Bacillus subtilis Spore Surface Display of Haloalkane Dehalogenase DhaA

The haloalkane dehalogenase DhaA can degrade sulfur mustard (2,2'-dichlorethyl sulfide; also known by its military designation HD) in a rapid and environmentally safe manner. However, DhaA is sensitive to temperature and pH, which limits its applications in natural or harsh environments. Spore surface display technology using resistant spores as a carrier to ensure enzymatic activity can reduce production costs and extend the range of applications of DhaA. To this end, we cloned recombinant Bacillus subtilis spores pHY300PLK-cotg-dhaa-6his/DB104(FH01) for the delivery of DhaA from Rhodococcus rhodochrous NCIMB 13064. A dot blotting showed that the fusion protein CotG-linker-DhaA accounted for 0.41% ± 0.03% (P < 0.01) of total spore coat proteins. Immunofluorescence analyses confirmed that DhaA was displayed on the spore surface. The hydrolyzing activity of DhaA displayed on spores towards the HD analog 2-chloroethyl ethylsulfide was 1.74 ± 0.06 U/mL (P < 0.01), with a specific activity was 0.34 ± 0.04 U/mg (P < 0.01). This is the first demonstration that DhaA displayed on the surface of B. subtilis spores retains enzymatic activity, which suggests that it can be used effectively in real-world applications including bioremediation of contaminated environments.