Structural Characterization of Lipopeptide Methyl Esters Produced by Bacillus licheniformis HSN 221

Structural Characterization of Lipopeptide Methyl Esters Produced byBacillus licheniformis HSN 221

by Yiming Li, Shizhong Yang, and Bozhong Mu*

State Key Laboratory of Bioreactor Engineering and Institute of Applied Chemistry, East ChinaUniversity of Science and Technology, Shanghai 200237, P. R. China

(phone: þ862164252063; fax: þ86 2164252485; e-mail: [email protected])

Lipopeptides and their analogues are of increasing interest due to their amphiphilic structures andpotential applications in various fields. Three purified lipopeptides analogues were obtained at the sametime after two-step column-chromatographic purification from cell-free broth cultivated by Bacilluslicheniformis HSN 221. Analysis by ESI-MS, GC/MS, HPLC, and Q-TOF MS/MS revealed their primarystructures as anteiso-C15- and iso-C15-b-hydroxy fatty acid-Gln-Leu-Leu-Val-MeAsp-Leu-Ile, anteiso-C15-and iso-C15-b-hydroxy fatty acid-MeGlu-Leu-Leu-Val-Asp-Leu-Ile and iso-C16-b-hydroxy fatty acid-Glu-Leu-Leu-Val-MeAsp-Leu-Ile, respectively. The production of two surfactin monomethyl esters and onelichenysin monomethyl ester directly from microorganisms is helpful to understand the variants ofmetabolites.

Introduction. – Biosurfactants are amphiphilic compounds produced by micro-organisms and consist of glycolipids, lipopeptides, polysaccharide – protein complexes,phospholipids, fatty acids, and neutral lipids [1]. They have superior advantages insurface and biological activities, including high biodegradability and compatibility, lowcritical micelle concentration, low toxicity, and low irritability of human skin [2] [3].Therefore, they have potential applications in food, cosmetic, medicine, and petroleumindustry.

Among these biosurfactants, lipopeptides are of increasing interest for researchers.They consist of a hydrophobic fatty acid moiety and a hydrophilic peptide moiety,which is mostly made up of a cyclic peptide with several amino acids in specialsequences. The fatty acid moiety includes n-, iso-, and anteiso-form fatty acids with 13–15 C-atoms [4] [5]. Although surfactin, one of the lipopeptides, was separated in 1968and characterized in 1969 [6] [7], there has been a revival of interest in thecharacterization of various lipopeptides only in recent years [8– 12]. It has been foundthat various isoforms of surfactin and lichenysin, which are attractive powerfullipopeptides, including [Val7]surfactin [13], lichenysin G, and its isoforms [14], and C12-lipopeptide [15], are produced by Bacillus genus. The free carboxy (COOH) groups inlipopeptides are closely related to their properties. Lichenysin with only one freeCOOH group has shown a significant surface activity compared to surfactin with twofree COOH groups in its peptide moiety [16].

Lipopeptide methyl esters have exhibited potential antiviral activities [17]. Theycan be produced by several chemical ways such as MeOH esterification of surfactin inthe presence of base or acid [9] [18] [19]. Here, we report the separation of three

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010) 2065

� 2010 Verlag Helvetica Chimica Acta AG, Z�rich

lipopeptide analogues naturally produced by Bacillus licheniformis HSN 221 andinvestigation of their structures.

Results and Discussion. – Crude lipopeptides were purified by two column steps.Three fractions purified by preparative RP-HPLC were then collected and labeled asFraction 11, Fraction 12, and Fraction 13 (Fig. 1). The molecular masses of theFractions 11– 13 detected by ESI-MS were 1048, 1049, and 1063, respectively.

1. Fatty Acid Part. The fatty acid parts were analyzed by GC/MS after hydrolysis andesterification. The fragment ion with m/z 103, which is specific for b-hydroxy fatty acids,was used to extract the chromatogram to detect their peaks (Fig. 2, a). It indicated acyclic peptide on the basis of the characterized peak, arising from the cleavage of anester bond connected to the cyclic peptides. From the MS results (Fig. 2, b and c), their[M�H]þ ion peaks were identified at m/z 271, 271, and 285, which were due the loss ofH from MeOH-esterified C16- and C17-b-hydroxy fatty acids. The relative values I43/I57,which are contributions of the cleavage of the i-Pr group divided by that of the anteiso-Pr group, were calculated to determine their structures in the branched chain [20]. Thehigher relative value of I43/I57 indicates the presence of the iso-form fatty acid, lower I43/I57 indicates the presence of anteiso-form fatty acid. On the basis of molecular-ion peaksin MS and their I43/I57 values, as well as the tR values of the peaks in the extracted ionchromatogram, the fatty acids were identified as anteiso-C15- and iso-C15-b-hydroxyfatty acids for Fraction 11 and 12, respectively, and as iso-C16-b-hydroxy fatty acid forFraction 13.

2. Amino Acid Part. The H2O phases remaining from the extraction of the fattyacids were analyzed by HPLC. Compared with a standard chromatogram of 18 aminoacids, three fractions were detected to contain Glu, Asp, Val, Ile, and Leu in the sameratio of 1 : 1 :1 :1 :3.

Amino acid- and fatty acid-part analyses, led to molecular weights of Fractions 11–13, 1035, 1035, and 1049, respectively, which are all 14 Da lower than the actual

Fig. 1. Preparative RP-C18-HPLC of lipopeptides produced by Bacillus licheniformis HSN 221

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)2066

molecular weights determined by ESI-MS. Therefore, 14-Da differences, whichresulted from the methylation of Glu and Asp, arose from the amino acid part.

The sequences of amino acids were obtained from Q-TOF MS/MS analyses. Themain sodiated fragments are listed based on the mechanism of the cleavage of fattyacids, cleavage by double H transfer (DHT), and simple cleavage of amino acids(Tables 1– 3) [19] [21]. The ions series with peaks at m/z 721.48, 608.40, 495.32, 382.22,and 267.11 in Fraction 11 are produced by the losses of C-terminal amino acids one byone from the sequence of Leu-Leu-Val-MeAsp-Leu-Ile. In an ester structure, the H2Oadded to C-terminal part in a cyclic lipopeptide would increase the mass by 18 as can be


Fig. 2. GC/MS Analysis of Fraction 11: a) gas chromatogram of extracted m/z 103.0; b) mass spectrum ofthe compound with tR between 14.45 and 14.48 min; c) mass spectrum of the compound with tR between

14.56 and 14.58 min


Tabl

e1.

Sodi

ated

Fra

gmen

tsfr

omF

ract

ion

11

Pea

ksan

dcl

eava

geby

doub

leH

tran

sfer

Pea

ksan

dcl

eava

gew

ith

fatt

yac

ids

Pea

ksan

dsi

mpl

ecl

eava

ge

267.

11a )

617.

4632

0.26

a )N

aþ

Leu

-Leuþ

H2O

Naþ

FA11

b)-

Gln

-Leu

-Leu

Naþ

Leu

-Leu

-Val�

CO

382.

2271

6.53

365.

29a )

Naþ

Val

-Asp

Me-

Leuþ

H2O

Naþ

FA11

b)-

Gln

-Leu

-Leu

-Val

Naþ

Leu

-Val

-Aspþ

NH

orN

aþ

Val

-Asp

-Leuþ

NH

495.

3284

5.57

392.

21N

aþ

Val

-Asp

Me-

Leu

-Ileþ

H2O

Naþ

FA11

b)-

Gln

-Leu

-Leu

-Val

-MeA

spN

aþ

Gln

-Leu

-Leuþ

NH

orN

aþ

Leu

-Val

-MeA

spþ

CO

608.

4095

8.66

405.

29a )

Naþ

Leu

-Val

-Asp

Me-

Leu

-Ileþ

H2O

Naþ

FA11

b)-

Gln

-Leu

-Leu

-Val

-MeA

sp-L

euN

aþ

Gln

-Leu

-Leuþ

CO

721.

4810

71.7

544

9.30

a )N

aþ

Leu

-Leu

-Val

-Asp

Me-

Leu

-Ileþ

H2O

Naþ

FA11

b)-

Gln

-Leu

-Leu

-Val

-MeA

sp-L

eu-I

leN

aþ

Leu

-Leu

-Val

-MeA

sp�

CO

orN

aþ

Leu

-Val

-M

eAsp

-Leu�

CO

orV

al-M

eAsp

-Leu

-Leu�

CO

912.

6850

5.29

Naþ

Mc )�

(Ileþ

COþ

H2O

)N

aþ

Leu

-Leu

-Val

-MeA

spþ

CO

orN

aþ

Leu

-Val

-M

eAsp

-Leuþ

CO

orV

al-M

eAsp

-Leu

-Leuþ

CO

799.

59N

aþ

Mc )�

(Leu

-Ileþ

COþ

H2O

)

670.

51N

aþ

Mc )�

(MeA

sp-L

eu-I

leþ

COþ

H2O

)

571.

44N

aþ

Mc )�

(Val

-MeA

sp-L

eu-I

leþ

COþ

H2O

)

a )P

eaks

not

labe

led

inth

eF

igur

es.

b)

FA:F

atty

acid

ofth

efr

acti

on.

c )M

:Mol

ecul

eof

the

frac

tion

.


Tabl

e2.

Sodi

ated

Fra

gmen

tsfr

omF

ract

ion

12

Pea

ksan

dcl

eava

geby

doub

leH

tran

sfer

Pea

ksan

dcl

eava

gew

ith

fatt

yac

ids

Pea

ksan

dsi

mpl

ecl

eava

ge

267.

1663

2.39

a )32

0.22

Naþ

Leu

-Leuþ

H2O

Naþ

FA12

b)-

MeG

lu-L

eu-L

euN

aþ

Leu

-Leu

-Val�

CO

382.

15a )

731.

4636

5.16

a )N

aþ

Asp

-Leu

-Leuþ

H2O

Naþ

FA12

b)-

MeG

lu-L

eu-L

eu-V

alN

aþ

Leu

-Val

-Aspþ

NH

orN

aþ

Val

-Asp

-Leuþ

NH

481.

2484

6.48

391.

25N

aþ

Val

-Asp

-Leu

-Leuþ

H2O

Naþ

FA12

b)-

MeG

lu-L

eu-L

eu-V

al-A

spN

aþ

Leu

-Leu

-Valþ

COþ

NH

594.

3395

9.54

463.

25N

aþ

Leu

-Val

-Asp

-Leu

-Leuþ

H2O

Naþ

FA12

b)-

MeG

lu-L

eu-L

eu-V

al-A

sp-L

euN

aþ

Leu

-Leu

-Val

-Asp

orN

aþ

Leu

-Val

-Asp

-Leu

orN

aþ

Val

-Asp

-Leu

-Leu

707.

10a )

1072

.58

Naþ

Leu

-Leu

-Val

-Asp

-Leu

-Leuþ

H2O

Naþ

FA12

b)-

MeG

lu-L

eu-L

eu-V

al-A

sp-L

eu-I

le

941.

51a )

Naþ

Mc )�

(Ileþ

H2O

)

828.

45N

aþ

Mc )�

(Leu

-Ileþ

H2O

)

685.

45N

aþ

Mc )�

(Asp

-Leu

-Ileþ

COþ

H2O

)

586.

39N

aþ

Mc )�

(Val

-Asp

-Leu

-Ileþ

COþ

H2O

)

473.

31N

aþ

Mc )�

(Leu

-Val

-Asp

-Leu

-Ileþ

COþ

H2O

)

360.

13a )

Naþ

Mc )�

(Leu

-Leu

-Val

-Asp

-Leu

-Ileþ

COþ

H2O

)

a )P

eaks

not

labe

led

inth

eF

igur

es.

b)

FA:F

atty

acid

ofth

efr

acti

on.

c )M

:Mol

ecul

eof

the

frac

tion

.


Tabl

e3.

Sodi

ated

Fra

gmen

tsfr

omF

ract

ion

13

Pea

ksan

dcl

eava

geby

doub

leH

tran

sfer

Pea

ksan

dcl

eava

gew

ith

fatt

yac

ids

Pea

ksan

dsi

mpl

ecl

eava

ge

267.

51a )

519.

34a )

320.

25N

aþ

Leu

-Leuþ

H2O

Naþ

FA13

b)-

Glu

-Leu

Naþ

Leu

-Leu

-Val�

CO

382.

2163

2.43

449.

29N

aþ

Val

-MeA

sp-L

euþ

H2O

Naþ

FA13

b)-

Glu

-Leu

-Leu

Naþ

Leu

-Leu

-Val

-Asp

Me�

CO

orN

aþ

Leu

-Val

-A

spM

e-L

eu�

CO

orV

al-A

spM

e-L

eu-L

eu�

CO

495.

2973

1.48

Naþ

Val

-MeA

sp-L

eu-L

euþ

H2O

Naþ

FA13

b)-

Glu

-Leu

-Leu

-Val

608.

3686

0.54

Naþ

Leu

-Val

-MeA

sp-L

eu-L

euþ

H2O

Naþ

FA13

b)-

Glu

-Leu

-Leu

-Val

-MeA

sp

721.

4797

3.64

Naþ

Leu

-Leu

-Val

-MeA

sp-L

eu-L

euþ

H2O

Naþ

FA13

b)-

Glu

-Leu

-Leu

-Val

-MeA

sp-L

eu

1086

.70

Naþ

FA13

b)-

Glu

-Leu

-Leu

-Val

-MeA

sp-L

eu-I

le

955.

66a )

Naþ

Mc )�

(Ileþ

H2O

)

842.

54N

aþ

Mc )�

(Leu

-Ileþ

H2O

)

685.

50N

aþ

Mc�

(Asp

Me-

Leu

-Ileþ

COþ

H2O

)

586.

44N

aþ

Mc )�

(Val

-Asp

Me-

Leu

-Ileþ

COþ

H2O

)

a )W

eak

peak

sno

tla

bele

din

the

Fig

ures

.b)

FA:F

atty

acid

ofth

efr

acti

on.

c )M

:Mol

ecul

eof

the

frac

tion

.

seen from the ion peak at m/z 267. The loss of Ile-Leu-H2O, in all three Fractionsestablish the presence of the C-terminal amino acids Ile-Leu, which are cleaved by theDHT mechanism. These amino acids in a cyclic peptide were connected to fatty acidpart by an ester bond. Moreover, ions with peaks at m/z 1071.75, 958.66, 845.57, 716.53,and 617.46 are fragment ions from the cleavage with fatty acids. The mass differencesbetween these peaks evidence the losses of amino acids in the sequence of Leu-Leu-Val-MeAsp-Leu-Ile from the C-terminus. Other fragment ion peaks of these aminoacids were detected at m/z 320.26, 365.29, 392.21, 405.29, 449.30, and 505.29, indicatinga simple cleavage. One molecule of Glu has been determined according to the aminoacids analysis. Based on the losses of amino acids from cleavage with fatty acids, Glushould be connected to the fatty acid as the N-terminus of the amino acid residues.Moreover, preferential cleavages of amide bonds adjacent to Asp and Glu residueshave been observed [22 –26]. The cleavage may easily occur at a peptide bond not onlybetween N-terminal amino acid and remaining amino acid residues, but also betweenneutral amino acids [19]. In addition, the 1-Da difference has been found in Fraction 11between the calculated molecular weight from the two-part analysis and the actualmolecular weight detected by ESI-MS. The reason might be hydrolysis of Gln,converting to Glu. Therefore, the sequence of amino acids in Fraction 11 wasdetermined as Gln-Leu-Leu-Val-MeAsp-Leu-Ile. Similar ion-peak series were found inFractions 12 and 13, indicating their amino acid sequences as MeGlu-Leu-Leu-Val-Asp-Leu-Ile and Glu-Leu-Leu-Val-MeAsp-Leu-Ile, respectively.

The peaks at m/z 449 and 505 of Fraction 11 and Fraction 13 are due simplecleavage of amino acids containing MeAsp. Two ion series with peaks at m/z 685.50,586.44, and m/z 670.51, 571.44 are produced by cleavage with fatty acids containingMeAsp. However, peaks corresponding to these ion series did not appear in the spectraof Fraction 12. Also molecular loss of Asp can be concluded from the ion peaks at m/z846.48 and 731.46, indicating that Glu has been methylated in Fraction 12 rather thanAsp.

Since MeAsp and Glu have same molecular masses, 129-Da loss of fragments in theMS/MS spectra can be ascribed to both of these two molecules. However, only one Gluand one Asp were detected after HPLC determination of three fractions. If MeAsp isreplaced by Glu, their positions should be changed considering the mechanism ofcleavage with fatty acids. It should be MeAsp instead of Glu in Fraction 11, becausepeaks at m/z 392 and 405 could be due fragments containing Gln rather than Asn. Thesame ion peaks at m/z 382, 495, 608, and 721 were detected in the spectra of bothFraction 11 and Fraction 13, indicating the presence of AspMe in the same position ofboth molecules.

The 14-Da difference of molecular mass has been produced by the following fourfactors in the molecule of lipopeptide. The first factor is the different fatty acid chain.Second, one amino acid is replaced by the other amino acid such as the smaller Valbeing replaced by Leu or Ile [13] [18] [27]. Third, an additional Me group may be linkedto one of the N-atoms or CH2 group of peptides [28] [29]. Fourth, one of the carboxygroups of amino acid residues such as Glu or Asp is methylated [18]. However, threefactors are eliminated on the basis of the results of fatty acid and amino acid analysis.Therefore, it is the CH2 group in Glu or Asp of lipopeptide methyl esters, which leads toa 14-Da difference.


3. Primary Structures. The primary structures of Fractions 11– 13 have beendetermined from their molecular weight, and fatty-acid, amino-acid composition, andsequence analysis as anteiso-C15- and iso-C15-fatty acid-Gln-Leu-Leu-Val-MeAsp-Leu-Ile, anteiso-C15- and iso-C15-fatty acid-MeGlu-Leu-Leu-Val-Asp-Leu-Ile, and iso-C16-fatty acid-Glu-Leu-Leu-Val-MeAsp-Leu-Ile, respectively (Fig. 3,a – c).

Among the known 23 groups of lipopeptides [30], three fractions have structuressimilar to those of [Ile7]surfactin and lichenysin. Accordingly, Fraction 1 is [Ile7]-lichenysin monomethyl ester, and Fractions 12 and 13 are [Ile7]surfactin monomethylesters.

Conclusions. – Lipopeptide methyl esters are usually produced by chemical ways[9] [18] [19]. In this work, the three fragments obtained from preparative RP-HPLCwere consistent independent of the extraction solvent such as MeOH and Et2O,indicating that the three lipopeptide methyl monoesters in this work are produceddirectly by microorganism, and they are new members of the known 23 groups of

Fig. 3. Primary structures of a) Fraction 11, b) Fraction 12, and c) Fraction 13


lipopeptides [30]. So far, there have been few reports on production of these newlipopeptides directly by microorganisms. They have enriched the family of lipopeptidesand will provide a significant help in the search for variants of lipopeptides and theirpotential applications.

This work was supported by the National Natural Science Foundation of China (No. 50744016), thegrant from the Ministry of Science and Technology of China (2007CB707801), and the ShanghaiMunicipal Science and Technology Commission (071607014).

Experimental Part

General. Column chromatography (CC): HPLC: JASCO UV-2075 ; columns: Hypersil ODS-A RP-C18, 50 mm, 100 mm�30 mm; ODS2 RP-C18, 5 mm, 21.2 mm�250 mm; Hypersil ODS2 RP-C18, 5 mm,4.6 mm�250 mm. Q-TOF MS-MS: Q-Tof Micromass Co., UK. ESI-MS: Micromass Co. LCT KC 317,UK. GC/MS: Agilent, USA, 6890 chromatograph with a model 5975 mass-selective detector and a HP-5MS cap. column, 30 m�0.25 mm�0.25 mm.

Microorganism and Growth Conditions. The microorganism, Bacillus licheniformis HSN 221, wasseparated from oil field and kept in our laboratory. It was grown aerobically on YPD medium containing(per l) 20 g of glucose, 20 g of peptone, and 10 g of yeast extract with a final pH of 7.2 adjusted with 6mNaOH. The fermentation was conducted at 408 under shaking (120 rpm) for 72 h.

Isolation of the Crude Lipopeptide. After fermentation, bacterial cells were removed from thelipopeptide-containing medium by centrifugation at 5000g for 20 min. The pH of the supernatant wasadjusted to 2.0 with 6m HCl and then stored overnight at 48. The precipitates were collected aftercentrifugation (5000g, 15 min) and were extracted by MeOH or AcOEt for several times. The crudelipopeptide was obtained after the evaporation of the solvent evaporated with a rotary evaporator undervacuum at 458.

Purification of Lipopeptides. The crude samples were dissolved in MeOH and purified by thefollowing two steps. First, ca. 0.6 g of crude sample dissolved in 0.5 ml MeOH was run on the normal-pressure C18 reversed-phase (RP) column (ODS-A, 50 mm, 100 mm�30 mm) at r.t. The column wasbalanced with 80% MeOH and 0.05% TFA (CF3COOH) in advance. It was then washed with 200 ml of80% MeOH at a flow rate of 6.0 ml min�1. The active lipopeptides were eluted with 200 ml of 100%MeOH and collected in ten tubes (20 ml per tube). The elution was monitored at a wavelength of 210 nmby a spectrophotometer.

Second, the prep. RP high-pressure liquid chromatography (prep.-RP-HPLC) was carried out. AHPLC system (JASCO, Japan) with a Hypersil ODS2 RP-C18 column (5 mm, 21.2 mm�250 mm) wasused in this step. The mobile phases were MeOH (A) and 0.05% TFA in doubly dist. H2O (B). About100 mg of sample dissolved in MeOH was filtered and then injected into the system. It was eluted at aflow rate of 15 ml min�1 with a gradient system starting with 90% A and maintained for 55 min, thengradually increased to 100% A in 5 min, and maintained at 100% A for 20 min. The elutions weredetected at 214 nm and collected in 75 tubes (15 ml per tube). Purified dry lipopeptides were obtainedafter evaporating the solvent by rotary evaporator under vacuum at 458.

Molecular-Weight Determinations. The molecular weights of the three fractions were accomplishedby electrospray ionization mass spectrometry (ESI-MS). About 1.0 mg of the dried pure lipopeptide wasdissolved in 1.0 ml of MeOH and submitted to ESI-MS (Micromass Co., LCT KC 317, UK) operated ationization source temp. of 808, electrolyte voltage of 200 V, and spray inlet temp. of 1208. The equipmentwas run in the pos.-ion mode.

Determination of Fatty Acid Parts. Fatty acid parts of lipopeptides were determined by GC/MS.About 2.0 mg of purified lipopeptide and 1.0 ml of 6m HCl soln. were added to a hydrolyzing tube. Thesealed tube was put into the oven at 1108 for 20 h. The mixture was then transferred into a 25-ml tube andwashed three times with doubly dist. H2O and Et2O, resp. The samples containing fatty acids were thencollected by extraction with 3 ml of Et2O for three times and dried samples were esterified in 1.0 ml of10% H2SO4/MeOH soln. at 558 for 6 h [31]. About 3 ml of deionized H2O was added to the mixture, and


MeOH-esterified fatty acid was obtained from the extraction of such a soln. with 3 ml of Et2O for threetimes. After evaporation of Et2O at r.t., the fatty acid esters was dissolved in 1.0 ml of MeOH andanalyzed by GC/MS (Agilent, USA, 6890 chromatograph with a model 5975 mass-selective detector and aHP-5MS cap. column, 30 m�0.25 mm�0.25 mm; flow rate of He 1.0 ml min�1). The column temp. wasinitially held at 1208 for 5 min and then increased to 2608 at a rate of 88 min�1. The post-run of the systemwas carried out at 2808 for 10 min. The injected sample volume was 1.0 ml with a split ratio of 10 : 1.

Determination of Amino Acids. Composition and ratio of amino acids in lipopeptides weredetermined according to a precolumn derivatization procedure with phenyl isothiocyanate (PhNCS) bymeans of HPLC [32]. The remaining H2O phase after extraction of fatty acid esters was collected, dried,and dissolved in 1.0 ml of H2O; 200 ml of this soln. was mixed with 100 ml of 0.1m PhSCN soln. in MeCNand 100 ml of 1.0m Et3N soln. in MeCN, and maintained at r.t. for 40 min. After addition of 400 ml ofhexane, the mixture was stirred for 5 min and let to stand for 10 min. The H2O phase was filtered (0.22-mm filter; Millipore, USA), and 10 ml of the soln. was submitted to HPLC analysis (Hypersil ODS2 RP-C18, 5 mm, 4.6 mm�250 mm; flow rate 1.0 ml min�1) for amino acid analysis. The HPLC spectra weredetected at 254 nm. The solvent systems were A) MeCN/H2O 4 : 1 (v/v) and B) 0.1m AcONa/MeCN 97 : 3(v/v). The sample was eluted by the following program: 0 min, 0% A ; 13 min, 7% A ; 23 min, 23% A ;29 min, 35% A ; 35 min, 40% A ; 40 min, 100% A ; 45 min, 100% A ; 47 min 0% A.

Sequence Analysis of Amino Acid. Sequences of amino acids in the lipopeptides were determined bymeans of Q-TOF MS/MS. About 1.0 mg of the dried pure lipopeptide was dissolved in 1.0 ml of MeOHand analyzed by Q-TOF MS-MS (Q-Tof micro, UK) equipped with an electrospray ion (ESI) source andoperated at the following conditions: cap. voltage 3200 V, sample cone voltage 70 V, and collision energy70 V.

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Received May 5, 2009


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Structural Characterization of Lipopeptide Methyl Esters Produced by Bacillus licheniformis HSN 221