(228q) Spore Surface Display of a Laccase to Enhance Its Robustness

Cost-effective deconstruction of lignocellulose is a major challenge to produce biofuel and other valuable fermentation products from renewable biomass. Large amounts of enzymes are needed due to the recalcitrant structure of lignocellulose, and the enzymatic degradation is often carried out under nonideal conditions owing to the pretreatment of the biomass. Inexpensive production of robust cellulolytic enzymes is critical to develop economically feasible biomass degradation processes. Extremophiles, growing under harsh conditions, provide a good resource and have been extensively explored to find robust enzymes. In this work, a thermostable laccase from thermophilic bacterium Geobacillus thermoglucosidasius was characterized and functionally displayed on the spore surface of Bacillus subtilis. It belongs to a newly discovered family of laccases, DUF152 family, and exhibits excellent activity and thermostability. The half-life of the purified laccase is about 3 hours at 80 °C. It was displayed on the spore surface to further enhance its robustness and reduce the production cost, because spores are able to survive various harsh environments and sporulation is well-established protocol and no protein purification is needed. A few spore surface proteins were explored as the anchor and different linkers were tested. The displayed laccase was shown to have a half-life at 80 °C about 1.5 times of the purified protein and improved solvent resistance. Co-expression of the protein together with the anchor-laccase fusion protein is critical to maximize the activity of dimers displayed on the spore surface. Overall, this work showed that spore surface display is a cost-effective protein expression and display platform that can enhance enzyme stability and complex protein such as multimers can be also functionally displayed on the spore surface.