An innovative method was developed for rapid sensitive detection and efficient structural characterization of lipopeptide biosurfactants by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry by using whole microbial cells and crude culture filtrates as targets in combination with surface tension measurements. This was done for a bacterial strain that was isolated from petroleum sludge and efficiently produces biosurfactants. This organism was identified by using biochemical, physiological, and genetic parameters as a Bacillus subtilis strain, designated B. subtilis C-1. This assignment was supported by a mass spectrometric investigation of the secondary metabolite spectrum determined by whole-cell MALDI-TOF mass spectrometry, which revealed three lipopeptide complexes, the surfactins, the iturins, and the fengycins, which are well-known biosurfactants produced by B. subtilis strains. These compounds were structurally characterized by in situ structure analysis by using postsource decay MALDI-TOF mass spectrometry. The isoforms were separated by miniaturized high-resolution reversed-phase high-performance liquid chromatography for mass spectrometric characterization. Iturin compounds which contain unusual fatty acid components were detected.Biosurfactants are a structurally diverse group of surfaceactive molecules synthesized by microorganisms (5,7,9,10,32,36). They have unique amphipathic properties derived from their complex structures, which include a hydrophilic moiety and a hydrophobic portion. The most efficient biosurfactants reduce the surface tension of water from 72 dynes/cm to values in the range of 25 to 30 dynes/cm. Biosurfactant production can be determined by measuring the change in surface tension of cell-free culture broth.Microbial surfactants have commonly been classified into the following categories: (i) glycolipids, (ii) lipopeptides, (iii) fatty acids, neutral lipids, and phospholipids, (iv) polymeric surfactants, and (v) particulate biosurfactants (5,7,9,10,32,36). The lipopeptides are an interesting class of microbial surfactants (36) because of their manifold attractive properties. Members of this group often possess antibiotic activity as well.Bacillus subtilis strains produce a broad spectrum of bioactive peptides with great potential for biotechnological and biopharmaceutical applications. A well-known class of such compounds includes the lipopeptides surfactin (1, 13, 14, 17, 18), fengycin (35), and the iturin compounds (3) (iturins [26], mycosubtilins [25], and bacillomycins [27]), which are amphiphilic membrane-active biosurfactants and peptide antibiotics with potent antimicrobial activities. All these agents occur as families of closely related isoforms which differ in the length and branching of the fatty acid side chains and in the amino acid substitutions in the peptide rings (20,36). The surfactin and iturin compounds are cyclic lipoheptapeptides which contain a ␤-hydroxy fatty acid and a ␤-amino fatty acid, respectively, as lipophilic co...

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOFMS) has been used for rapid and efficient typing of Bacillus subtilis strains according to their secondary metabolite products using whole intact cells as targets. Cells were grown either in liquid media or on agar plates, respectively. Strains were classified by their unique spectra of bioactive peptides in the m/z range from 500 to 5000. The lipopeptide products surfactin, fengycin and the members of the iturin family were structurally characterized in situ by post-source decay (PSD) analysis. The potential of MALDI-MS for taxonomic studies, as well as for the investigation of the regulation and compartimentalization of secondary metabolites in the bacterial cell, is demonstrated. These achievements qualify this methodology for rapid screening of microorganisms to exploit bioactive compounds for biotechnological and pharmaceutical applications. Figure 4. MALDI-TOF post-source decay mass spectrometric analysis of surfactin from intact cells of B. subtilis 21332. The parent ion at m/z 1061.1 corresponds to the Na adduct of surfactin with a C-15 fatty acid side chain. The b 1 -b 5 ions of the protonated fragments and the b* 1 -b* 6 ions of the Na-fragments are indicated in the spectrum.The insert in the spectrum shows the corresponding structure of surfactin. It is not possible to distinguish Ile and Leu by this technique.

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOFMS) has been used for rapid and efficient typing of Bacillus subtilis strains according to their secondary metabolite products using whole intact cells as targets. Cells were grown either in liquid media or on agar plates, respectively. Strains were classified by their unique spectra of bioactive peptides in the m/z range from 500 to 5000. The lipopeptide products surfactin, fengycin and the members of the iturin family were structurally characterized in situ by post-source decay (PSD) analysis. The potential of MALDI-MS for taxonomic studies, as well as for the investigation of the regulation and compartimentalization of secondary metabolites in the bacterial cell, is demonstrated. These achievements qualify this methodology for rapid screening of microorganisms to exploit bioactive compounds for biotechnological and pharmaceutical applications. Copyright # 1999 John Wiley & Sons, Ltd. Received 3 March 1999; Revised 22 March 1999; Accepted 23 March 1999Recently matrix-assisted laser desorption/ionization (MAL-DI) has been introduced as a novel technique for a rapid efficient typing of microorganisms 1-5 as well as for the characterization of eukaryotic cells. 6,7 This methodology is in many respects superior to classic techniques for taxonomic studies of microorganisms. It avoids laborious and time consuming sample preparation. Convincing advantages of MALDI-MS are its simplicity, its high sensitivity providing data in minutes and allowing detection in the pico-and even femtomole range, its relative ruggedness concerning complex mixtures containing high salt and detergents, and the potential for autosampling. Previous MALDI mass spectrometric fingerprinting of bacteria by species-and strain-specific biomarkers were mainly based on the identification of cell wall constituents 1 and specific protein patterns in the mass range up to approx. 40 kDa.2-5 Research performed so far demonstrates the ability of this technique to discriminate between pathogenic and non-pathogenic bacteria, 8 to detect food-borne pathogens, 3 to characterize the structure of cellular components and metabolites under in vivo conditions, 1,9 to profile biological cell reactions and to evaluate cellular productivity. 7 In addition, in the context of the increasing appearance of antibiotic resistance, MALDI-MS seems to be qualified for the fast and reliable screening for resistant strains of pathogens.

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