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Bacillus subtilis Spore Surface Display of Haloalkane Dehalogenase DhaA

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Abstract

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.

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Acknowledgements

This work is supported by the State Key Laboratory of NBC Protection for Civilian (SKLNBC2013-05). The authors are grateful to Prof. Jing Yuan at the University of Delaware for the plasmid pCEL15.

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Authors and Affiliations

  1. Research Institute of Chemical Defense, Academy of Military Sciences, Beijing, 102205, People’s Republic of China

    Fuli Wang, Tianyu Song, Hui Jiang, Chengxin Pei, Qibin Huang & Hailing Xi

  2. State Key Laboratory of NBC Protection for Civilian, Academy of Military Sciences, Beijing, 102205, People’s Republic of China

    Fuli Wang, Tianyu Song, Hui Jiang, Chengxin Pei, Qibin Huang & Hailing Xi

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  1. Fuli Wang
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Wang, F., Song, T., Jiang, H. et al. Bacillus subtilis Spore Surface Display of Haloalkane Dehalogenase DhaA. Curr Microbiol 76, 1161–1167 (2019). https://doi.org/10.1007/s00284-019-01723-7

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