Directed evolution of CotA laccase for increased substrate specificity using Bacillus subtilis spores

Directed evolution is an effective strategy to engineer and optimize protein properties, and microbial cell-surface display is a successful method to screen protein libraries. Protein surface display on Bacillus subtilis spores is demonstrated as a tool for screening protein libraries for the first time. Spore display offers advantages over more commonly utilized microbe cell-surface display systems, which include gram-negative bacteria, phage and yeast. For instance, protein-folding problems associated with the expressed recombinant polypeptide crossing membranes are avoided. Hence, a different region of protein space can be explored that previously was not accessible. In addition, spores tolerate many physical/chemical extremes; hence, the displayed proteins are "preimmobilized" on the inherently inert spore surface. Immobilized proteins have several advantages when used in industrial processes. The protein stability is increased and separations are simplified. Finally, immobilized proteins can be used in a wide array of simple device applications and configurations. The substrate specificity of the enzyme CotA is narrowed. CotA is a laccase and it occurs naturally on the outer coat of B. subtilis spores. A library of CotA genes were expressed in the spore coat, and it was screened for activity toward ABTS [diammonium 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)] over SGZ (4-hydroxy-3,5-dimethoxy-benzaldehyde azine). A mutant CotA was found to be 120-fold more specific for ABTS. This research demonstrates that B. subtilis spores can be a useful platform for screen protein libraries.