CAREER: Rational Engineering of an Ionic Liquid Tolerant Cellulase Cocktail

摘要：利用各种可再生生物质材料作为替代能源是当今研究的一个重要焦点。纤维素是一种有机化合物，是一种由几百到几千个连接的d -葡萄糖单元组成的线性链的多糖。纤维素是绿色植物和多种藻类初代细胞壁的重要结构成分。例如，木材的纤维素含量为40 ~ 50%。纤维素酶是一种主要由真菌、细菌和原生动物产生的酶，能催化纤维素水解、分解纤维素和一些相关的多糖。纤维素酶将纤维素分子分解成单糖（“单糖”），如-葡萄糖，或较短的多糖和低聚糖。离子液体（在环境温度下熔化的盐状物质）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。特别有趣的是，通过适当的正离子和阴离子选择，il可以合理地调节来溶解高浓度的未经处理的结晶纤维素。IL和纤维素之间氢键的形成破坏了内部氢键网络，导致纤维素链紧密地聚集在一起。除了降低纤维素的结晶度外，选择ILs还可以使生物质内的基质成分松动，从而增加纤维素的整体顽固性。此外，由于液态纤维素的热稳定性和不易挥发性，液态纤维素是一种对环境有吸引力的解决方案，可以满足纤维素加工对清洁介质的需求。用于溶解纤维素的传统溶剂含有无机盐、酸、碱和金属配合物，大部分有毒且污染环境。然而，由于纤维素酶在这些溶剂中广泛失活，il的吸引力被否定了。因此，为了充分利用脂质酶处理整个生物质，需要调节脂质酶对纤维素酶的溶剂效应。这个综合研究和教学项目的目的是开发一种方法，通过工程酶电荷来提高纤维素酶对离子液体（ILs）的耐受性，用于生物质加工。该研究将专门验证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，从而影响纤维素酶的稳定性的假设。作为纤维素材料的溶剂，由于其吉祥的性质，包括溶解大量生物质的独特能力，il提供了相当大的机会。在这个项目中，改变酶荷对纤维素酶变性的影响将通过核磁共振波谱和分子动力学（MD）模拟以前所未有的分辨率直接探测。研究人员将利用定点诱变技术改变酸性溶纤维素菌的内切葡聚糖酶EI、热胞梭菌的外切葡聚糖酶CbhA和β -葡萄糖苷酶JMB19063 GH3的电荷。此外，表面静电在整个生物质转化过程中防止不溶性基质组分抑制纤维素酶的作用也将得到阐明。具体的研究目标是：1)通过NMR和MD识别EI、CbhA和GH3中被il干扰的结构位点；2)合理设计和测试EI、CbhA和GH3表面电荷改变的变体，以提高对il诱导失活的耐受性；3)表征位点定向电荷突变对非生产木质素对EI、CbhA和GH3吸附的影响。教育目标是：1)通过参与博尔德谷学区研究研讨会计划，为当地高中生提供研究机会；2)通过iGEM计划的发展，为本科生创造研究机会；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.