Bacillus subtilis Spore Surface Display of Haloalkane Dehalogenase DhaA

  • Fuli Wang
  • Tianyu Song
  • Hui Jiang
  • Chengxin Pei
  • Qibin Huang
  • Hailing XiEmail author


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.



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.

Compliance with Ethical Standards

Conflict of interest

The authors have no conflicts of interest to declare.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Fuli Wang
    • 1
    • 2
  • Tianyu Song
    • 1
    • 2
  • Hui Jiang
    • 1
    • 2
  • Chengxin Pei
    • 1
    • 2
  • Qibin Huang
    • 1
    • 2
  • Hailing Xi
    • 1
    • 2
    Email author
  1. 1.Research Institute of Chemical DefenseAcademy of Military SciencesBeijingPeople’s Republic of China
  2. 2.State Key Laboratory of NBC Protection for CivilianAcademy of Military SciencesBeijingPeople’s Republic of China

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