(489b) A Novel Self-Assembled Protein Nanostructure as Multifunctional Catalyst for Xylan Hydrolysis

Cellulosome, a multi-protein nanostructure (18 nm), is an active nanosystem naturally made by many anaerobic microbes to assimilate recalcitrant cellulose. This self-assembled system brings multiple enzymes to proximity of the substrate, and provides a structure that ensures a high local concentration and correct ratio/orders of the components, thereby maximizing synergy. Consequently, it has much higher catalysis efficiency than soluble enzymes present in a non-organized fashion. The molecular architecture of a cellulosome consists of a structural scaffold protein (or scaffoldin) containing multiple repeating units called cohesins, into which multiple enzymes, each tagged with a complementary end (called dockerin), can dock and self-assemble into a multi-protein complex. The specific protein-protein, or complementary cohesin-dockerin interaction, provides the mechanism for position-specific self-assembly.

Using cellulosome architecture as design template, we have designed a two-unit nano protein structure, named xylosome, for xylan hydrolysis. The xylosome consists of a two-cohesin scaffold protein and two xylan-degrading enzymes (a xylanase and a xylosidase) each tagged with a dockerin. Xylanase from Clostridium thermocellum with a dockerin complementary to the first cohesin unit was cloned from and overexpressed in E. coli. Other components of the nanostructure are being constructed with gene fusion strategy. These components, after isolation and purification, will be allowed to self-assemble. A range of environmental conditions, such as pH, ionic strength, calcium concentrations, temperatures, the presence of other proteins, and target protein concentration, will be investigated in order to understand how they influence self-assembly, stability, and functionality of the nanostructure. The performance of xylosome will be evaluated with xylan substrates.

The presentation will detail the molecular design, construction, and characterization of this novel protein nanostructure and highlight its application as a high-performance multifunctional catalyst in hydrolysis of xylan.