymes in the crystal scaffolds will also be probed to understand the performance of the enzymes in the developed biomaterials. These efforts will not only produce biomaterials to efficiently and sustainably break down plant skins to allow for the extraction of valuable energy resources and materials but also understand how enzymes function in these biomaterials. The demonstrated strategy will also improve the progress of science because it can be adapted to breaking down of other natural sources which contain valuable components and require multiple enzymes to cooperate. The research efforts will be bridged with an educational plan to bring research opportunities to local underrepresented groups by involving them in green chemistry research. This principle investigator will also develop an educational program called Green Chemistry- Green Planet in order to introduce the green chemistry concept to local elementary students through established programs such as the North Dakota (ND) 4-H and Operation Military Kids programs in ND.Technical summary/abstractPlants are sustainable sources of energy and materials, yet the valuable components are protected by plant cell walls. The challenges to break down these cell walls are the difficulty in degrading the intense cellulose network, the major cause of cell wall stiffness, and complexities due to other components such as lipids and glycoproteins, which also enhance viscosity and stiffness. Using cellulases and accessory enzymes to degrade cellulose and other components is the green choice of plant biomass degradation due to their specificity and biocompatibility, yet the challenge is the need of multiple enzymes including proteases which damage the partner enzymes. This project will overcome this challenge by immobilizing three celluases, a lipase, and a protease on Metal-Organic Materials (MOM) via enzyme-MOM co-crystallization. In the resultant co-crystals, each enzyme is partially exposed to the reaction medium for substrate contact while partially buried under MOM surfaces for enzyme protection to reduce proteolytic damage. The developed biocatalysts will be demonstrated on the degradation of a model plant biomass, followed by probing the structure-property relationship of the resultant multi-enzyme/MOM biocatalysts differing in MOM ligands using Electron Paramagnetic Resonance spectroscopy. The hypotheses are 1) simultaneous immobilization of 5 enzymes enhances the cost efficiency of and accelerate plant biomass degradation with reduced proteolytic damage and 2) the biocatalytic performance of the developed biocatalysts depends on the structural basis of enzyme exposure on MOM surfaces and MOM ligands. These hypotheses will be tested through three objectives: 1) develop a 5-in-1/Ca-BDC biocatalyst for the rapid biodegradation of plant biomass, 2) determine the structural basis of the 5-in-1/Ca-BDC biocatalyst, and 3) establish the structure-property relationship of the 5-in-1/Ca-MOMs the biocatalytic performance of the developed biocatalysts. The educational plan of this project will provide opportunities for averagely 1 Native American student, 2 undergraduate students, and 1 local high school student per year to participate in green chemistry research. In addition, an educational program called Green Chemistry - Green Planet will be developed to enhance the science knowledge of numerous K-12 students via the North Dakota (ND) 4-H system and the Operation Military Kids program in ND.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.