Production of D-Allulose with D-Psicose 3-Epimerase Expressed and Displayed on the Surface of Bacillus subtilis Spores

The production of D-allulose is usually conducted via isolated-enzyme or whole-cell biocatalysis reactions. In the present study, an innovative biocatalyst, D-psicose-3-epimerasel (DPEase) from Clostridium scindens. ATCC 35704, presented on the surface of Bacillus subtilis spores, was applied for D-allulose production. DPEase was fused at the C-terminus of the anchoring protein, CotZ, via a peptide linker, and trophic genes were used as selection markers during the chromosomal integration. The optimal temperature and pH of the fusion protein CotZ-DPEase were 55 degrees C and pH 7.5-8.0, respectively, and the anchored DPEase exhibited high thermostability. Under optimal conditions, 30 g/L of recombinant spores can produce 85 g/L D-allulose from 500 g/L D-fructose after 12 h, and 60% of the yield was maintained after five cycles of utilization. Therefore, this biocatalyst system, capable of expressing and immobilizing DPEase on the spore surface of B. subtilis, was an appropriate alternative for D-allulose production.