Substrate Binding Effects of Carbohydrate Binding Modules on the Catalytic Activity of a Multifunctional Cellulase

CelE from Clostridium thermocellum is a glycoside hydrolase family 5 enzyme that has broad specificity for hydrolysis of plant biomass polysaccharides, particularly cellulose, xylan, and mannan. A fusion of CelE with the well-studied carbohydrate binding module CBM3a was created, CelE_CBM3a, and this construct alone was able to give high-yield hydrolysis of both cellulose and hemicellulose in pretreated plant biomass. In this study, thirty-nine different CBMs from the C. thermocellum genome were fused to GFP, and the specific binding interactions of the robotic cell-free translated GFP_CBM constructs were determined with a panel of soluble and insoluble substrates. Most CBMs tested were able to bind at least one substrate, and several GFP_CBMs displayed unanticipated binding capabilities. We also asked whether these CBMs could yield different catalytic capabilities when fused to the multifunctional enzyme, CelE. Different CelE_CBM fusions were also expressed using robotic cell-free translation, and our results show that some CBMs enhanced cellulose hydrolysis through specific interaction between the CBM domain and polysaccharide. In addition, different CelE_CBM fusions gave lichenan-, xylan-, mannan-, and biomass-specific improvements in the catalytic activities. The methods described here provide a useful system that can be used to test the function of other CBM domains and to assist in the design and testing of new engineered enzyme formats.This work was supported by the DOE Great Lakes Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494) and the University of Wisconsin-Madison's Science and Medicine Graduate Research Scholars Advanced Opportunity Fellowship (SciMed GRS AOF) Program (J.A.W.), the National Institute of General Medical Sciences Molecular Biophysics Training Program (NIH T32 GM08293, J.A.W.), and the National Science Foundation Graduate Research Fellowship Program (DGE-1256259 to J.A.W.).