A Multiscale Approach to Characterizing Interfacial Carbohydrate-Active Enzymes

该项目研究了酶如何将不溶性纤维素生物材料分解为可溶性可发酵糖（如葡萄糖）的分子基础，以及如何使它们更有效地实现工业规模的生物燃料生产。 这项研究的结果将有助于开发低成本的生物工艺，从纤维素生物质生产可再生燃料，同时提供广泛的教育和推广活动，促进培训和普遍认识的潜力，生物燃料提供了一个可持续的和可再生的能源未来。具有不同表面化学性质的纤维素酶与结晶纤维素表面的生产性停滞和结合相互作用，目的是工程化更有活性的酶变体。 实现这一目标的具体目标包括：1）使用基于细胞的和无细胞的蛋白质表达系统设计和生产具有非天然表面化学的纤维素酶变体，2）估计纤维素酶变体对结晶纤维素的结合亲和力及其与整体平均体积纤维素酶比活性的关系，和3）测定纤维素酶变体的界面结合的单分子表面运动性，并将单分子事件与整体结合和比活性测量相关联。 将进行高通量无细胞蛋白表达和组合纤维素水解活性测定以鉴定改进的纤维素酶变体。 单分子纤维素酶结合和运动性测量将使用光镊力谱技术进行。 连同整体平均体积测量，该研究将提供新的见解，蛋白质表面残基和离散的单酶生物力学步骤参与非生产性的界面结合和催化活性的结晶纤维素表面结合的纤维素酶，从而绘制一个基因工程路径，使成本更低，更有效的生物转化过程中，使纤维素生物燃料。 研究人员将让各级学生以及高中教师参与适合年龄的活动，从针对研究生和本科生的纤维素酶工程和单分子生物物理学的深入培训，到针对年轻学生的示范项目，以及通过实地考察当地能源行业来提高生物燃料意识。

The project investigates the molecular underpinnings of how enzymes break down insoluble cellulosic biomaterials to soluble fermentable sugars like glucose and how to make them more efficient to enable industrial-scale biofuel production. The results of the study will aid the development of lower-cost bioprocesses for producing renewable fuels from cellulosic biomass while providing a broad range of educational and outreach activities promoting training and general awareness of the potential that biofuels offer toward a sustainable and renewable energy future.The study will examine the relationship between non-productive stalling and binding interactions of cellulases with varying surface chemistry to crystalline cellulose surfaces with the goal of engineering more active enzyme variants. Specific aims toward achieving this goal involve: 1) design and production of cellulase variants with non-native surface chemistry using both cell-based and cell-free protein expression systems, 2) estimates of the binding affinity of cellulase variants to crystalline cellulose and its relationship to ensemble-averaged bulk cellulase specific activity, and 3) determine the interfacially bound single-molecule surface motility for cellulase variants and correlating single-molecule events to bulk ensemble binding and specific activity measurements. High throughput cell-free protein expression and combinatorial cellulose hydrolytic activity assays will be carried out to identify improved cellulase variants. Single-molecule cellulase binding and motility measurements will be conducted using an optical tweezers force spectroscopy technique. Together with ensemble-averaged bulk measurements, the study will provide new insight into both the protein surface residues and discrete single-enzyme biomechanical steps involved in the non-productive interfacial binding and catalytic activity of crystalline cellulose surface bound cellulases, thereby charting a genetic engineering path that enables a lower cost and more efficient bioconversion process for making cellulosic biofuels. The researchers will engage students at all levels as well as high school teachers in age-appropriate activities ranging from in-depth training in cellulase engineering and single-molecule biophysics for graduate and undergraduate students to demonstration programs for younger students and generating biofuels awareness through field-trips to local energy industries.This project is co-funded by the Division of Materials Research through the BioMaPs funds.