CAREER: Rational Engineering of an Ionic Liquid Tolerant Cellulase Cocktail

摘要：Joel L.KaarProposal#：1454379机构：科罗拉多大学博尔德分校使用各种可再生生物质材料作为替代能源是当今研究的重要焦点。纤维素是一种有机化合物，是一种由数百到数千个相连的D-葡萄糖单元组成的线性链组成的多糖。纤维素是绿色植物和多种藻类初级细胞壁的重要结构成分。例如，木材的纤维素含量为40%至50%。纤维素酶是主要由真菌、细菌和原生动物产生的几种酶中的任何一种，它们催化纤维素的分解、纤维素和一些相关多糖的分解。纤维素酶将纤维素分子分解成单糖(单糖)，如β-葡萄糖，或较短的多糖和低聚糖。离子液体(在常温下熔化的离子液体类盐类物质)作为溶解生物质的替代溶剂的出现，为纤维素材料的转化提供了相当大的机会。特别值得注意的是，通过适当选择阳离子和阴离子，ILS可以合理地调节以溶解高浓度的未经处理的结晶纤维素。IL和纤维素之间的氢键的形成破坏了导致纤维素链紧密堆积在一起的内部氢键网络。除了降低纤维素的结晶度外，精选ILS还可以疏松生物质中的基质成分，从而增加纤维素的整体阻力。此外，由于ILS的热稳定性和非挥发性，ILS构成了对环境有吸引力的解决方案，从而解决了对用于纤维素加工的清洁介质的需求。用于溶解纤维素的传统溶剂含有无机盐、酸、碱和金属络合物，具有很大的毒性和环境污染。然而，ILS的诱人性质被这些溶剂中纤维素酶的广泛失活所否定。因此，为了充分利用离子液体来加工整个生物质，需要调节离子液体对纤维素酶的溶剂效应。本研究和教学项目的目的是开发一种通过工程酶充电来提高纤维素酶对离子液体(ILS)耐受性的方法。这项研究将专门测试这样一个假设，即通过定点突变改变纤维素酶的表面电荷可以调节与ILS的相互作用，从而影响纤维素酶的稳定性。作为纤维素材料的溶剂，离子液体因其优良的性质而提供了相当多的机会，包括独特的溶解大量生物质的能力。在这个项目中，改变酶电荷对ILS变性纤维素酶的影响将以前所未有的分辨率通过核磁共振波谱和分子动力学(MD)模拟来直接探测。这种影响将在使用定点突变改变嗜热酸杆菌具有良好特性的内切葡聚糖酶Ei、热梭状芽胞杆菌的外切葡聚糖酶CbhA和β-葡萄糖苷酶JMB19063 GH3的同时进行研究。此外，在整个生物质转化过程中，表面静电在防止不溶基质组分对纤维素酶抑制方面的作用将被阐明。具体的研究目标是：1)通过核磁共振和MD确定EI、CbhA和GH3中被ILS结构扰动的位置；2)合理设计和测试表面电荷改变的EI、CbhA和GH3变体，以提高对IL诱导的失活的耐受性；以及3)表征定点电荷突变对木质素在EI、CbhA和GH3上非生产性吸附的影响。教育目标是：1)通过参与巨石谷学区研究研讨会计划，促进当地高中生的研究机会；2)通过iGEM计划的发展，为本科生研究创造机会；3)为本科生课程创建和实施生物催化实验模块。

AbstractPI: Joel L. KaarProposal #: 1454379Institution: University of Colorado at BoulderThe use of various renewable biomass materials as an alternate source of energy is an important focus of research today. Cellulose is an organic compound, a polysaccharide consisting of a linear chain of several hundred to many thousands of linked D-glucose units. Cellulose is an important structural component of the primary cell wall of among others, green plants and many forms of algae. For example, the cellulose content of wood is 40 to 50%. Cellulase is any of several enzymes produced chiefly by fungi, bacteria, and protozoans that catalyze cellulolysis, the decomposition of cellulose and of some related polysaccharides. Cellulases break down the cellulose molecule into monosaccharides ("simple sugars") such as beta-glucose, or shorter polysaccharides and oligosaccharides. The emergence of ionic liquids (ILs - salt-like substances that melt at ambient temperature) as alternative solvents for the dissolution of biomass presents considerable opportunities for the conversion of cellulosic materials. Of particular interest, with the appropriate selection of cation and anion, ILs can be rationally tuned to dissolve high concentrations of untreated crystalline cellulose. The formation of hydrogen bonds between the IL and cellulose disrupts the internal hydrogen bonding network that causes cellulose chains to pack tightly together. In addition to lowering the degree of crystallinity of cellulose, select ILs can loosen the matrix components within biomass that add to the overall recalcitrance of cellulose. Furthermore, due to the thermal stability and non-volatile nature of ILs, ILs constitute and environmentally attractive solution to the need for cleaner media for cellulose processing. Conventional solvents used to dissolve cellulose, which contain inorganic salts, acids, bases, and metal complexes, are largely toxic and environmentally polluting. However, the attractive properties of ILs are negated by the broad inactivation of cellulases in these solvents. Consequently, for ILs to be fully exploited for processing whole biomass, the solvent effects of ILs on cellulases needs to be mediated.The aim of this integrated research and teaching project is to develop an approach to enhance the tolerance of cellulases to ionic liquids (ILs) for biomass processing via engineering enzyme charge. The research will specifically test the hypothesis that changing the surface charge of cellulases by site-directed mutagenesis can mediate interactions with ILs, which impact cellulase stability. As solvents for cellulosic materials, ILs present considerable opportunities due to their auspicious properties, including the unique capacity to solubilize large amounts of biomass. In this project, the impact of altering enzyme charge on the denaturation of cellulase by ILs will be directly probed by nuclear magnetic resonance spectroscopy and molecular dynamics (MD) simulations with unprecedented resolution. This impact will be investigated while using site-directed mutagenesis to alter the charge of the well-characterized endoglucanase EI from Acidothermus cellulolyticus, exoglucanase CbhA from Clostridium thermocellum, and beta-glucosidase JMB19063 GH3. Additionally, the role of surface electrostatics on the prevention of cellulase inhibition by insoluble matrix components in the conversion of whole biomass will be elucidated. The specific research objectives are to: 1) identify sites in EI, CbhA, and GH3 that are structurally perturbed by ILs by NMR and MD, 2) rationally design and test EI, CbhA, and GH3 variants with altered surface charge for improved tolerance to IL-induced inactivation, and 3) characterize the effect of site-directed charge mutations on non-productive lignin adsorption to EI, CbhA, and GH3. The educational objectives are to: 1) promote research opportunities for local high school students through involvement in Boulder Valley School District Research Seminar Program, 2) create opportunities for undergraduate research via growth of iGEM program, and 3) create and implement laboratory modules on biocatalysis for undergraduate curriculum.