Engineering cellulases for enhanced degradation of insoluble substrates

There is tremendous social and political interest in the production of sustainable and carbon neutral liquid fuels that can meet our transportation energy demands. Second-generation biofuels, derived from cellulosic non-edible plant matter (biomass), represent a possible solution to this issue. The US Department of Energy and the Department of Agriculture estimate that the US can produce the energy equivalent 42 percent of the total US annual transportation consumption from cellulosic biomass, without having a grossly negative impact on food supply. Biomass can be deconstructed to glucose, a type of sugar that can be fermented to make the biofuels. Glucose monomers are located within a strong crystalline polymer called cellulose, which is surrounded by other complex polymeric structures called hemicellulose and lignin. In order to obtain this sugar the biomass therefore has to pass through mechanical, physicochemical, and enzymatic treatments. The mechanical and physicochemical stages of the process increase the accessibility of cellulose to cellulolytic enzymes responsible of converting the cellulose to glucose. Optimization of these enzymes can have a significant impact on the economics and feasibility of biofuels. A detailed understanding of how carbohydrate-binding domains (CBMs) increase the activity of cellulases remains elusive. We have completed a series of studies that shed light on how certain CBMs affect the activity of cellulases from three different perspectives: 1) Specific enzyme-substrate interactions: Neutron reflectivity is used to analyze the effect of endocellulases on cellulose film density and thickness. Two endocellulases, each tethered or untethered to a single CBM, are compared in this study. 2) General synergy between CBMs from three different families and a specific cellulase: We set out to determine if there is any correlation between CBM family and activity level or substrate preference, as measured by enhanced activity of a single cellulase. From a library of chimeric enzymes tested for activity on crystalline and non-crystalline substrates, we chose to examine in more detail three enzymes that exhibited approximately equal activity enhancements on amorphous cellulose. These chimeras consisted of a model endocellulase tethered to a CBM belonging to family one, two or three. Neutron reflectivity was used to study the interaction of the three chimeric endocellulases with amorphous cellulose model films. 3) The effect of enzymatic ratios in simple cellulolytic cocktails and the impact of CBM-containing enzymes on those ratios: We focused on the optimization of simple cellulolytic cocktails for real-world operational conditions. With basic conditions established, optimization is achieved by changing the ratios of the different components within the cocktail. The resulting optimal ratios and efficiency of glucose release was examined for cocktails comprising either commercial enzymes or enzymes identified to be active under extreme, but industrially relevant conditions

Addition of a carbohydrate-binding module enhances cellulase penetration into cellulose substrates

Introduction. Cellulases are of great interest for application in biomass degradation, yet the molecular details of the mode of action of glycoside hydrolases during degradation of insoluble cellulose remain elusive. To further improve these enzymes for application at industrial conditions, it is critical to gain a better understanding of not only the details of the degradation process, but also the function of accessory modules. Method. We fused a carbohydrate-binding module (CBM) from family 2a to two thermophilic endoglucanases. We then applied neutron reflectometry to determine the mechanism of the resulting enhancements. Results: Catalytic activity of the chimeric enzymes was enhanced up to three fold on insoluble cellulose substrates as compared to wild type. Importantly, we demonstrate that the wild type enzymes affect primarily the surface properties of an amorphous cellulose film, while the chimeras containing a CBM alter the bulk properties of the amorphous film. Conclusion: Our findings suggest that the CBM improves the efficiency of these cellulases by enabling digestion within the bulk of the film

Development of a High Throughput Platform for Screening Glycoside Hydrolases Based on Oxime-NIMS.

Cost-effective hydrolysis of biomass into sugars for biofuel production requires high-performance low-cost glycoside hydrolase (GH) cocktails that are active under demanding process conditions. Improving the performance of GH cocktails depends on knowledge of many critical parameters, including individual enzyme stabilities, optimal reaction conditions, kinetics, and specificity of reaction. With this information, rate- and/or yield-limiting reactions can be potentially improved through substitution, synergistic complementation, or protein engineering. Given the wide range of substrates and methods used for GH characterization, it is difficult to compare results across a myriad of approaches to identify high performance and synergistic combinations of enzymes. Here, we describe a platform for systematic screening of GH activities using automatic biomass handling, bioconjugate chemistry, robotic liquid handling, and nanostructure-initiator mass spectrometry (NIMS). Twelve well-characterized substrates spanning the types of glycosidic linkages found in plant cell walls are included in the experimental workflow. To test the application of this platform and substrate panel, we studied the reactivity of three engineered cellulases and their synergy of combination across a range of reaction conditions and enzyme concentrations. We anticipate that large-scale screening using the standardized platform and substrates will generate critical datasets to enable direct comparison of enzyme activities for cocktail design