Expression and secretion of active Moringa oleifera coagulant protein in Bacillus subtilis

  • Clint ArnettEmail author
  • Justin Lange
  • Ashley Boyd
  • Martin Page
  • Donald Cropek
Biotechnological products and process engineering


Cationic polypeptide proteins found in the seeds of the tropical plant Moringa oleifera have coagulation efficiencies similar to aluminum and ferric sulfates without their recalcitrant nature. Although these proteins possess great potential to augment or replace traditional coagulants in water treatment, harvesting active protein from seeds is laborious and not cost-effective. Here, we describe an alternative method to express and secrete active M. oleifera coagulant protein (MO) in Bacillus subtilis. A plasmid library containing the MO gene and 173 different types of secretory signal peptides was created and cloned into B. subtilis strain RIK1285. Fourteen of 440 clones screened were capable of secreting MO with yields ranging from 55 to 122 mg/L of growth medium. The coagulant activity of the highest MO secreting clone was evaluated when grown on Luria broth, and cell-free medium from the culture was shown to reduce turbidity in a buffered kaolin suspension by approximately 90% compared with controls without the MO gene. The clone was also capable of secreting active MO when grown on a defined synthetic wastewater supplemented with 0.5% tryptone. Cell-free medium from the strain harboring the MO gene demonstrated more than a 2-fold reduction in turbidity compared with controls. Additionally, no significant amount of MO was observed without the addition of the synthetic wastewater, suggesting that it served as a source of nutrients for the effective expression and translocation of MO into the medium.


Bacillus subtilis Biocoagulant Coagulant protein Moringa oleifera Water treatment Water turbidity 



This research was supported in part by an appointment at the Research Participation Program administered by the Oak Ridge Institute for Science and Education through a cooperative agreement between the US Department of Energy and the US Army Construction Engineering Research Laboratory. Although this research was sponsored by the Department of Defense, the content of the information does not necessary reflect the position or policy of the government and no official endorsement should be inferred.

Compliance with ethical standards

The authors of this article did not perform any studies with animals or human subjects.

Conflict of interest

The authors declare that they have no conflicts of interest.


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Copyright information

© This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2019

Authors and Affiliations

  • Clint Arnett
    • 1
    Email author
  • Justin Lange
    • 2
  • Ashley Boyd
    • 1
  • Martin Page
    • 1
  • Donald Cropek
    • 1
  1. 1.Construction Engineering Research Laboratory (CERL)U.S. Army Engineer Research and Development Center (ERDC)ChampaignUSA
  2. 2.Oak Ridge Associated Universities (ORAU)Oak Ridge Institute for Science and Education (ORISE)Oak RidgeUSA

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