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High-Frequency Occurrence of Surfactin Monomethyl Isoforms in the Ferment Broth of a Bacillus Subtilis Strain Revealed by Ion Trap Mass Spectrometry

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High-Frequency Occurrence of Surfactin Monomethyl Isoforms in the Ferment Broth of a Bacillus Subtilis Strain Revealed by Ion Trap Mass Spectrometry molecules Article High-Frequency Occurrence of Surfactin Monomethyl Isoforms in the Ferment Broth of a Bacillus subtilis Strain Revealed by Ion Trap Mass Spectrometry Anita Kecskeméti 1, Attila Bartal 1, Bettina Bóka 1,László Kredics 1 ID ,László Manczinger 1, Kadaikunnan Shine 2, Naiyf S. Alharby 2, Jamal M. Khaled 2,Mónika Varga 1, Csaba Vágvölgyi 1,2 and András Szekeres 1,* 1 Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Közép fasor 52, H-6726 Szeged, Hungary; [email protected] (A.K.); [email protected] (A.B.); [email protected] (B.B.); [email protected] (L.K.); [email protected] (L.M.); [email protected] (M.V.); [email protected] (C.V.) 2 Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia; [email protected] (K.S.); [email protected] (N.S.A.); [email protected] (J.M.K.) * Correspondence: [email protected]; Tel.: +36-62-544-516 Received: 23 July 2018; Accepted: 30 August 2018; Published: 1 September 2018 Abstract: Surfactins are cyclic lipopeptides consisting of a β-hydroxy fatty acid of various chain length and a peptide ring of seven amino acids linked together by a lactone bridge, forming the cyclic structure of the peptide chain. These compounds are produced mainly by Bacillus species and possess numerous biological effects such as antimicrobial (antiviral, antibacterial, and antifungal) activities. A mixture of surfactins extracted from Bacillus subtilis strain SZMC 6179J was examined by HPLC-ESI-IT-MS technique, enhancing their separation to reveal novel lipopeptide varieties with higher masses and to characterize their structures. During the MS2 spectra analyses of their sodiated molecular ions [M + Na]+, a previously rarely encountered group of surfactins was detected and two novel types of the group were discovered containing methyl esterified aspartic acid residue in their fifth amino acid position. The relative amounts of these monomethyl isoforms exceeded 35% of the produced surfactin in total. In the m/z value of 1114, all the detected isoforms possessed aspartic acid 4-methyl ester residue in their fifth amino acid position (C17-[Lxx4, AME5], C18-[AME5]), offering an opportunity to separate a pure fraction of the compound and to study its biological activities in the future. Keywords: Bacillus; cyclic lipopeptides; HPLC-ESI-IT-MS; surfactin 1. Introduction Surfactins are cyclic lipopeptide-type biosurfactants produced mainly by Bacillus subtilis, a Gram-positive bacterium [1]. It consists of a hydrophobic β-hydroxy fatty acid chain of various length (C12–C15) linked to a ring of seven amino acids (Figure1). The cyclic structure is formed by a lactone bridge between the β-hydroxyl functional group of the fatty acid and the C-terminal of the heptapeptide [2]. Because of its potent surface activity properties [2], surfactin exhibits various biological activities such as antiviral, antitumor, and antimicrobial effects [3], which makes this heptapeptide the subject of various environmental, industrial, and therapeutical investigations [4,5]. The length of the fatty acid chain and the amino acid sequence varies, resulting in numerous isoforms of the compound listed and characterized in previous works [2] (Table1). Although most of these variants were reported in the 1990s, a recent study from our group introducing a novel surfactin Molecules 2018, 23, 2224; doi:10.3390/molecules23092224 www.mdpi.com/journal/molecules MoleculesMolecules 20182018, ,2323, ,x 2224 FOR PEER REVIEW 22 of of 12 12 variants were reported in the 1990s, a recent study from our group introducing a novel surfactin moleculemolecule containing containing Val Val in in the the second second amino amino acid acid position position [6] [6 ](Figure (Figure 1)1) proved that there may still bebe surfactin surfactin isoforms isoforms that that are are yet yet to to be be discovered. discovered. Moreover, Moreover, the the incorporations incorporations of esterified of esterified form form of Aspof Asp (Asp (Asp--O-Me,O-Me, aspartic aspartic acid acid 4-methyl 4-methyl est ester—AME)er—AME) and and Glu Glu (Glu (Glu--OO-Me,-Me, glutamic glutamic acid acid 5 5-methyl-methyl esterester—GME)—GME) were were also also reported reported occasionally occasionally [7 [7––9].9]. In In the the present present study, study, a ann HPLC HPLC gradient gradient elution elution methodmethod was was applied applied that that was was capable capable of of separating separating all all lipopeptide lipopeptide components components in in our our sample sample and and to to 2 examineexamine the the separated separated,, previously hidden,hidden, moremore non-polarnon-polar fractions fractions by by MS MS2measurements measurements in in order order to toidentify identify the the various various surfactin surfactin molecules, molecules, with with different different amino amino acid acid sequences sequences and aliphaticand aliphatic acid chainacid chainlengths, lengths, produced produced by B. by subtilis B. subtilisSZMC SZMC 6179J 6179J to the to full the extent. full extent. Figure 1. The structure of the surfactin variant [Val2] described by Bóka et al. [6]. R = C3H7–C5H11. Figure 1. The structure of the surfactin variant [Val2] described by Bóka et al. [6]. R = C3H7–C5H11. Table 1. Natural surfactin peptide sequence variants including the recently found isoforms containing Table 1. Natural surfactin peptide sequence variants including the recently found isoforms containing AMEAME in in the the fifth fifth position. position. Peptide Nomenclature of Surfactin References AA1 AA2 AA3 PeptideAA4 AA5 AA6 AA7 Nomenclature of Surfactin References [Sur] surfactin Glu Leu Leu Val Asp Leu Leu [10] AA1 AA2 AA3 AA4 AA5 AA6 AA7 [Val2] surfactin Glu Val Lxx Val Asp Lxx Lxx [6] [Ala4] surfactin[Sur] surfactin Glu Glu Leu Leu Leu ValAla AspAsp LeuLeu LeuLeu [10] [11] [Leu4] surfactin[Val2] surfactin Glu Glu Leu Val Leu Lxx ValLeu AspAsp LxxLeu LxxLeu [6] [12] [Ile4] surfactin[Ala4] surfactinGlu Glu Leu Leu Leu AlaIle AspAsp LeuLeu LeuLeu [11] [12] [Ile2,4] surfactin[Leu4] surfactin Glu Glu Ile Leu Leu LeuIle AspAsp LeuLeu LeuLeu [12] [13] [Val7] surfactin[Ile4] surfactin Glu Glu Leu Leu Leu IleVal AspAsp LeuLeu LeuVal [12][14,15] [Ile7] surfactin[Ile2,4] surfactinGlu Glu Leu Ile Leu IleVal AspAsp LeuLeu LeuIle [13] [14] [Val2,7] [Val7]surfactin surfactin Glu Glu Val Leu Leu ValVal AspAsp LeuLeu ValVal [14,15][13] [Val2, Ile7][Ile7] surfactin surfactin Glu Glu Val Leu Leu ValVal AspAsp LeuLeu IleIle [14] [16] [Ile2, Val7][Val2,7] surfactin surfactin Glu Glu Ile Val Leu ValVal AspAsp LeuLeu ValVal [13] [16] [Ile2,4,7][Val2, surfactin Ile7] surfactinGlu Glu Ile Val Leu ValIle AspAsp LeuLeu IleIle [16] [13] [GME1,AME5][Ile2, Val7] surfactin surfactin GME Glu Leu Ile Leu ValVal AspAME LeuLeu ValLeu [16] [7] [GME1,Val7][Ile2,4,7] surfactin surfactin GME Glu Leu Ile Leu IleVal AspAsp LeuLeu IleVal [13] [7] [GME1, [GME1,AME5]Ile7] surfactin surfactinGME GME Leu Leu Leu ValVal AMEAsp LeuLeu LeuIle [7] [7] [AME5] [GME1,Val7]surfactin surfactinGlu GME Leu Leu Leu ValVal AspAME LeuLeu ValLeu [7] [8] [AME5,Val7][GME1, surfactin Ile7] surfactinGlu GME Lxx Leu Lxx Leu ValVal AspAME LeuLxx IleVal [7This] study [Lxx4, AME5][AME5] surfactin surfactin Glu Glu Lxx Leu Lxx Leu ValLxx AMEAME LeuLxx LeuLxx [8This] study [AME5,Val7]GME surfactin—glutamic acid Glu 5-methyl Lxx ester, Lxx AME— Valaspartic AME acid 4- Lxxmethyl Valester. This study [Lxx4, AME5] surfactin Glu Lxx Lxx Lxx AME Lxx Lxx This study GME—glutamic acid 5-methyl ester, AME—aspartic acid 4-methyl ester. Molecules 2018, 23, 2224 3 of 12 2. Results and Discussions Molecules 2018, 23, x FOR PEER REVIEW 3 of 12 2.1. Separation of Lipopeptides 2. Results and Discussions The starting point for the separation of lipopeptides was our previous study, where isocratic separations2.1. Separation were applied of Lipopeptides [6] based on the method described by Akpa et al. [17]. Within the isocratic elution of the water/acetonitrile mixture (20:80, v/v), the surfactins with identical molecular weights The starting point for the separation of lipopeptides was our previous study, where isocratic elutedseparations in well-defined were applied time windows[6] based on according the method to described their polarities by Akpa andet al. increased [17]. Within molecular the isocratic masses in theelution following of the order: water/acetonitrilem/z 1016, 15–27mixture min; (20:80,m /v/zv),1030, the surfactins 16–36 min; with 1044 identicalm/z ,molecular 23–44 min; weights 1058 m/z, 31–55eluted min. Thisin well method‐defined allowed time windows the detection according of to surfactinstheir polarities up and to m/z increased1058, molecular but the last masses lipopeptide in (C16-[Val7])the following already order: eluted m/z 1016, near 15–27 to the min; end m/z of 1030, the 16–36 isocratic min; 1044 chromatographic m/z, 23–44 min; run,1058 m suggesting/z, 31–55 the presencemin. of This other method surfactin allowed molecules the detection with of highersurfactins molecular up to m/z weight1058, but [6 the]. The last lipopeptide previously (C16 separated‐ and identified[Val7]) already isoforms eluted represented near to the end three of typesthe isocratic of known chromatographic surfactin variants run, suggesting and a fourth, the presence previously of other surfactin molecules with higher molecular weight [6]. The previously separated and unknown, group, which was characterised by the replacement of the leucine residue by valine in identified isoforms represented three types of known surfactin variants and a fourth, previously the aminounknown, acid group, position which 2. Therefore,was characterised it was by assumed the replacement that the of the prolonged leucine residue separation by valine is able in the to lead to gatheramino mass acid spectrometricposition 2.
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