(399c) Novel Self-Assembled Protein Nanostructures for Xylan Bioprocessing

Lignocellulose, present in numerous industrial waste and agriculture residues, can be used for production of a wide variety of molecules including biofuels, commodity chemicals and materials. However, processing the recalcitrant material in a cost-effective and environmental-friendly manner presents significant engineering challenges. Bioprocessing of hemicellulose is one example that illustrates such challenges. Its complete hydrolysis requires not only backbone-acting enzymes but also the synergistic action of several side-chain acting enzymes. To effectively hydrolyze the target substrates, cellulolytic aerobic microbes secrete copious amounts of hemicellulases into the extracellular environment, whereas anaerobic organisms organize enzymes in cell-associated, self-assembled protein nanostructures, known as cellulosomes. Cellulosomes are constructed using a scaffoldin protein with repeating cohesin domains and enzymes tagged with corresponding dockerins. The specific protein-protein interaction between cohesins and dockerins provide the method of self-assembly. These protein structures position multiple proteins in close proximity to the substrate, resulting in greater enzymatic synergy and improved catalytic efficiency compared to unorganized, free enzymes.

Mimicking the architecture of cellulosomes, we constructed four two-unit nanostructures named xylanosomes for enhancing xylan hydrolysis. Four different enzymes with either a Clostridium thermocellum or Clostridium cellulovorans dockerin domain were cloned and expressed in E. coli to cover four out of the six required activities to fully hydrolyze xylan: xylanase, feruloyl esterase, arabinofuranosidase, and beta-xylosidase. These enzymes were combined with a complementary chimeric two-cohesin scaffoldin protein to create four possible enzyme combinations. The performance of both free enzyme mixtures and self-assembled structures was investigated on a hemicellulose substrate, wheat arabinoxylan. Results reveal that select enzyme combinations show synergy in these structures giving up to 3 times greater release of reducing sugars compared to a free system. Conversely, some structures gave negative synergies or showed no improvement. Additionally, different synergies were observed in combining either family 10 or family 11 xylanases with feruloyl esterases.

The presentation will detail the construction of these xylanosomes and their ability to selectively improve xylan hydroysis compared to the corresponding free enzyme system. Possible explanations for the observed synergies or lack thereof will be discussed.