Abstract
A three-stage linear gradient strategy using reverse-phase high-performance liquid chromatography (HPLC) was optimized for rapid, high-quality, and simultaneous purification of the lipopeptide isoforms of iturin, fengycin, and surfactin, which may differ in composition by only a single amino acid and/or the fatty acid residue. Matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) was applied to detect the lipopeptides harvested from each reversed-phase HPLC peak. Amino acid analysis based on phenyl isothiocyanate derivatization was further used for confirmation of the amino acid species and molar ratio in a certain HPLC fraction. By this MALDI-TOF-MS/MS coupled with amino acid analysis, it was revealed that iturin at m/z 1,043 consists of a circular Asn-Tyr-Asn-Gln-Pro-Asn-Ser peptide and C14 β-OH fatty acid. Surfactin homologs from Bacillus subtilis THY-7 at m/z 1,030, 1,044, 1,058, and 1,072 possess a circular Glu-Leu-Leu-Val-Asp-Leu-Leu peptide and the β-OH fatty acid with a different length (C13–C16). Fengycin species at m/z 1,463 and 1,477 are homologs possessing the circular peptide Glu-Orn-Tyr-Thr-Glu-Ala-Pro-Gln-Tyr-Ile linked to a C16 or C17 γ-OH fatty acid, whereas fengycin at m/z 1,505 contains a Glu-Orn-Tyr-Thr-Glu-Val-Pro-Gln-Tyr-Ile sequence with a Val instead of Ala at position 6. The method developed in this work provided an efficient approach for characterization of diverse lipopeptide isoforms from the iturin, fengycin, and surfactin families.
Similar content being viewed by others
References
Liu XY, Yang SZ, Mu BZ (2009) Production and characterization of a C-15-surfactin-O-methyl ester by a lipopeptide producing strain Bacillus subtilis HSO121. Process Biochem 44(10):1144–1151. doi:10.1016/j.procbio.2009.06.014
Arima K, Kakinuma A, Tamura G (1968) Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation. Biochem Biophys Res Commun 31(3):488–494. doi:10.1016/0006-291x(68)90503-2
Chen H, Wang L, Su CX, Gong GH, Wang P, Yu ZL (2008) Isolation and characterization of lipopeptide antibiotics produced by Bacillus subtilis. Lett Appl Microbiol 47(3):180–186. doi:10.1111/j.1472-765X.2008.02412.x
Wakayama S, Ishikawa F, Oishi K (1984) Mycocerein, a novel antifungal peptide antibiotic produced by Bacillus cereus. Antimicrob Agents Chemother 26(6):939–940
Lee SC, Kim SH, Park IH, Chung SY, Choi YL (2007) Isolation and structural analysis of bamylocin A, novel lipopeptide from Bacillus amyloliquefaciens LP03 having antagonistic and crude oil-emulsifying activity. Arch Microbiol 188(4):307–312. doi:10.1007/s00203-007-0250-9
Desai JD, Banat IM (1997) Microbial production of surfactants and their commercial potential. Microbiol Mol Biol Rev 61(1):47–64
Banat IM, Makkar RS, Cameotra SS (2000) Potential commercial applications of microbial surfactants. Appl Microbiol Biotechnol 53(5):495–508
Das P, Mukherjee S, Sen R (2008) Antimicrobial potential of a lipopeptide biosurfactant derived from a marine Bacillus circulans. J Appl Microbiol 104(6):1675–1684. doi:10.1111/j.1365-2672.2007.03701.x
Hiradate S, Yoshida S, Sugie H, Yada H, Fujii Y (2002) Mulberry anthracnose antagonists (iturins) produced by Bacillus amyloliquefaciens RC-2. Phytochemistry 61(6):693–698. doi:10.1016/s0031-9422(02)00365-5
Yu GY, Sinclair JB, Hartman GL, Bertagnolli BL (2002) Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani. Soil Biol Biochem 34(7):955–963. doi:10.1016/s0038-0717(02)00027-5
Vanittanakom N, Loeffler W, Koch U, Jung G (1986) Fengycin - a novel antifungal lipopeptide antibiotic produced by Bacillus subtilis F-29-3. J Antibiot 39(7):888–901
Villegas-Escobar V, Ceballos I, Mira JJ, Argel LE, Peralta SO, Romero-Tabarez M (2013) Fengycin C Produced by Bacillus subtilis EA-CB0015. J Nat Prod 76(4):503–509. doi:10.1021/np300574v
Bonmatin JM, Laprevote O, Peypoux F (2003) Diversity among microbial cyclic lipopeptides: Iturins and surfactins. Activity-structure relationships to design new bioactive agents. Comb Chem High Throughput Screen 6(6):541–556
Hathout Y, Ho YP, Ryzhov V, Demirev P, Fenselau C (2000) Kurstakins: a new class of lipopeptides isolated from Bacillus thuringiensis. J Nat Prod 63(11):1492–1496. doi:10.1021/np000169q
Peypoux F, Pommier MT, Marion D, Ptak M, Das BC, Michel G (1986) Revised structure of mycosubtilin, a peptidolipid antibiotic from Bacillus subtilis. J Antibiot 39(5):636–641
Volpon L, Besson F, Lancelin JM (1999) NMR structure of active and inactive forms of the sterol-dependent antifungal antibiotic bacillomycin L. Eur J Biochem 264(1):200–210. doi:10.1046/j.1432-1327.1999.00605.x
Li YM, Yang SZ, Mu BZ (2010) The surfactin and lichenysin isoforms produced by Bacillus licheniformis HSN 221. Anal Lett 43(6):929–940. doi:10.1080/00032710903491047
Bonmatin JM, Labbe H, Grangemard I, Peypoux F, Magetdana R, Ptak M, Michel G (1995) Production, isolation and characterization of [Leu4]- and [Ile4]surfactins from Bacillus subtilis. Lett Pept Sci 2(1):41–47. doi:10.1007/bf00122922
Volpon L, Tsan P, Majer Z, Vass E, Hollosi M, Noguera V, Lancelin JM, Besson F (2007) NMR structure determination of a synthetic analogue of bacillomycin Lc reveals the strategic role of L-Asn1 in the natural iturinic antibiotics. Spectrochim Acta Part A 67(5):1374–1381. doi:10.1016/j.saa.2006.10.027
Vater J, Kablitz B, Wilde C, Franke P, Mehta N, Cameotra SS (2002) Matrix-assisted laser desorption ionization-time of flight mass spectrometry of lipopeptide biosurfactants in whole cells and culture filtrates of Bacillus subtilis C-1 isolated from petroleum sludge. Appl Environ Microbiol 68(12):6210–6219. doi:10.1128/aem. 68.12.6210-6219.2002
Sun LJ, Lu ZX, Bie XM, Lu FX, Yang SY (2006) Isolation and characterization of a co-producer of fengycins and surfactins, endophytic Bacillus amyloliquefaciens ES-2, from Scutellaria baicalensis Georgi. World J Microbiol Biotechnol 22(12):1259–1266. doi:10.1007/s11274-006-9170-0
Finking R, Marahiel MA (2004) Biosynthesis of nonribosomal peptides. Annu Rev Microbiol 58:453–488. doi:10.1146/annurev.micro.58.030603.123615
Marahiel MA (2009) Working outside the protein-synthesis rules: insights into non-ribosomal peptide synthesis. J Pept Sci 15(12):799–807. doi:10.1002/psc.1183
Chen CY, Baker SC, Darton RC (2006) Batch production of biosurfactant with foam fractionation. J Chem Technol Biotechnol 81(12):1923–1931. doi:10.1002/jctb.1625
Mata-Sandoval JC, Karns J, Torrents A (1999) High-performance liquid chromatography method for the characterization of rhamnolipid mixtures produced by Pseudomonas aeruginosa UG2 on corn oil. J Chromatogr A 864(2):211–220. doi:10.1016/s0021-9673(99)00979-6
Haddad NIA, Wang J, Mu BZ (2008) Isolation and characterization of a biosurfactant producing strain, Brevibacilis brevis HOB1. J Ind Microbiol Biotechnol 35(12):1597–1604. doi:10.1007/s10295-008-0403-0
Yuan J, Raza W, Huang QW, Shen QR (2011) Quantification of the antifungal lipopeptide iturin A by high performance liquid chromatography coupled with aqueous two-phase extraction. Jo Chromatogr B 879(26):2746–2750. doi:10.1016/j.jchromb.2011.07.041
Bidlingmeyer BA, Cohen SA, Tarvin TL (1984) Rapid analysis of amino acids using pre-column derivatization. J Chromatogr 336(1):93–104. doi:10.1016/s0378-4347(00)85133-6
Cohen SA, Bidlingmeyer BA, Tarvin TL (1986) PITC derivatives in amino acid analysis. Nature 320(6064):769–770. doi:10.1038/320769a0
Campanella L, Crescentini G, Avino P (1999) Simultaneous determination of cysteine, cystine and 18 other amino acids in various matrices by high-performance liquid chromatography. J Chromatogr A 833(2):137–145. doi:10.1016/s0021-9673(98)01023-1
Harvey DJ (2003) Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates and glycoconjugates. Int J Mass Spectrom 226(1):1–35. doi:10.1016/s1387-3806(02)00968-5
Sivapathasekaran C, Mukherjee S, Samanta R, Sen R (2009) High-performance liquid chromatography purification of biosurfactant isoforms produced by a marine bacterium. Anal Bioanal Chem 395(3):845–854. doi:10.1007/s00216-009-3023-2
Yanagi M, Yamasato K (1993) Phylogenetic analysis of the family Rhizobiaceae and related bacteria by sequencing of 16S rRNA gene using PCR and DNA sequencer. FEMS Microbiol Lett 107(1):115–120
Yang J, Sun L, Bai X, Zhou H (2002) Simultaneous determination of 18 amino acids by reversed-phase high performance liquid chromatography with precolumn phenylisothiocyanate derivatization. Se Pu 20(4):369–371
Liu X-Y, Yang S-Z, Mu B-Z (2008) Isolation and characterization of a C(12)-lipopeptide produced by Bacillus subtilis HSO 121. J Pept Sci 14(7):864–875. doi:10.1002/psc.1017
Zabet-Moghaddam M, Heinzle E, Tholey A (2004) Qualitative and quantitative analysis of low molecular weight compounds by ultraviolet matrix-assisted laser desorption/ionization mass spectrometry using ionic liquid matrices. Rapid Commun Mass Spectrom 18(2):141–148. doi:10.1002/rcm.1293
Harrison AG (2003) Fragmentation reactions of protonated peptides containing glutamine or glutamic acid. J Mass Spectrom 38(2):174–187. doi:10.1002/jms.427
Paizs B, Suhai S (2005) Fragmentation pathways of protonated peptides. Mass Spectrom Rev 24(4):508–548. doi:10.1002/mas.20024
Wysocki VH, Tsaprailis G, Smith LL, Breci LA (2000) Special feature: commentary - mobile and localized protons: a framework for understanding peptide dissociation. J Mass Spectrom 35(12):1399–1406. doi:10.1002/1096-9888(200012)35:12<1399::aid-jms86>3.0.co;2-r
Madonna AJ, Voorhees KJ, Taranenko NI, Laiko VV, Doroshenko VM (2003) Detection of cyclic lipopeptide biomarkers from Bacillus species using atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry. Anal Chem 75(7):1628–1637. doi:10.1021/ac020506v
Sen R (2008) Biotechnology in petroleum recovery: the microbial EOR. Prog Energy Combust 34(6):714–724. doi:10.1016/j.pecs.2008.05.001
Bachmann RT, Johnson AC, Edyvean RGJ (2014) Biotechnology in the petroleum industry: an overview. Int Biodeterior Biodegrad 86:225–237. doi:10.1016/j.ibiod.2013.09.011
Sachdev DP, Cameotra SS (2013) Biosurfactants in agriculture. Appl Microbiol Biotechnol 97(3):1005–1016. doi:10.1007/s00253-012-4641-8
Mandal SM, Barbosa A, Franco OL (2013) Lipopeptides in microbial infection control: scope and reality for industry. Biotechnol Adv 31(2):338–345. doi:10.1016/j.biotechadv.2013.01.004
Bockmuhl D (2012) Biosurfactants as antimicrobial ingredients for cleaning products and cosmetics. Tenside Surfactants Deterg 49(3):196–198
Whang LM, Liu PWG, Ma CC, Cheng SS (2009) Application of rhamnolipid and surfactin for enhanced diesel biodegradation-effects of pH and ammonium addition. J Hazard Mater 164(2–3):1045–1050. doi:10.1016/j.jhazmat.2008.09.006
Ongena M, Jacques P, Toure Y, Destain J, Jabrane A, Thonart P (2005) Involvement of fengycin-type lipopeptides in the multifaceted biocontrol potential of Bacillus subtilis. Appl Microbiol Biotechnol 69(1):29–38. doi:10.1007/s00253-005-1940-3
Ongena M, Jacques P (2008) Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends Microbiol 16(3):115–125. doi:10.1016/j.tim.2007.12.009
Kracht M, Rokos H, Ozel M, Kowall M, Pauli G, Vater J (1999) Antiviral and hemolytic activities of surfactin isoforms and their methyl ester derivatives. J Antibiot 52(7):613–619
Dufour S, Deleu M, Nott K, Wathelet B, Thonart P, Paquot M (2005) Hemolytic activity of new linear surfactin analogs in relation to their physico-chemical properties. Biochimi Biophys Acta 1726(1):87–95. doi:10.1016/j.bbagen.2005.06.015
Yang SZ, Wei DZ, Mu BZ (2006) Determination of the amino acid sequence in a cyclic lipopeptide using MS with DHT mechanism. J Biochem Biophys Methods 68(1):69–74. doi:10.1016/j.jbbm.2006.03.008
Kwanyuen P, Burton JW (2010) A modified amino acid analysis using PITC derivatization for soybeans with accurate determination of cysteine and half-cystine. J Am Oil Chem Soc 87(2):127–132. doi:10.1007/s11746-009-1484-2
Acknowledgments
This work was supported by the 973 National Key Basic Research Project (2013CB733600), the National Natural Science Foundation (no. 21176143), and the Tsinghua University Initiative Scientific Research Program (no. 20111081120).
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
ESM 1
(PDF 435 kb)
Rights and permissions
About this article
Cite this article
Yang, H., Li, X., Li, X. et al. Identification of lipopeptide isoforms by MALDI-TOF-MS/MS based on the simultaneous purification of iturin, fengycin, and surfactin by RP-HPLC. Anal Bioanal Chem 407, 2529–2542 (2015). https://doi.org/10.1007/s00216-015-8486-8
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-015-8486-8