Display of Escherichia coli Phytase on the Surface of Bacillus subtilis Spore Using CotG as an Anchor Protein

Escherichia coli phytase (AppA) has been widely used as an exogenous feed enzyme for monogastric animals; however, the production of this enzyme has been examined primarily in E. coli and yeast expression systems. As an alternative to production of soluble phytase, an enzyme immobilization method using the Bacillus subtilis spore outer-coat protein CotG as an anchoring motif for the display of the AppA was attempted. Using this motif, AppA was successfully produced on the spore surface of B. subtilis as verified by Western blot analysis and phytase activity measurements. Analysis of the pH stability indicated that more than 50% activity was retained after incubation at four different pH values (2.0, 4.0, 7.0, and 8.0) for up to 12h, with maximum activity observed at pH4.5. The highest enzyme activity seen at 55 degrees C and thermal stability measurements demonstrated that more than 30% activity remained after 30min incubation at 60 degrees C. The spore surface-displayed AppA was resistant to pepsin, and more stable than phytase produced previously using a yeast expression system. Furthermore, we present data indicating that the use of peptide linkers may help improve the bioactivity of displayed enzymes on the spore surface of B. subtilis.