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Analysis of free amino acids during fermentation by Bacillus subtilis using capillary electrophoresis

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Abstract

A high performance capillary electrophoresis (HPCE) method was presented to identify and quantitate free amino acids during fermentation by Bacillus subtilis. Amino acids, pre-column derivatized with phenylisothicyanate, were separated and characterized by HPCE. In order to optimize separation conditions, the assay was developed by varying the β-cyclodextrin concentration and pH of the background electrolyte. A buffer system comprising 30 mM phosphate and 3 mM β-cyclodextrin at pH 7.0, voltage of 20 kV and detection wavelength of 254 nm showed the best results, with 17 out of 20 phenylthioncarbamyl amino acids in a solution adequately separated. For quantification, p-aminobenzoic acid was added as an internal standard. Analysis of free amino acids in Bacillus subtilis culture medium using this method revealed good consistency with the values obtained using conventional ninhydrin-based amino acid analyzer. Four free amino acids (aspartic acid, glutamic acid, proline, and tyrosine) concentration in an extracellular matrix during fermentation by Bacillus subtilis were mainly monitored using this method.

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References

  1. Ajesh, K. and K. Sreejith (2009) Peptide antibiotics: An alternative and effective antimicrobial strategy to circumvent fungal infections. Peptides. 30: 999–1006.

    Article  CAS  Google Scholar 

  2. Kim, P. I., J. Ryu, Y. H. Kim, and Y. T. Chl (2010) Production of biosurfactant lipopeptides Iturin A, Fengycin, and Surfactin A from Bacillus subtilis CMB32 for control of colletotrichum gloeosporioides. J. Microbiol. Biotechnol. 20: 138–145.

    CAS  Google Scholar 

  3. Oleinikova, G. K., T. A. Kuznetsova, F. Huth, H. Laatsch, V. V. Isakov, L. S. Shevchenko, and G. B. Elyakov (2001) Cyclic lipopeptides with fungicidal activityfrom the sea isolate of the bacterium Bacillus subtilis. Russ. Chem. Bull. 50: 2231–2235.

    Article  CAS  Google Scholar 

  4. Cho, S. J., S. K. Lee, B. J. Cha, Y. H. Kim, and K. S. Shin (2003) Detection and characterization of the Gloeosporium gloeosporioides growth inhibitory compound iturin A from Bacillus subtilis strain KS03. FEMS Microbiol. Lett. 223: 47–51.

    Article  CAS  Google Scholar 

  5. Dunlap, C. A., D. A. Schisler, N. P. Price, and S. F. Vaughn (2011) Cyclic lipopeptide profile of three bacillus subtilis strains; Antagonists of fusarium head blight. J. Microbiol. 49: 603–609.

    Article  CAS  Google Scholar 

  6. Besson, F., M. L. Hourdou, and G. Michel (1990) Studies on the biosyntheses of iturin, an antibiotic of bacillus-subtilis, and a lipopeptide containing beta-hydroxy fatty-acids. Biochim. Biophys. Acta. 1036: 101–106.

    Article  CAS  Google Scholar 

  7. Hourdou, M. L., F. Besson, and G. Michel (1988) Studies on the biosynthesis of beta-amino acids, the lipid moiety of iturins A, in Bacillus subtilis. J. Antibiot. 41: 207–211.

    Article  CAS  Google Scholar 

  8. Iwase, N., M. Rahman, and T. Ano (2009) Production of iturin A homologues under different culture conditions. J. Environ. Sci. 21: 28–32.

    Article  Google Scholar 

  9. Kaspar, H., K. Dettmer, Q. Chan, S. Daniels, S. Nimkar, M. L. Daviglus, J. Stamler, P. Elliott, and P. J. Oefner (2009) Urinary amino acid analysis: A comparison of iTRAQ (R)-LC-MS/MS, GC-MS, and amino acid analyzer. J. Chromatogr. B. 877: 1838–1846.

    Article  CAS  Google Scholar 

  10. Bernal, J. L., M. J. Nozal, L. Toribio, J. C. Diego, and A. Ruiz (2005) A comparative study of several HPLC methods for determining free amino acid profiles in honey. J. Sep. Sci. 28: 1039–1047.

    Article  CAS  Google Scholar 

  11. Herbert, P., P. Barros, N. Ratola, and A. Alves (2000) HPLC determination of amino acids in musts and port wine using OPA/ FMOC derivatives. J. Food Sci. 65: 1130–1133.

    Article  CAS  Google Scholar 

  12. Senden, M., A. Van Der Meer, J. Limborgh, and H. Wolterbeek (1992) Analysis of major tomato xylem organic acids and PITCderivatives of amino acids by RP-HPLC and UV detection. Plant Soil. 142: 81–89.

    CAS  Google Scholar 

  13. Callejon, R. M., W. Tesfaye, M. J. Torija, A. Mas, A. M. Troncoso, and M. L. Morales (2008) HPLC determination of amino acids with AQC derivatization in vinegars along submerged and surface acetifications and its relation to the microbiota. Eur. Food Res. Technol. 227: 93–102.

    Article  CAS  Google Scholar 

  14. Aoyama, C., T. Santa, M. Tsunoda, T. Fukushima, C. Kitada, and K. Imai (2004) A fully automated amino acid analyzer using NBD-F as a uorescent derivatization reagent. Biomed. Chromatogr. 18: 630–636.

    Article  CAS  Google Scholar 

  15. Chen, F., S. Wang, W. Guo, and M. Hu (2005) Determination of amino acids in Sargassum fusiforme by high performance capillary electrophoresis. Talanta. 66: 755–761.

    Article  CAS  Google Scholar 

  16. Dube, S., M. Khumalo, N. Torto, and J. Nyati (2006) Characterization of amino acids in silk sericin protein from Gonometa rufobrunnae by MEKC with phenyl isothiocyanate derivatization. J. Sep. Sci. 29: 1245–1250.

    Article  CAS  Google Scholar 

  17. Ptolemy, A., L. Tran, and P. Britz-McKibbin (2006) Single-step enantioselective amino acid flux analysis by capillary electrophoresis using on-line sample preconcentration with chemical derivatization. Anal. Biochem. 354: 192–204.

    Article  CAS  Google Scholar 

  18. Imai, K., H. Matsunaga, T. Santa, and H. Homma (1995) Availability of phenylisothiocyanate for the amino-acid-sequence configuration determination of peptides containing D/L-amino acids. Biomed. Chromatogr. 9: 195–196.

    Article  CAS  Google Scholar 

  19. Fisher, G. H., I. Arias, I. Quesada, S. D’Aniello, F. Errico, M. M. Di Fiore, and A. D’Aniello (2001) A fast and sensitive method for measuring picomole levels of total free amino acids in very small amounts of biological tissues. Amino Acids. 20: 163–173.

    Article  CAS  Google Scholar 

  20. Negro, A., S. Garbisa, L. Gotte, and M. Spina (1987) The use of reverse-phase high-performance liquid-chromatography and precolumn derivatization with dansyl chloride for quantitation of specific amino-acids in collagen and elastin. Anal. Biochem. 160: 39–46.

    Article  CAS  Google Scholar 

  21. Herrero, M., E. Ibanez, S. Fanali, and A. Cifuentes (2007) Quantitation of chiral amino acids from microalgae by MEKC and LIF detection. Electrophoresis. 28: 2701–2709.

    Article  CAS  Google Scholar 

  22. Edman, P. (1956) On the mechanism of the phenyl isothiocyanate degradation of peptides. Acta Chem. Scand. 10: 761–768.

    Article  Google Scholar 

  23. Tarr, G. E. (1977) Improved manual sequencing methods. Methods Enzymol. 47: 335–357.

    Article  CAS  Google Scholar 

  24. Jing, Y., L. G. Sun, X. Z. Bai, and H. T. Zhou (2002) Simultaneous determination of 18 amino acids by reversed-phase high performance liquid chromatography with precolumn phenylisothiocyanate derivatization. Chromatography. 20: 369–371.

    Google Scholar 

  25. Zhang, X. Y., J. Y. Zhou, W. Fu, Z. D. Li, J. A. Zhong, J. Yang, L. A. Xiao, and H. Tan (2010) Response surface methodology used for statistical optimization of jiean-peptide production by Bacillus subtilis. Electron. J. Biotechnol. 13. doi: 10.2225/vol13-issue4-fulltext-5.

  26. Lin, G., K. Y. Shen, W. J. Zhao, and J. C. Jiang (2007) Determination of the metabolism of amino acids during fermentation of bacitracin by capillary electrophoresis. Chin. J. Pharm. 38: 515–518.

    CAS  Google Scholar 

  27. Komarova, N., J. Kamentsev, A. Solomonova, and R. Anufrieva (2004) Determination of amino acids in fodders and raw materials using capillary zone electrophoresis. J. Chromatogr. B. 800: 135–143.

    Article  CAS  Google Scholar 

  28. Zahou, E., H. Jornvall, and T. Bergman (2000) Amino acid analysis by capillary electrophoresis after phenylthiocarbamylation. Anal. Biochem. 281: 115–122.

    Article  CAS  Google Scholar 

  29. Zhang, L. Y. and M. X. Sun (2009) Fast determination of glutathione by capillary electrophoresis with fluorescence detection using beta-cyclodextrin as modifier. J. Chromatogr. B. 877: 4051–4054.

    Article  CAS  Google Scholar 

  30. Lee, Y. H. and T. I. Lin (1995) Capillary electrophoretic determination of amino-acids — improvement by cyclodextrin additives. J. Chromatogr. A. 716: 335–346.

    Article  CAS  Google Scholar 

  31. Shen, Z., Z. Sun, L. Wu, K. Wu, S. Sun, and Z. Huang (2002) Rapid method for the determination of amino acids in serum by capillary electrophoresis. J. Chromatogr. A. 979: 227–232.

    Article  CAS  Google Scholar 

  32. Ummadi, M. and B. Weimer (2002) Use of capillary electrophoresis and laser-induced fluorescence for attomole detection of amino acids. J. Chromatogr. A. 964: 243–253.

    Article  CAS  Google Scholar 

  33. Thormann, W., F. Prost, and A. Prochazkova (2002) Capillary electrophoresis with (R)-(-)-N-(3,5-dinitrobenzoyl)-alpha-phenylglycine as chiral selector for separation of albendazole sulfoxide enantiomers and their analysis in human plasma. J. Pharm. Biomed. Anal. 27: 555–567.

    Article  CAS  Google Scholar 

  34. Parsons, H. M., D. R. Ekman, T. W. Collette, and M. R. Viant (2009) Spectral relative standard deviation: A practical benchmark in metabolomics. Analyst. 134: 478–485.

    Article  CAS  Google Scholar 

  35. López-Cervantes, J., D. Sánchez-Machado, and J. Rosas-Rodríguez (2006) Analysis of free amino acids in fermented shrimp waste by high-performance liquid chromatography. J. Chromatogr. A. 1105: 106–110.

    Article  Google Scholar 

  36. Arrebola, E., R. Jacobs, and L. Korsten (2010) Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens. J. Appl. Microbiol. 108: 386–395.

    Article  CAS  Google Scholar 

  37. Valles, E. S., N. P. Garcia, R. R. Madrera, and A. P. Lobo (2005) Influence of yeast strain and aging time on free amino acid changes in sparkling ciders. J. Agr. Food Chem. 53: 6408–6413.

    Article  Google Scholar 

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Authors and Affiliations

  1. Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, 610-041, China

    Yanli Ren, Jinyan Zhou, Xiaoyong Zhang, Zhidong Li, Juan Zhong, Jie Yang, Tan Xu & Hong Tan

  2. The Key Laboratory of Environmental and Applied Microbiology, Chinese Academy of Sciences, Chengdu, 610-041, China

    Yanli Ren, Jinyan Zhou, Xiaoyong Zhang, Zhidong Li, Juan Zhong, Jie Yang, Tan Xu & Hong Tan

  3. The Key Laboratory of Environmental Microbiology, Sichuan Province, Chengdu, 610-041, China

    Yanli Ren, Jinyan Zhou, Xiaoyong Zhang, Zhidong Li, Juan Zhong, Jie Yang, Tan Xu & Hong Tan

  4. Graduate University of Chinese Academy of Sciences, Beijing, 100-049, China

    Yanli Ren

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  1. Yanli Ren
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Correspondence to Hong Tan.

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Ren, Y., Zhou, J., Zhang, X. et al. Analysis of free amino acids during fermentation by Bacillus subtilis using capillary electrophoresis. Biotechnol Bioproc E 17, 1244–1251 (2012). https://doi.org/10.1007/s12257-012-0292-y

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  • DOI: https://doi.org/10.1007/s12257-012-0292-y

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