Hanen, 2014, B Mojavensis

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Process Biochemistry 49 (2014)16991707

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Process Biochemistry

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Identification and biochemical characteristics oflipopeptides fromBacillus mojavensis A21

Hanen Ben Ayed a, Noomen Hmideta,, Max Bchetb, Marlne Cholletb,Gabrielle Chataignb, Valrie Leclreb, PhilippeJacquesb, MoncefNasri a

a Laboratoire de Gnie Enzymatique et deMicrobiologie, Universit de Sfax, Ecole Nationale dIngnieurs de Sfax, B.P. 1173-3038 Sfax, Tunisiab ProBioGEM EA1026, PolytechLille/IUTA, Universit Lille-Nord de France, F-59655 Villeneuve dAscq, France

a r t i c l e i n f o

Article history:

Received 25 March 2014Received in revised form 30 June 2014Accepted 3 July 2014Available online 11 July 2014

Keywords:

NRPSPCRMALDI-TOF-MSFengycinSurfactinPumilacidin

a b s t r a c t

This study reports the potential ofa marine bacterium, Bacillus mojavensis A21, to produce lipopeptidebiosurfactants. The crude lipopeptide mixture was found to be very effective in reducing surface ten-sion to 31mNm1 . PCRexperiments using degenerate primers revealed the presence ofnonribosomalpeptide synthetases genes implied in the biosyntheses offengycin and surfactin. Matrix-Assisted LaserDesorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) performed on whole cells ofB.mojavensisA21 confirmedthe presenceoflipopeptides identifiedas membersofsurfactin and fengycinfamilies. Further, a detailed analysisperformed by MALDI-TOF-TOFrevealed the presence ofpumilacidincompounds. The crude lipopeptide mixture was tested for its inhibitory activity against Gram-positiveand Gram-negative bacteria, and fungal strains. It was found to display significantantimicrobial activity.Strain A21 lipopeptide mixture was insensitive to proteolytic enzymes, stable between pH 3.0 and 11.0,andresistantto high temperature. Production oflipopeptides is a characteristicofseveralBacillus species,but toour knowledge this isthefirst report involving identificationofpumilacidin, surfactinand fengycinisoforms in a B. mojavensis strain.

2014 Elsevier Ltd. All rights reserved.

1. Introduction

The genus Bacillus is known to produce a broad spec-trum of biologically active molecules with great potentialfor medical and biotechnological applications. Among thesemolecules, biosurfactants have received great attention in differ-ent fields, including phytosanitary sector, medicine, cosmetics,food and feed additives, bioremediation, etc., [1]. Structurally,biosurfactants are amphiphilic molecules and comprise variousdifferent chemical structures, such as glycolipids, lipopeptides,polysaccharideprotein complexes, phospholipids, fatty acids andneutral lipids [2]. Compared to chemical surfactants, biosur-factants have several advantages, including low toxicity, highbiodegradability under natural conditions, ecological acceptabilityand effectiveness at extreme temperatures and pH values [3].

Lipopeptides areamong the most commonly isolated and char-acterized biosurfactants. They have received great attention dueto their medical, food and biotechnological applications. Further,theywere foundto removeefficientlypetroleumhydrocarbonsand

Corresponding author. Tel.: +216 22 76 31 14;fax: +216 74 275 595.E-mail address: hmidet [email protected] (N. Hmidet).

heavy metals from contaminated soils [4]. The lipopeptides pro-duced by numerous Bacillus spp. are classified into three familiesdepending on their amino acids sequence: surfactins, iturins andfengycins [5] and are considered as safe. These advantages makelipopeptides potential alternatives to chemically synthesized sur-factants. From another side, the fast progress of biotechnology hasaccelerated the research and development of new and more effec-tive lipopeptides.

These molecules are synthesized by multimodular enzymescomplexes known as nonribosomal peptides synthetases (NRPSs)[5,6]. Lipopeptides contain hydrophilic peptides, which differ inamino acid composition and sequence (seven to ten amino acids)linked to a hydrophobic fatty acid with different chain lengths andisomeries [5].

Among theBacillus species, Bacillus subtilis is bestknown for theproduction of lipopeptides, mainly surfactin and fengycin [7]. Sur-factin is oneof the most effectivebiosurfactants and shows severalpharmacological activities including the antimicrobial, antiviral,antitumoral and antifibrinolytic ones. It is a cyclic lipoheptapep-tide which contains a -hydroxy-fatty acid with a chain length of1315 carbon atoms [8]. Several variants of surfactin have beendescribed such as lichenysin from Bacillus licheniformis or pumi-lacidin from Bacillus pumilus. In addition to surfactin, fengycin is

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1700 H. Ben Ayed et al./ Process Biochemistry 49 (2014) 16991707

another best-known lipopeptide, which exhibits several biologicalactivities, includingactivityagainstfilamentous fungi [9]. Fengycinis a cyclic lipodecapeptide containing a -hydroxy-fatty acid witha length of 1518 carbon atoms [9]. Two variants of fengycins (Aand B) have been already described in the literature. They differby the amino acid residue in position 6. Few studies reported theproduction of lipopeptides by Bacillusmojavensis strains [10,11].

In this study, B. mojavensis A21 isolated on the basis of its pro-teolytic activity [12] was also found to produce high amounts ofbiosurfactants(basedon their abilitytoreducesurface tensionfrom71 to 31mNm1) [13]. The biosurfactant mixture showed highphysico-chemical properties in terms of the surface activities andemulsification index. Further, the mixture was found to removediesel more effectively than synthetic surfactants. Therefore, thisstudy reports on (i) the detection of NRPS genes by PCR and (ii)structural characterization of lipopeptides by MALDI-TOF-MS. Inaddition, the stability of the produced lipopeptides under extremeconditions was investigated in order to estimate their potentialapplications.

2. Materials and methods

2.1. Bacterial strain and lipopeptides production

The microorganism used in this study was isolated in our lab-oratory from marine water in Sfax, Tunisia. It was identified as B.mojavensis A21 based on its biochemical and physiological char-acteristics, and on the 16S rRNA gene sequence analysis. It wasassigned the accession number EU366229 [12].

The strain B. mojavensis A21 was inoculated in 250mLErlen-meyer flask containing 25mLLuriaBertani (LB) broth medium(10 g/L tryptone, 5g/L yeast extract, 5g/L NaCl) and cultivated at37 C for 24h under agitation at 200rpm. For lipopeptides pro-duction, culture was conducted in 1L Erlenmeyer flask containing100mLof Landy medium [14] consisting of: glucose, 20g/L; l-glutamicacid,5g/L;yeastextract,1g/L;K2HPO4,1g/L;MgSO47H2O,0.5g/L; KCl, 0.5g/L; CuSO4, 1.6mg/L; Fe2(SO4)3, 1.2mg/L andMnSO4, 0.4mg/L. The medium was complemented by 100mMMOPS and the initial pH was adjusted to7.0 with3M KOH. Culturewas carried out for 72h at 30 C under shaking at 160 rpm. Afterfermentation, the culture broth was centrifuged at 13,000g for30min at 4 C, and the supernatant containing the crude lipopep-tide was collected. Lipopeptide molecules were partially purifiedfrom the cell-free supernatant by different steps of ultrafiltra-tion/diafiltration. The crude lipopeptide mixture obtained wasevaluated for its antimicrobial activity and its stability againstextremeconditions. All experiments werecarried out in triplicates.

Lipopeptide production was also analyzed by measurement ofsurface tension during the growth of the strain. The surface ten-sion of the cell-free supernatant was determined according to the

Du Noy ring method in a TDI tensiometer (Lauda, Knigshofen,Germany) as described by Leclre et al. [15]. The values obtainedare the mean of three measurements.

Glucose concentration was analyzed in the filtered samples byHPLC Spectra SYSTEM P1000 XR supplied by Thermoelectron Cor-poration (ThermoFisher Scientific Inc., Waltham,MA, USA)using aFast Fruit Juice column (1507.8mm, Waters Corp., Milford, MA,USA). The flow rate is set at 0.8ml/min and the column was main-tainedat 55 C. Elution wasachievedunder0.05% H3PO4. Detectionwas carried out by using a refractometer Spectra System RI-150.

2.2. Extraction and quantification of lipopeptides

Lipopeptides were extracted from culture supernatant by solidphase extraction using C18 Maxi clean cartridges (Extract CleanSPE500mg,GraceDavison-Alltech,Deerfield, IL, USA)accordingtoGuez et al. [16]. The supernatant was applied to a C18 cartridge,which retained lipopeptides. The cartridge was rinsedwith 8 mLofbi-distilled water and lipopeptides were then eluted with 8 mL of100%methanol.After evaporationof thesolvent,the crudelipopep-tide mixture was dissolved in 200L methanol.

Lipopeptides were analyzed and quantified by reversed-phasehigh-performance liquid chromatography (600s, Waters Corp.)using a C18column (5m, 250mm4.6mm, 218 TP,VYDAC,Hes-peria, CA, USA).

2.3. Detection of NRPS genes by PCR and DNA sequencing

Twopairsof degenerate primers (Af2-F/Tf1-RandAs1-F/Ts2-R),designed using thealignment of adenylation or thiolation domainsthat compose lipopeptide synthetases,were used for the detectionby PCR of the NRPS genes (Table 1) [17]. DNA was extracted fromovernight culture of A21 strain using the Wizard Genomic DNAPurification Kit and protocol (Promega Corp., Madison, USA).

The PCRthermal cycle program included an initial denaturationstep at 94 C for 3min, followed by 30 cycles, with denaturationat 94 C for 1min, annealing at 43 C for 30s, with As1-F/Ts2-R,and at 45 C with Af2-F/Tf1-R primers, and extension at 72C for45s. Final extension was performed at 72C for 10min. The Taqpolymerase used was Master Mix (Thermo Scientific Fermentas,Illkirch, France) with a final primer concentration of 1.2M. PCRproducts were analyzed by 0.7% (w/v) agarose gel electrophoresis.

PCR products were purified with QIAquick Gel Extraction Kit(Qiagen, Hilden, Germany)and then clonedinto pGEM-TEasy Vec-tor (Promega Corp.). Recombinant plasmids were introduced intoEscherichia coliJM109 cells by heat shock, according to the man-ufacturers protocol (Promega). Transformants were selected onLuriaBertani (LB) solid medium supplemented with ampicillin,IPTG (isopropyl--d-thiogalactopyranoside) and X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) at final concentrationsof 100g/mL, 200g/mL and 20g/mL, respectively. Whitecolonies were picked and then cultivated in LB medium supple-mented with 100g/mL ampicillin for 24h at 37 C. Plasmidswere isolated from the transformed cells using the QIAprep

Spin Miniprep kit (Qiagen, Germany). Cloned PCR products weresequenced using the universal primers pUC-M13-R/F (EurofinsMWGOperon,Ebersberg,Germany).DNAsequenceswereanalyzed

Table 1

List and characteristics of primers used in this study.

Primer n ames Sequence o f primersa HyCb Expectedfragment size(bp) Identifiednonribosomallipopeptides References

Af2-FTf1-R

GAATAYMTCGGMCGTMTKGAGCTTTWADKGAATSBCCGCC

3472

443, 452,455 Fengycins [17]

As1-FTs2-R

CGCGGMTACCGVATYGAGCATBCCTTTBTWDGAATGTCCGCC

1236

419, 422, 425, 431 Surfactins [17]

bp, base pair.a Using IUPAC DNA code: Y=C or T, M=A or C, K=G or T, W=A or T, D=G,A or T, S=G or C, B=G,TorC,R =A or G.b

Coefficient of hybridization (HyC) calculated as described by Tapi et al. [17].


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H. Ben Ayed et al./ Process Biochemistry 49 (2014) 16991707 1701

withtheGenBankdatabasesusingtheBLAST(BasicLocalAlignmentSearch Tools) software provided online by the National Center forBiotechnology Information (Bethesda, MD, USA).

2.4. Lipopeptides detection by MALDI-TOF-MS analysis

MALDI-TOF-MS was used to detect and characterize lipopep-tides from whole cells of strain A21. Individual colonies, grown

on LB agar plate at 30

C for 72h, were carefully suspended inEppendorf tube containing a matrix solution (10mg/mLcyano-4-hydroxycinnamicacidin 70%water,30%acetonitrile,and 0.1%TFA).The sample was homogenized and then centrifuged at 5000rpm.Forclassical analysis, 1L of the sample solution was spotted ontoa MALDI-TOF MTP384target plate (BrukerDaltonikGmbH, Leipzig,Germany) and let dry before analysis.

Mass profiles experiments were also analyzedwith an UltraflexMALDI-TOF/TOF mass spectrometer (Bruker, Bremen, Germany)equipped with a smartbeam laser. Samples were analyzed usingan accelerating voltage of 25kV and matrix suppression in deflex-ion mode at m/z750. The laser power was set to just above thethreshold of ionization (around 35%). Spectra were acquired inreflector positive mode in the range from 800 to 3000Da. Each

spectrum was the result of 1000 laser shots per m/zsegment persample delivered in 10 sets of 50 shots distributed in three differ-entlocations on thesurface of the matrix spot. The instrument wasexternally calibrated in positive reflector mode using Bradykinin[M+H]+ 757.3991, Angiotensin II [M+H]+ 1046.5418, AngiotensinI [M+H]+ 1296.6848, Substance P [M+H]+ 1347.7354, Bombesin[M+H]+ 1619.8223, and ACTH [M+H]+ 2093.0862.

MS/MS spectra were obtained using the LIFT techniquedescribed elsewhere (LIFT-TOF/TOF). In brief, fragment ions aregeneratedby a selectionof theprecursor ions produced duringtheMALDI process, similar to MS analysis; the isolation mass windowwas set to 1% of parent mass and the laser power boost to inducefragmentation was 80%.

2.5. Antimicrobial activity

Antibacterial activities were tested against Gram-positive andGram-negative bacterial strains. The bacteria used were Staphy-lococcus aureus (ATCC 25923), Bacillus cereus (ATCC 11778),Enterococcus faecalis (ATCC29212),Micrococcus luteus (ATCC4698),Pseudomonas aeruginosa (ATCC 27853), Salmonella typhimurium(ATCC19430),Klebsiellapneumoniae(ATCC13883)andE. coli (ATCC25922).AntifungalactivitiesweretestedagainstAspergillusnigerI1,Mucor rouxii DSM 1191 and Botrytis cinerea.

Antimicrobial activity ofB. mojavensis A21 lipopeptides wasassessed by the agar well diffusion method [18]. Culture suspen-sion (100L) of the indicated strains (about 106 colony formingunits (Cfu)/mL for bacterial cells and 5104 spores/mL for fungal

strains)were spread overLuria-Bertani(LB) agaror Sabourauddex-trose agar, respectively. Then, wells (3mm depth, 6 mm diameter)were made in the agar plates using a sterile borer. A 60L sam-ple of the crude lipopeptides mixture was loaded into the wells.The plates were then incubated for 24h at 37C for bacteria and72h at 30 C for fungal strains. The diameter of the inhibition zonewas measuredandtheresults reported inmm.Theexperiment wasconducted in triplicate.

2.6. Effects of heat, pH, proteolytic enzymes and organic solvents

on crude lipopeptide antibacterial activity

To investigate thermal stability, the crude lipopeptide mixturewas incubated in waterbath at different temperatures (60100 C)

for15min. After cooling the treated samples to room temperature,

residual antibacterialactivityusing S. aureusas indicatorstrain wasmeasured by theagar well diffusion method.

The effect of pH on lipopeptide activity was examined byassaying antimicrobial activity using S. aureus after incubation for2 h a t 4 C in the pH range of 3.011.0. Samples were neutralizedto pH 7.0 before measurement of the antimicrobial activity. Thefollowing buffer systems were used: 100 mM glycineHCl buffer,pH 2.04.0; 100mM acetate buffer, pH 4.06.0; 100mM TrisHClbuffer, pH 7.08.0 and100mM glycineNaOHbuffer, pH 9.011.0.

In order to evaluate stability to proteolytic enzymes, the crudelipopeptide was incubated with each enzyme for 2h at 37C with1 mg/mL (final concentration) of the following enzymes: trypsin,chymotrypsin,bromelainandalcalasein 0.05M TrisHClbuffer (pH8.0) and pepsin in 0.05M glycineHCl buffer (pH 2.0). The residualantibacterialactivitywas thenevaluatedby theagar-welldiffusionmethod against S. aureus.

Organic solvent stability of the crude lipopeptide A21 was car-ried out by incubating the lipopeptide solution for 1h at 37C inthepresenceofvariousorganicsolvents(50%,v/v). Residualactivitywas then determined against S. aureus as indicator strain.

3. Results and discussion

3.1. Detection by PCR of NRPS lipopeptides genes in B. mojavensis

A21

LipopeptidesaresynthesizedbyNRPSsencodedbyoperonswithdifferentopen reading frames. Forexample, the operonof surfactinfamily contains four open reading frames (ORFs) coding for sur-factin synthetase, which are designated srfA-A, srfA-B, srfA-C andsrfA-D.Thecomparativebioinformaticsanalysesofeachoperon ledto the design of different primer pairs for the three families takinginto account the differences between open reading frames of eachsynthetase gene [17].

In this study, PCR, with two primers pairs (Af2-F/Tf1-R andAs1-F/Ts2-R), was used initially for the detection of NRPS genes,

involvedin thebiosynthesis of fengycinandsurfactin, respectively.Both primers pairs gave amplicons with the expected sizes (Fig. 1).The PCR products were cloned into pGEM-T Easy vector and thensequenced as described in Section 2. The Blast n results obtainedwith the twosequenced fragments are presented in Table 2.

Fig. 1. PCR amplification by degenerate primers pairs with B. mojavensis A21. M:

molecular weightmarker, Af2-F/Tf1-R (lane1), As1-F/Ts2-R (lane2).


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Table 2

Blast results of thesequenced products obtained from PCRamplification with degenerate primers.

Primer names Product size (bp) GenBank accession number Probable proteins Identity (%)

Af2-FTf1-R

443 AAB80955.2 Fengycin synthetase of B. subtilis. subsp. str 168 97

As1-FTs2-R

421 ZP03590011.1 Surfactin synthetase of B. subtilis. 89

The sequence of the fragmentamplified by Af2-F/Tf1-R primersshowed high homology (97%) with the plipastatin synthetaseoperon ofB. subtilis subsp. subtilis strain 168. Further, As1-F/Ts2-Rprimers amplify fragmentwhichshoweda high similarity with thesurfactinsynthetaseoperonofB.subtilis168(89%).Thehighhomol-ogy indicated that surfactin and fengycin synthetases are nearlyidentical between Bacillus strains.

Few studies reported the production of lipopeptides by B.mojavensis strains. Snook et al. [11] reported the production, fromtheendophyticbacteriumB.mojavensisRRC1001,of Leu7-surfactinwhichwas found to suppress thegrowth ofFusariumverticillioides.More recently, a marine-derived bacterium B. mojavensis B0621Awas foundtoproduce a newiturinic lipopeptide calledmojavensinA [12]. The presence of surfactin and fengycin synthetase genes

was also detected in several Bacillus strains [7]. The production ofthis lipopeptidemixture by thesamestraincouldbe advantageous,since a synergistic effect may occur and improve the biologicalproperties.

3.2. Detection of lipopeptides production by MALDI-TOF analyses

MALDI-TOF mass spectrometryhasbeen largely used as an effi-cient tool for the detection and identification of lipid moleculesfrom whole microbial cells cultivated on a solid medium [19,20].Therefore, in order to check that the presence of both surfactinand fengycin operons detected by PCR was related to an effectiveexpression, MALDI-TOF-MSanalysiswasperformedon whole cellsofB.mojavensisA21 cultivated onLBsolidmedium. Fig.2showstheMALDI-TOF mass spectra of intact cells of this strain. Mass spec-

tra analysis showed two well resolved clusters of peaks, one atm/z values between 1045 and 1080 (Fig. 2A) and the other one inthe range of 14801550 (Fig. 2B). By comparing the mass with themass numbers reported for the lipopeptide complexes from otherBacillus strains [21], the first group of peaks could be attributedto surfactin isomers, while the second cluster of peaks could beassigned to fengycin isomers. Our results are in line with previousworksreported by Coutteet al. [22] andKim etal. [23] that showedthe co-production of different homologous compounds for eachlipopeptide family. These isoforms vary according to the lengthof their fatty acid side chains as well as the peptide amino acidcomposition.

The mass spectra reported in Fig. 2A revealed the presence ofthree major [M+K]+ peaks at m/z 1046.66, 1060.69 and 1074.73.

The peak with a m/z1046.66 corresponded to the mass of [M+K]+ion of surfactin with a fatty acid chain length of 13 carbon atoms,or pumilacidin C12. The peaks at m/z1060.69 and 1074.73 couldbe assigned as potassium adduct of surfactin C14 and C15, respec-tively, or pumilacidin C13 and C14, respectively (Fig. 2A).

On the other hand, the mass spectra reported in Fig. 2B showthe presence of 4 major peaks at m/z1487.14, 1501.83, 1515.88and 1529.88. These peaks could be attributed to [M+K]+ formsof fengycin AC15, AC16/BC14, AC17/BC15 and AC18/BC16, respec-tively (Fig. 2B). For each group, the different peaks differ by 14Da,which corresponds to the molecular weight of one CH2 group.

The results obtained with the PCR and MALDI-TOF-MS tech-niques confirmed the complementarities of these approaches.This is in contrast with results obtained by Leenders et al. [24]

who detected by PCR conserved genes encoding surfactin and

fengycin synthetase in B. subtilis 168; however, no lipopeptideswere detected using MALDI-TOF-MS, owing to the presence of amutated sfp- gene.

3.3. Characterization of surfactin/pumilacidin lipopeptides by

MS-MS analysis

MALDI-TOF MS2 analysis was also used in order to obtain moreprecise information on the chemical structure of some lipopep-tides. The fragment ion patterns of the parent ions atm/z1060.77and 1074.68, reported in Fig. 3, shows fragments that can cor-respond to differences among some amino acids in the peptidemoiety. A set of daughter ions was observed in the MS/MS spec-trum of the parent ion atm/z1060.77. The fragmentation patternof the peak 1060.77, illustrated in Fig. 3A, resulted in the appear-

ance of product ions at m/z 947.68 [M(A7 =113)] and 834.68[M(A7+ A6)]. These two peaks are the results of the consecu-tive losses of two Leu (or Ile) residues. Therefore, the amino acidresidue at position 7 is a Leu or Ile and not a Val residue. Otherpeaks were observed at m/z 737.64 [M(AA1+ FA)] and 624.73[M(AA1+ AA2+ FA)]. The obtained results indicatedthat the peakat m/z1060.77 is unambiguously a pumilacidin, a cyclic lipopep-tide with a fatty acid chain of 13 carbons, and Leu or Ile residue atposition 7.

However, there is more ambiguity with the parent ion at m/z1074.68. Indeed, on the basis of the obtained fragmentation, dis-played in Fig. 3B, this peak could be made up of three differentmolecules: surfactin Ile7/Leu7 C15, pumilacidin Ile7/Leu7 C14 andpumilacidin Val7 C15 (Fig. 3B).

The results obtained with the PCR and MALDI-TOF-MS tech-niques demonstrated that the strain B. mojavensis A21 producestwo families of lipopeptides with different homologous com-pounds. Lipopeptides belonging to surfactin and fengycin familieswere reported both in B. subtilis and Bacillus amyloliquefaciensstrains, together often with a third lipopeptide of the iturinsgroup. The co-production of surfactins and fengycins families byB. mojavensis A21 is an interesting characteristic which could sup-port their potential applications in many biotechnological fields[25]. Further, the structural diversity of the produced lipopep-tides may offer several potential applications. Indeed, it is wellknown that different isoforms and homologues exhibit differentproperties and activities, which depend in particular on the chainlength. Surfactins are known for their ability to act in a syner-

gistic manner with fengycin which may improve their activities[25]. The increasing interest in surfactinand fengycin is because oftheir amphiphilic character, which is responsible for their excel-lent surface-active properties. Surfactin, one of the most effectivebiosurfactants, showed several pharmacological activities includ-ing, antimicrobial, antiviral, antitumoral and antifibrinolytic [26].Fengycin shows specific antifungal activity against filamentousfungi andinhibitsphospholipaseA2 [27]. In anotherstudyreportedby Ongena et al. [28] the lipopeptide mixture contained surfactinand fengycin was found to be effective in the induction of sys-temic resistance in plants which makes them interesting for plantprotection.

Several works reported also the co-production of differentlipopeptides families. In this context, B. subtilis strains are known

to produce up to three families of lipopeptides [5]. For example,


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Fig. 2. Mass spectroscopy(MALDI-TOF-MS) spectra of molecular mass of strain A21 lipopeptides. Spectra of surfactin(A) and fengycin (B)produced by B. mojavensisA21.

B. subtilis ATCC 6633 and its derivative BBG100 were found toproduce surfactins and mycosubtilin, while ATCC 9943 secretesthree lipopeptides families: surfactins, iturin A and fengycins [15].The simultaneous production of two different lipopeptides hasalso been reported by Ahimou et al. [29] for B. subtilis. AnotherB. amyloliquefaciens strainwasalso found to produce surfactinanditurin-like compounds [30].

Although the surfactin production by B. mojavensis strain hasbeen reported [10], the coproductionof surfactinand fengycin hasbeen reported only byfewspecies, inparticular byB. subtilis strains[31]. Both of these lipopeptides are of great pharmaceutical andbiotechnological interest [26,32].

3.4. Lipopeptide production, antimicrobial activity and stability

studies of the crude lipopeptide mixture

3.4.1. Lipopeptide production

B. mojavensis A21 was cultivated in Landy medium for 72h at30 C. In this medium, glucose is used mainly as carbon sourcefor bacteriagrowth and lipopeptides production. The fermentationdata with respect to time indicating the changes in glucose con-centration, biomass, and products, as well as surface tension wasshown in Fig. 4. A direct relationship between microbial growth,surface tension reduction, glucose consumption and lipopeptideproductionwas observed. In fact, thesurface tension of theculture


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Fig. 3. MALDI-TOF MS/MS spectrum of lipopeptide produced by B. mojavensisA21. (A) MALDI-TOF MS/MS at m/z= 1074.9; and (B)MALDI-TOF MS/MS atm/z=1060.88.


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Fig. 4. Kinetics of growth, surface tension reduction, lipopeptide productionand glucose consumption by B. mojavensisA21strain. Culture wasperformed in Landymediumat 30 C for72h.

was reduced from 71 at the beginning of growth to 31mNm1

after 48h of cultivation and then remained nearly constant untilthe endof fermentation(72h).Based on HPLC analysis the concen-tration of fengycin produced was clearly higher than the surfactinone. Indeed, fengycin concentration was about 2.5 fold higherthansurfactin after 72h of cultivation. The surfactin production wasgrowth-associated and began early in the exponential phase up tothestationaryphase. However, thefengycinproductionbegan after12h till the end of the culture.

Thegrowth-associated relationship in surfactinproduction wasreported in some other B. subtilis strains [33]. Deleu et al. [34]

showed that surfactin is usually produced earlier than fengycin.A direct relationship between glucose consumption andlipopeptide production was observed and at the end of fermen-tation (72 h), the total amounts of glucose was consumed. Indeed,thecomplete consumption of glucose occurred coinciding withthemaximum production of lipopeptide.

In this study, lipopeptidesproduced by B. mojavensis A21 strainwere isolated from the cell-free supernatant by different stepsof ultrafiltration/diafiltration based on their aggregation behavior.Ultrafiltration (UF) wascarried outwith 10kDa cut-off membrane.The partially purified lipopeptide was tested for its antimicrobialactivity and its stability against extreme conditions was investi-gated.

3.4.2. Antimicrobial activityThe antibacterial activity of the crude lipopeptide produced

by B. mojavensis A21 was tested against a variety of microor-ganisms. Inhibition zones diameters were measured using theagar well diffusion method. Table 3 summarizes the antimicrobialactivity spectrum of the crude partially purified lipopeptide. Thecrude lipopeptide exhibited varying degrees of antibacterial activ-ity against all strains tested. Further, this inhibitory activity wasmore effective against Gram-positive bacteria compared to Gram-negative bacteria. Indeed, the diameters of the inhibition zoneswere in the range of 711mm and 1322 mm with Gram-negativeand Gram-positive bacteria, respectively. M. luteus and S. aureuswerethemostsensitivebacteria.Furthermore, lipopeptideA21wasfound toexhibit inhibitoryactivityagainstM. rouxiiandA. niger,but

had no effect onB. cinerea.

Table 3

Antimicrobial activity spectrum ofB. mojavensisA21 crude lipopeptides.

Indicator organism Inhibition Zonediameter (mm)

Gram (+) S. aureus (ATCC25923)B. cereus (ATCC 11778)E. faecalis (ATCC29212)M. luteus (ATCC4698)

211.0131.0162.0221.0

Gram () P. aeruginosa (ATCC 27853)K. pneumoniae (ATCC 13883)E. coli (ATCC 29212)S. typhimurium (ATCC 19430)

72.091.0111.0111.1

Fungi A. niger

B. cinereaM. rouxii

+

++

Determinations were performed in triplicate and data correspond to mean val-ues standard deviations.

Although antimicrobial activity was detected for most ofBacil-lus strains, there was considerable difference in their spectra anddegrees of inhibition. The highantibacterial and anti-fungal activi-ties of A21 crude lipopeptide may be related to a synergistic effectof both surfactins and fengycins.

3.4.3. Effects of proteolytic enzymes, heat, pH and organic

solvents on antibacterial lipopeptide activity

The resistance of lipopeptides against proteases and extremeconditions, including pH and temperature was a prerequisitefor their potential biomedical and biotechnological applications.Therefore, to check the stability, the crude lipopeptide mixturewas subjected to different conditions and its residual antimicro-bial activity was evaluated using S. aureus as indicator strain. Theobtained results are summarized in Table 4 and Fig. S1. The sta-bility of the crude lipopeptide against several proteolytic enzymeswas evaluated. Interestingly, all proteases tested had no effect onthe antimicrobial activity of the crude lipopeptide after incuba-tion for 2h at 37 C, since there was no significant difference inthe inhibition zone diameter compared to control (without enzy-matic treatment). This indicates that theantimicrobial compoundscouldbe cyclic peptidescontainingunusual amino acids [35]. These

results suggest that A21 lipopeptides possibly can survive at the


8/9


Table 4

Effects of enzymes, heat and organic solvents on antimicrobialactivity.

Treatment Inhibition Zone diameter (mm)

Enzyme (1mg/ml)

Trypsin 21 1.0Pepsin 19 1.0Chymotrypsin 18 1.0Bromelain 19 2.0Alcalase 17 1.1

Temperature (C)a

60 21 2.070 21 1.080 19 1.090 18 1.0100 18 2.0Freeze-dried 21 1.0

Organic solvents

Chloroform 13 2.0Acetonitrile 21 1.0Methanol 21 1.0

Determinations were performed in triplicate and data correspond to mean val-ues standard deviations.

a Incubations at different temperaturewere realized during 15min.

intestinal environment and could be administered with feed. Theobtained results exclude the eventual existence of bacteriocin-likesubstances, which may contribute to the inhibitory activities;since the crude lipopeptide mixture was resistant to proteolyticenzymes. Further, the stability of lipopeptides against proteolyticenzymes is also supportedby itshigh stabilityagainst proteases(atleast six) produced by A21 strain [12].

Moreover, one of the significant findings of this study was thethermostability of the antimicrobial activity of lipopeptides pro-duced by B. mojavensis A21. Antimicrobial activity of the crudelipopeptide against S. aureus remained highly stable after 15minincubation attemperatures from 60to 80 C,whilea slight decreasein activity was observed at 90 and 100 C (Table 4).

The activity of lipopeptides A21 at different pH values was also

determined (Fig.S1). The obtained results suggestedthatpHhasnosignificant effect on the antimicrobial activity. The highest activ-ity was observed at pH 7.0 and 8.0. The activity decreased slightlybelow pH 6.0 and above pH 9.0. A similar stability profile wasalso observed for lipopeptide biosurfactant produced by Serratiamarcescens NSK-1 [36].

In order to provide some information about the organic solventwhich can be used for lipopeptide extraction, the crude lipopep-tide produced by B. mojavensis A21 was incubated in the presenceof several organic solvents. It was observed that the addition ofmethanol and acetonitrile, during 15min at room temperature,showed no appreciable effect on lipopeptide activity.However, theactivity decreased after incubation with chloroform, with an inhi-bition zone of 16mm, while that of control was 21mm (Table 4).

Further, lipopeptides produced by B. mojavensis A21 exhib-ited high pH and thermal stability. Heat stability is potentially auseful characteristicduring food preservation because many food-processing procedures involve a heating step.

4. Conclusion

The presenceofNRPS geneswas detectedbyPCRusing degener-ate primers. In addition, the produced lipopeptides were detectedand their structures were more precisely characterized by MALDI-TOF-MS onwholecellsof A21strain.Thecrudelipopeptidemixturewas found to be mainlyconstituted of surfactins, pumilacidins andfengycins.

The lipopeptide mixture exhibited a strong antimicrobial activ-

ity. Further, lipopeptides showed high stability under various

extreme conditions. They were quite stable over a wide temper-ature range from 60 to 100 C, and were highly active over a widerange of pH from 3.0 to 11.0. In addition, theantimicrobial activitywas not affected by exposure to organic solvents for 1h at 37C.These results indicated the robust stability of lipopeptides A21under extreme conditions.

In the light of all these results, the lipopeptides from strain A21could be excellent candidates for application in food and pharma-ceuticals industries.

Acknowledgement

This work was funded by the Ministry of Higher Education andScientific Research-Tunisia.

Appendix A. Supplementary data

Supplementary data associated with this article canbe found, inthe online version, at doi:10.1016/j.procbio.2014.07.001.

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Hanen, 2014, B Mojavensis