Abstract
Bacillus amyloliquefaciens strain LP03 isolated from soil, produced an antagonistic compound that strongly inhibited the growth of plant-pathogenic fungi and a lipopeptide biosurfactant. Also, isolated strain LP03 had a marked crude oil-emulsifying activity as it developed a clear zone around the colony after incubation for 24 h at 37°C. LP03 was identified as Bacillus amyloliquefaciens by analysis of partial 16 S rRNA gene and partial gyrA gene sequence. The lipopeptide was purified by acid precipitation of cell-free culture broth, extraction of the precipitates with methanol, silica gel column chromatography, and reverse-phase, high-pressure liquid chromatography. The purified biosurfactant was analyzed biochemical structure by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and electrospray ionization mass spectrometry/mass spectrometry (ESI-MS/MS). The masses of the two peaks were observed by HPLC chromatography. Their masses were determined to be 1,044 and 1,058 m/z with MALDI-TOF mass spectrometry. As constituents of the peptide and lipophilic part of the m/z 1,022.6, seven amino acids (Glu-Leu-Met-Leu-Pro-Leu-Leu) and β-hydroxy-C13 fatty acid were determined by ESI-MS/MS. The lipopeptide of 1,022.6 Da differed from surfactins in the substitution of leucine, valine and aspartic acid in positions 3, 4, and 5 by methionine, leucine, and proline, respectively. Novel lipopeptide was designated as bamylocin A.
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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 31:488–494
Banat IM (1995) Characterization of biosurfactants and their use in pollution removal-state of the art. Acta Biotechnol 15:251–267
Banat IM, Makkar RS, Cameotra SS (2000) Potential applications of microbial surfactants. Appl Microbiol Biotechnol 53:495–508
Chun J, Bae KS (2000) Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA gene sequence. Antonie van Leeuwenhoek 78:123–127
Chiou AL, Wu WS (2003) Formulation of Bacillus amyloliquefaciens B190 for Control of Lily Grey Mould (Botrytis elliptica). J Phytopathol 151:13–18
Cutting SM, Vander HPB (1990) Genetic analysis. In: Harwood CR, Cutting SM (eds) Molecular biological methods for bacillus. Wiley, Chichester, pp 27–74
Itokawa H, Miyashita T, Morita H, Takeya K, Hirano T, Homma M, Oka K (1994) Structural and conformational studies of [Ile7] and [Leu7] surfactins from Bacillus subtilis natto. Chem Pharm Bull 42:604–607
Kalinovskaya NI, Kuznetsova TA, Ivanova EP, Romanenko LA, Voinov VG, Huth F, Laatsch H (2002) Characterization of surfactin-like cyclic depsipeptides synthesized by Bacillus pumilus from Ascidian Halocynthia aurantium. Mar Biotechnol 4:179–188
Kim HS, Lee CH, Suh HH, Ahn KH, Oh HM, Kwon GS, Yang JW, Yoon BD (1997) A lipopeptide biosurfactant produced by Bacillus subtilis C9 selected through the Oil film-collapsing assay. J Microbiol Biotechnol 7:180–188
Kretschner A, Bock H, Wagnee F (1982) Chemical and physical characterization of interfacial-active lipids from Rhodococcus erythropolis grown on n-alkane. Appl Environ Microbiol 44:864–870
Magaritis A, Kennedy K, Zajic JE, Gerson DF (1979) Biosurfactant production by Nocardia erythropolis. Dev Ind Microbiol 20:623–630
Nakayama S, Takahashi S, Hirai M, Shoda M (1997) Isolation of new variants of surfactin by a recombinant Bacillus subtilis. Appl Microbiol Biotechnol 48:80–82
Naruse N, Tenmyo O, Kobaru S, Kamei H, Miyaki T, Konishi M, Oki T (1990) Pumilacidin, a complex of new antiviral antibiotics: production, isolation, chemical properties, structure and biological activity. J Antibiot 43:267–280
Noudeh GD, Housaindokht M, Bazzaz BSF (2005) Isolation, characterization, and investigation of surface and hemolytic activities of a lipopeptide biosurfactant produced by Bacillus subtilis ATCC 6633. J Microbiol 43:272–276
Peypoux F, Bonmatin JM, Labbe H, Das B, Ptak M, Michel G (1991) Isolation and characterization of a new variant of surfactin, the [Val7]surfactin. Eur J Biochem 202:101–106
Ron EZ, Rosenberg E (2001) Natural roles of biosurfactant. Environ Microbiol 3:229–236
Roongsawang N, Thaniyavarn J, Thaniyavarn S, Kameyama T, Haruki M, Imanaka T, Moriawa M, Kanaya S (2002) Isolation and characterization of a halotolerant Bacillus subtilis BBK-1 which produces three kinds of lipopeptides: bacillomycin L, plipastatin, and surfactin. Estremophiles 6:499–506
Rosenberg E, Ron EZ (1999) High- and low-molecular-mass microbial surfactants. Appl Microbiol Biotechnol 52:154–162
Steller S, Vater J (2000) Purification of the fengycin synthetase multienzyme system from Bacillus subtilis b213. J Chromatogr B 737:267–275
Sun L, Lu Z, Bie X, Lu F, Yang S (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. doi:10.1007/s11274-006-9170-0
Symmank H, Franke P, Saenger W, Bernhard F (2002) Modification of biologically activepeptides: production of novel lipohexapeptide after engineering of Bacillus subtilis surfactin synthetase. Protein Eng 15:913–921
Trischman JA, Jensen PR, Fenical W (1994) Halobacillin: a cytotoxic cyclic acylpeptide of the iturin class produced by a marine Bacillus. Tetrahedron Lett 35:5571–5574
Tsuge K, Ano T, Shoda M (1996) Isolation of a gene essential for biosynthesis of the lipopeptide antibiotics plipastatin B1 and surfactin in Bacillus subtilis. YB8 165:243–251
Vater J, Kablitz B, Wilde C, Franke P, Mehta N, Cameotra SS (2002) Matrix-assisted laser desertion 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 68:6210–6219
Yakimov MM, Timmis KN, Wray V, Fredrickson HL (1995) Characterization of a new lipopeptide surfactant produced by thermotolerant and halotolerant subsurface Bacillus licheniformis BAS50. Appl Environ Microbiol 61:1706–1713
Yu GY, Sinclair JB, Hartman GL, Bertagnolli BL (2002) Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani. Soil Biol Biochem 34:955–963
Zajic JE, Guignard H, Gerson DF (1977) Properties and biodegradation of a bioemulsifier from Corynebacterium hydrocarboclatus. Biotechnol Bioeng 19:1303–1302
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This paper was partially supported by the Dong-a University Research Fund in 2006, Sang-cheol Lee, Sun-Hee Kim, and In-Hye Park was supported by Brain Korea 21 project.
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Communicated by Erko Stackebrandt.
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Lee, SC., Kim, SH., Park, IH. et al. Isolation and structural analysis of bamylocin A, novel lipopeptide from Bacillus amyloliquefaciens LP03 having antagonistic and crude oil-emulsifying activity. Arch Microbiol 188, 307–312 (2007). https://doi.org/10.1007/s00203-007-0250-9
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DOI: https://doi.org/10.1007/s00203-007-0250-9