CAREER: Rational Engineering of an Ionic Liquid Tolerant Cellulase Cocktail

职业：离子液体耐受纤维素酶混合物的合理工程

• 批准号: 1454379
• 负责人: Joel Kaar
• 金额: $ 50万
• 依托单位: University of Colorado at Boulder
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1454379&HistoricalAwards=false
• 关键词: CAREER; Rational; Engineering; Ionic; Liquid

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.

摘要：Joel L. Kaarpropopals＃：1454379 Institution：科罗拉多大学博尔德大学的使用各种可再生生物量的材料作为替代能源的替代来源是当今研究的重要重点。纤维素是一种有机化合物，一种多糖由数百至数千个连接的D-葡萄糖单元组成的线性链组成。纤维素是原代细胞壁的重要结构成分，绿色植物和多种形式的藻类。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，纤维素的分解和一些相关的多糖。纤维素酶将纤维素分子分解为单糖（“简单糖”），例如β-葡萄糖或较短的多糖和寡糖。离子液体（在环境温度下融化的盐样物质）的出现作为溶解生物质的替代溶剂，这为纤维素材料的转化带来了相当大的机会。特别有趣的是，通过适当选择阳离子和阴离子，可以合理调整IL，以溶解高浓度的未处理的晶体纤维素。 IL和纤维素之间的氢键的形成破坏了内部氢键网络，从而导致纤维素链紧紧堆积在一起。除了降低纤维素的结晶度外，选择的IL还可以松开生物量中的基质成分，从而增加了纤维素的整体顽固性。此外，由于ILS的热稳定性和非挥发性性质，ILS构成了对需要更清洁培养基进行纤维素加工的环境吸引力的解决方案。用于溶解纤维素的常规溶剂，其中含有无机盐，酸，碱和金属配合物，在很大程度上是有毒的，并且在环境中受到污染。但是，通过这些溶剂中纤维素酶的广泛失活而否定了IL的吸引力。因此，要使IL完全利用用于处理整个生物量的IL，需要介导IL对纤维素酶的溶剂作用。该综合研究和教学项目的目的是开发一种方法，以增强对离子液体（ILS）对生物量处理工程工程的耐离子液体（IL）的耐受性。这项研究将特异性检验以下假设：通过定点诱变改变纤维素酶的表面电荷可以介导与IL的相互作用，从而影响纤维素酶稳定性。作为纤维素材料的溶剂，ILS由于其吉祥的特性而提供了相当大的机会，包括溶解大量生物质的独特能力。在该项目中，通过前所未有的分辨率，将直接探测酶荷兰对ILS变性的影响。在使用定位诱变时，将研究这种影响，以改变来自酸性纤维溶解度的特征良好的内生糖酶EI的电荷，来自热门梭状芽胞杆菌和β-葡萄糖苷酶JMB19063 GH3的酸葡聚糖CBHA，exogolucanase cbha和β-葡萄糖苷酶JMB19063 GH3。此外，将阐明表面静电学在预防不溶性基质成分抑制纤维素酶在整个生物质转化中的作用。具体的研究目标是：1）识别EI，CBHA和GH3中的地点在结构上受到NMR和MD的IL结构扰动，2）在理性的设计和测试EI，CBHA和GH3变体中具有改变地表电荷的变化，以改善对IL诱导的灭活量的耐受性，并表现出无效的效果，并表现出3）的效果，以表征有效的效率，以表征有效的效率，以实现效应的效果。 EI，CBHA和GH3。教育目标是：1）通过参与Boulder Valley学区研究研讨会计划，为当地高中学生促进研究机会，2）通过IGEM计划的增长创造本科研究的机会，3）创建和实施实验室模块，以实验本科课程的生物催化。