A Multiscale Approach to Characterizing Interfacial Carbohydrate-Active Enzymes

该项目研究酶如何将不溶的纤维素生物材料分解为葡萄糖等可溶性可发酵糖的分子基础，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从纤维素生物质生产可再生燃料的低成本生物工艺，同时提供广泛的教育和推广活动，促进培训和普遍认识生物燃料提供的潜力，以实现可持续和可再生能源的未来。研究将探讨非生产性停滞和具有不同表面化学的纤维素酶与结晶纤维素表面结合作用之间的关系，目的是设计更活跃的酶变体。实现这一目标的具体目标包括：1)使用基于细胞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)估计纤维素酶变体与结晶纤维素的结合亲和力及其与总体平均整体纤维素酶比活力的关系，以及3)确定纤维素酶变体的界面结合单分子表面能动性，并将单分子事件与整体系综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酶活性分析，以确定改进的纤维素酶变体。单分子纤维素酶的结合和运动性测量将使用光镊力光谱技术进行。结合整体平均批量测量，这项研究将为蛋白质表面残基和离散的单酶生物力学步骤提供新的见解，这些步骤涉及结晶纤维素表面结合纤维素酶的非生产性界面结合和催化活性，从而绘制出一条基因工程路线，使制造纤维素生物燃料的生物转化过程能够实现更低成本和更高效。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，范围从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，到为年轻学生提供的示范项目，以及通过实地考察当地能源行业提高对生物燃料的认识。该项目由材料研究部通过BioMaps基金共同资助。

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.