Expression of Laccase from Geobacillus

High enzyme cost for hydrolysis of lignocellulosic biomass is a major challenge for next generation fuel and chemical production from renewable biomass. Large amounts of enzymes are needed and many problems exist, such as high production cost, poor stability, and low tolerance to inhibitors generated during pretreatment and hydrolysis. Bacterial enzymes, especially the ones from thermophiles, are generally more robust compared with enzymes from fungi. Some Geobacilli, a genus of thermophilic bacteria, have demonstrated lignocellulolytic activities. In this study, we have characterized a novel laccase from Geobacilli with ligninolytic activities. To further reduce the production cost and enhance the enzyme stability, two strategies are used to express the laccase. First, the laccase was expressed and secreted in B. subtilis to reduce the purification costs. Signal peptides and copper chaperons are tuned to enhance the functional expression and secretion of laccase. These results will aid in the understanding of the mechanisms of copper binding, laccase folding and secretion. In addition, using laccase as an example, we demonstrate functional secretion of otherwise intracellular enzymes by signal peptide engineering. Another strategy is to develop a spore surface display system to enhance its function under unfavorable conditions. Laccase expressed on the B. subtilis spore surface shows improved stability. This system also provides a platform to investigate the synergism between laccase and other lignocellulolytic enzymes in biomass deconstruction.