A Multiscale Approach to Characterizing Interfacial Carbohydrate-Active Enzymes

表征界面碳水化合物活性酶的多尺度方法

基本信息

批准号：
1604421
负责人：
Shishir Chundawat
金额：
$ 44.97万
依托单位：
Rutgers University New Brunswick
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-01 至 2023-02-28
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1604421&HistoricalAwards=false
关键词：
Multiscale Approach Characterizing Interfacial Carbohydrate

项目摘要

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.

该项目调查了酶如何将不溶性纤维素生物材料分解为可溶性发酵糖（如葡萄糖）以及如何使它们更有效地启用工业规模生物燃料生产的糖。该研究的结果将有助于开发较低成本的生物处理，以生产纤维素生物量的可再生燃料，同时提供广泛的教育和外展活动，以促进培训以及对生物油的一般意识，并普遍意识到生物油器为可持续性和可再生能源提供的潜在。表面的目的是工程更活跃的酶变体。实现这一目标的具体目的涉及：1）使用基于细胞和无细胞的蛋白质表达系统的非本地表面化学的纤维素酶变体的设计和生产，2）纤维素酶变异纤维素与结晶纤维素的结合亲和力的估计及其与整体纤维化酶特异性活动的关系及其与整体纤维化酶特异性活动的关系，以及3）确定单分子事件以进行整体结合和特定的活性测量。将进行高吞吐细胞的蛋白质表达和联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测量将使用光学镊子力光谱技术进行。该研究将与整体平均大量测量一起提供新的洞察力，以了解蛋白质表面残基和离散的单酶生物力学步骤，参与了晶体纤维素纤维素结合的非生产性互起结合和催化活性，从而使生物效率和更有效的生物效率能够启用较低的成本化过程。研究人员将与各个层次的学生以及高中教师一起参加适合年龄的活动，从纤维素酶工程的深入培训和毕业生和本科生的单分子生物物理学到年轻学生的示范计划，以及通过现场能源工业的生物燃料的认识，通过现场能量工业来创造生物燃料的意识，由本地项目进行。