Abstract 1849 Enzyme engineering for improved biofuel production from plant sources

Biofuels can be generated using the conversion of plant biomass to fermentable sugars and are a promising sustainable fuel resource. Lignocellulose is one major structural component of plant cell walls but is difficult to break down, resulting in less efficient biofuel yields. Efforts have been made to enhance lignocellulose bioconversion efficiency, including biomass pretreatments and enzymatic pre-processing. Although naturally occurring enzymes have been utilized for preprocessing, they are not very efficient for lignocellulose breakdown and are also often not thermostable enough for commercial use. Comparison of many enzymes previously tested as ligninases led us to select a bifunctional chimeric enzyme with both glucanase and a laccase for improvement through enzyme engineering methods. A predicted 3D protein structure for the chimeric protein was generated using AlphaFold2 and compared with solved structures for each individual domain. Both domains appear to be able to fold correctly and should thus retain their individual functions when combined in the chimeric protein. Next multiple sites for mutations that could improve protein stability and thus possibly also improve function were identified using a machine learning algorithm. Future experiments will focus on overexpressing, purifying, and characterizing the unmodified chimeric enzyme in vitro, for comparison with the different mutants.