3 Biotech

, 8:392 | Cite as

Fe(III)-based immobilized metal–affinity chromatography (IMAC) method for the separation of the catechol siderophore from Bacillus tequilensis CD36

  • Yunya Li
  • Wei Jiang
  • Ruijie Gao
  • Yujie Cai
  • Zhengbing Guan
  • Xiangru LiaoEmail author
Short Reports


Catechol siderophore plays an important role in microbial ecology, agriculture, and medicine, but its research is often limited by the difficulty in acquisition of it in large quantities. Based on evidence from the coordination chemistry and chemical biology, catechol siderophore could chelate Fe3+ with high affinity. Therefore, Fe(III)-based immobilized metal–affinity chromatography (IMAC) was applied to capture siderophore from the culture filtrate of Bacillus tequilensis CD36. The ethanol-precipitated sample and the separated sample from Fe(III)-based IMAC were analyzed by liquid chromatography–mass spectrometry. According to the result, the pure siderophore DHB-Gly-Thr could be extracted from the ethanol-precipitated sample. Compared with other purifications, Fe(III)-based IMAC was convenient and had fewer steps. In addition, it also reduced the use of toxic chemical solvents in some traditional extraction process, such as extraction and ion exchange chromatography. Fe(III)-based IMAC was successfully used in separation of the catechol siderophore from B. tequilensis CD36. The results revealed that Fe(III)-based IMAC was an efficient and environmentally friendly method for the separation and purification of catechol siderophore.


Fe(III)-based IMAC Catechol siderophore LC–MS Bacillus tequilensis 



This work was financially supported by the Collaborative Innovation Involving Production, Teaching and Research Funds of Jiangsu Province (BY2014023-28) and the Agricultural Support Project, Wuxi Science and Technology Development (CLE01N1310). This research was also funded by the Program of Marine Biological Resources Exploitation and Utilization of Science and Technology Innovation Team of Taizhou (no. 1302ky08).

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest.


  1. Ahire JJ, Patil KP, Chaudhari BL, Chincholkar SB (2011) Bacillus spp. of human origin: a potential siderophoregenic probiotic bacteria. Appl Biochem Biotechnol 164(3):386–400. CrossRefPubMedGoogle Scholar
  2. Barnouin KN, Hart SR, Thompson AJ, Okuyama M, Waterfield M, Cramer R (2005) Enhanced phosphopeptide isolation by Fe(III)-IMAC using 1,1,1,3,3,3-hexafluoroisopropanol. Proteomics 5(17):4376–4388. CrossRefPubMedGoogle Scholar
  3. Braich N, Codd R (2008) Immobilised metal affinity chromatography for the capture of hydroxamate-containing siderophores and other Fe(III)-binding metabolites directly from bacterial culture supernatants. Analyst 133(7):877–880. CrossRefPubMedGoogle Scholar
  4. Butler A (2005) Marine siderophores and microbial iron mobilization. Biometals 18(4):369–374. CrossRefPubMedGoogle Scholar
  5. Ding S, Xu D, Li B, Fan C, Zhang C (2010) Improvement of 31P NMR spectral resolution by 8-hydroxyquinoline precipitation of paramagnetic Fe and Mn in environmental samples. Environ Sci Technol 44(7):2555–2561. CrossRefPubMedGoogle Scholar
  6. Ejje N, Soe CZ, Gu J, Codd R (2013) The variable hydroxamic acid siderophore metabolome of the marine actinomycete Salinispora tropica CNB-440. Metallomics 5(11):1519. CrossRefPubMedGoogle Scholar
  7. Gaberc-Porekar V, Menart V (2001) Perspectives of immobilized-metal affinity chromatography. J Biochem Biophys Methods 49(1–3):335–360. CrossRefPubMedGoogle Scholar
  8. Gordon AS, Donat JR, Kango RA, Dyer BJ, Stuart LM (2000) Dissolved copper-complexing ligands in cultures of marine bacteria and estuarine water. Mar Chem 70(1–3):149–160. CrossRefGoogle Scholar
  9. Hart SR, Waterfield MD, Burlingame A, Cramer R (2002) Factors governing the solubilization of phosphopeptides retained on ferric NTA IMAC beads and their analysis by MALDI TOFMS. J Am Soc Mass Spectr 13(9):1042–1051. CrossRefGoogle Scholar
  10. Haupt K, Roy F, Vijayalakshmi MA (1996) Immobilized metal ion affinity capillary electrophoresis of proteins—a model for affinity capillary electrophoresis using soluble polymer-supported ligands. Anal Biochem 234(2):149–154. CrossRefPubMedGoogle Scholar
  11. He XM, Yuan BF, Feng YQ (2017) Facial synthesis of nickel(II)-immobilized carboxyl cotton chelator for purification of histidine-tagged proteins. J Chromatogr B 1043:122–127. CrossRefGoogle Scholar
  12. Hertlein G, Muller S, Garcia-Gonzalez E, Poppingal L, Sussmuth RD, Genersch E (2014) Production of the catechol type siderophore bacillibactin by the honey bee pathogen Paenibacillus larvae. PLoS One 9(9):12. CrossRefGoogle Scholar
  13. Li J, Liu S, Jiang Z, Sun C (2017) Catechol amide iron chelators produced by a mangrove-derived Bacillus subtilis. Tetrahedron 73(35):5245–5252. CrossRefGoogle Scholar
  14. Liu X, Yang GM, Guan DX, Ghosh P, Ma LQ (2015) Catecholate-siderophore produced by As-resistant bacterium effectively dissolved FeAsO4 and promoted Pteris vittata growth. Environ Pollut 206:376–381. CrossRefPubMedGoogle Scholar
  15. Miethke M, Klotz O, Linne U, May JJ, Beckering CL, Marahiel MA (2006) Ferri-bacillibactin uptake and hydrolysis in Bacillus subtilis. Mol Microbiol 61(6):1413–1427. CrossRefPubMedGoogle Scholar
  16. Nixon RL, Ross ARS (2016) Evaluation of immobilized metal-ion affinity chromatography and electrospray ionization tandem mass spectrometry for recovery and identification of copper(II)-binding ligands in seawater using the model ligand 8-hydroxyquinoline. Front Mar Sci 3:246. CrossRefGoogle Scholar
  17. Porath J, Carlsson J, Olsson I, Belfrage G (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258(5536):598–599. CrossRefPubMedGoogle Scholar
  18. Saha M, Sarkar S, Sarkar B, Sharma BK, Bhattacharjee S, Tribedi P (2016) Microbial siderophores and their potential applications: a review. Environ Sci Pollut R 23(5):3984–3999. CrossRefGoogle Scholar
  19. Simionato AVC, Silva-Stenico ME, Tsai SM, Carrilho E (2010) Evidences of siderophores synthesis by grapevine Xylella fastidiosa, causal agent of pierce’s disease, through instrumental approaches. J Braz Chem Soc 21(4):635–641. CrossRefGoogle Scholar
  20. Stintzi A, Barnes C, Xu L, Raymond KN (2000) Microbial iron transport via a siderophore shuttle: a membrane ion transport paradigm. Proc Natl Acad Sci USA 97(20):10691–10696. CrossRefPubMedGoogle Scholar
  21. Yu X, Ai C, Xin L, Zhou G (2011) The siderophore-producing bacterium, Bacillus subtilis CAS15, has a biocontrol effect on Fusarium wilt and promotes the growth of pepper. Eur J Soil Biol 47(2):138–145. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Yunya Li
    • 1
  • Wei Jiang
    • 1
  • Ruijie Gao
    • 1
  • Yujie Cai
    • 1
  • Zhengbing Guan
    • 1
  • Xiangru Liao
    • 1
    Email author
  1. 1.Key Laboratory of Industrial Biotechnology, Ministry of Education, School of BiotechnologyJiangnan UniversityWuxiChina

Personalised recommendations