A MULTISCALE APPROACH TO CHARACTERIZING INTERFACIAL CARBOHYDRATE-ACTIVE ENZYMES

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.