Lipopeptide surfactants: Production, recovery and pore forming capacity

Accepted Manuscript Title: Lipopeptide surfactants: Production, Recovery and Pore Forming Capacity Author: Mnif In`es Ghribi Dhouha PII: DOI: Reference:

S0196-9781(15)00202-8 http://dx.doi.org/doi:10.1016/j.peptides.2015.07.006 PEP 69509

To appear in:

Peptides

Received date: Revised date: Accepted date:

13-3-2015 30-6-2015 3-7-2015

Please cite this article as: Ingravees Mnif, surfactants: Production, Recovery and Pore http://dx.doi.org/10.1016/j.peptides.2015.07.006

Dhouha Ghribi.Lipopeptide Forming Capacity.Peptides

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Lipopeptide surfactants: Production, Recovery and Pore Forming Capacity Mnif Inès 1,2,*; Ghribi Dhouha 1,2 1

Higher Institute of Biotechnology

2

Unit Enzymes and Bioconversion, National School of Engineers

*Correspondence to: Inès Mnif, Unité « Enzyme et Bioconversion », ENIS, BP W 3038 Sfax, Tunisia E-mail address: [email protected] Tel: 216 74674364, Fax: 216 74675055

Runing title: Lipoprotein Surfactants Production and Applications

Highlights

Lipopeptides structural diversity (Surfactin, Iturin, Fengycin …) Main factors affecting Lipoptides production Lipopeptides extraction and purification Pore Forming Capacity and related applications of lipopeptides Antimicrobial, hemolytic and antitumor activities of lipopeptides Potential applications of lipopeptides in biomediacal, pharmaceutic and agriculture fields Abstract

Lipopeptides are microbial surface active compounds produced by a wide variety of bacteria, fungi and yeast. They are characterized by highly structural diversity and have the ability to decrease the surface and interfacial tension at the surface and interface, respectively. Surfactin, Iturin and Fengycin of Bacillus subtilis are among the most studied lipopeptides. This review will present the main factors encountering lipopeptides production along with the techniques developed for their extraction and purification. Moreover, we will discuss their ability to form pores and destabilize biological membrane permiting their use as antimicrobial, hemolytic and antitumor agents. These open great potential applications in biomediacal, pharmaceutic and agriculture fields. Keywords: Lipopeptides; Surfactants; Production; Biosynthesis; Extraction; Purification; Biological activities; Antimicrobials

Introduction Biosurfactants are microbial derived surface-active compounds produced by a wide variety of microorganisms. They decrease the surface and interfacial tension between individual molecules in the surface and interface, respectively, and they are endowed with diverse biological activities (such as antimicrobial; antiviral; hemolytic and insecticide) [1]. They are amphiphilic compounds with hydrophilic (amino-acid or peptides; di- or polysaccharides; anions or cations) and hydrophobic moieties (saturated or unsaturated fatty acid). Rhamnolipids of P. aeruginosa, Surfactin of B. subtilis, Emulsan of Acinetobacter

calcoaceticus and sophorolipids of Candida bombicola are among the most recognized biosurfactants [1]. Lipopeptides are extensively studied. Structurally, they are constituted by a fatty acid in combination with a peptide moiety and correspond to a group of isoforms that differs by the composition of the peptide moiety, the length of the fatty acid chain and the link between the two parts (Figure 1). Several isoforms can be produced by the same strain. For example, Sang et al. [2] reported the coproduction of Surfactin, Iturin and Fengycin by B. subtilis JKK328 and Xia et al. [3] depicted their co-production by Pseudomonas sp. WJ6. The halophilic strain B. subtilis BBK-1 coproduced Surfactin, Plipastatin and Bacillomycin L [4] and B. licheniformis F2.2 coproduced Plipastatin and Surfactin [5]. Bacillus related lipopeptides [6] and Pseudomonas related lipopeptides [7] are the most studied. In addition, lipopeptides can be produced by actinomycete [8,9] and diverse fungal strains [10,11]. Surfactin, Iturin, Fengycin and Lichenysin are among the most documented lipopeptides. Also, Viscosin, Tensin, Arthrofactin, Pseudofactin and Syringomycin mainly produced by Pseudomonas isolates are widely described in literature reviews. Generally, lipopeptides surfactants are characterized by their low critical micelle concentration (CMC), the concentration of detergent above which monomers self-assemble into non-covalent aggregates (called micelles) and an abrupt decrease of surface tension occurred [12]. They are also characterized by diverse functional properties (such as emulsification/de-emulsification, dispersing, foaming, viscosity reducers, solubilizing and mobilizing agents and pore forming capacity) permitting their use in many domains [1]. Besides, lipopeptides biosurfactants have very advantageous over synthetic emulsifiers; low toxicity, higher biodegradability and higher efficiency towards extreme temperature, pH and salinity offering great opportunities as replacements for chemical surfactants [13].

Owing their attractive functional properties and their various biological activities enlarging their use in numerous domains, scientists were interested in lipopeptides production. They studied the main factors affecting their production yield along with the developed strategies allowing biosurfactant production improvement. Furthermore, research interest has focused on lipopeptide recovery. This review deals in its first part with the latest research and development in lipopeptides type surfactants, including nutritional and physicochemical factors affecting their production, means of production improvement, mechanisms of biosynthesis and techniques of their extraction and purification. In a second part, we will concentrate on lipopeptide pore forming capacities along with potential biological activities including their antimicrobial, antitumor and hemolytic potencies.

Lipopeptide surfactants production Microbial derived surface active lipopeptides are generally produced by fermentation using micro-organisms on water miscible or immiscible substrates. They can be produced by bacteria, molds or yeast. For increasing biosurfactant yield, optimal addition of media components and selection of the optimal culture conditions will induce the maximum or the optimum productivity. In fact, nutritional and physicochemical parameters like pH, temperature and aeration can influence lipopetides production and therefore the fermentation cost. Also, the production on low cost substrates permitted the reduction of the production cost. The enhancement of lipopeptide production can be achieved by means of the optimization of lipopeptide production using response surface methodology and the development of overproducing microbial mutants. Generally, lipopeptide surfactants are produced by aerobic micro-organisms; bacteria [14–18], mods [19–22], yeasts [23] and actinomycetes [8,9]. Surfactin [24], Iturin [25,26] and Fengycin [15,27] derived from Bacillus species are the most recognized. Also,

Pseudomonas species produce a wide variety of lipopeptides compounds like Tensin [28], Pseudophomin [29] and Massetolid [30] of P. fluorescens, Pseudodesmin of P. tolaassii [31], Xantholysin of P. putida [32] and Syringomycin of P. syringae [33]. Similarly, Polymyxin derived from Paenibacillus species are well reported in previous studies [34,35]. Moreover, actinomycete species were documented to produce a wide variety of antimicrobial lipopeptides [36]. Amphotericin, Laspartomycin, Aspartocin, Daptomycin and Amphomycin derived from Streptomyces species and Friulimicin derived from Actinoplanes are well recognized by their antimicrobial activities [36]. Lipopeptide surfactants are naturally produced like mixture of various macromolecules belonging to the same family or class. In fact, B. subtilis JKK328 [2] and B. subtilis S499 [43] can produce the three types of homologous (Surfactin, Iturin and Fengycin). B. subtilis BBK1 coproduces Surfactin, Plipastatin and Bacillomycin L [4], B. licheniformis F2.2 strain coproduce Plipastatin and Surfactin [5] and B. subtilis JKK238 coproduce Iturin, Fengycin and Surfactin [2]. Generally, nutritional parameters can influence the nature of the produced lipopeptide [44]. Lipopeptides can be produced on media containing hydrophilic carbon sources like carbohydrates [37,38]. However, they can be produced by certain microorganisms during their growth on water immiscible substrates like hydrocarbons [37,39–41] and vegetable oils [23]. In fact, they are produced to permit micro-organisms growth on water immiscible substrates. Having the ability to reduce interfacial tension between water and the hydrophobic substrates, lipopeptides enhanced their uptake [42]. In addition to the carbon source, divalent cations addition was reported to have a great influence on lipopeptides production. Abushady et al. [45] described that the addition of Mn2+ and Fe2+ stimulated Surfactin production by B. subtilis. Similarly, it was demonstrated that

Surfactin production by B. subtilis ATCC 21332 can be enhanced from 0.33 g/l to 2.6 g/l by the addition of 0.01 mM Mn2+ in the medium culture [46]. Also, Hong et al. [47] described the stimulation of Surfactin production by the addition of 4 mM Fe2+. Moreover, the presence of 1 mM ZnSO4, 1 mM FeCl3 and 0.1 mM MnSO4 increased enormously Surfactin production by B. subtilis BS5 [48]. Lin et al. [49] reported a dose dependant manner stimulation of Iturin by the addition of divalent cations Fe2+ and Mg2+. Certain physicochemical factors like temperature of incubation, initial pH of the culture medium and the speed of agitation can affect lipopeptides production. In fact, lipopeptides production can be accomplished in temperature ranging from 25 to 45 °C. Temperatures of about 30 °C and 28 °C favoured a maximum of Surfactin production by B. subtilis [45] and B. coagulus [50], respectively. For B. natto, Surfactin production was better at 37 °C [51]. Also, the temperature of incubation can affect the nature of the isomers produced. For B. subtilis RB14, an optimum of Iturin A was at 25 °C, however, at 37 °C, Surfactin was produced by the same strain [52]. Similarly, temperature can have an effect on the nature of the homologues produced. In fact, when cultivating B. subtilis RB14, an increase of temperature induced the production of Iturin A with a fatty acid chain length of 16 carbons [52]. The initial pH value of the culture medium can also have a great effect on lipopeptide production by some strains. The control of pH during fermentation is an essential criterion to maintain an optimal production rate [53]. Generally, a neutral pH favoured an optimum lipopeptide production. For B. subtilis BS5, it’s between 6.5 and 6.8 [48]. However, Surfactin production by B. coagulans can be realized at pH ranging from 4 to 7.5 [50]. Enhancement of lipopeptide surfactants production Use of low-cost raw materials

Enhancement of the production yield is the major bottleneck in lipopeptide surfactants production, as in the case with most biotechnological processes. Often the amount and type of a raw material an contribute considerably to the production cost; it is estimated that raw materials account for 10–30 % of the total production cost in most biotechnological processes [1]. Thus the use of low-cost agro-based byproducts can reduce considerably the production cost. A variety of cheap raw materials, including plant-derived oils, oil wastes, starchy substances, lactic whey and distillery wastes have been reported to support biosurfactant production. Previous studies reported the use of rice bran [54], soy bean flour [55,56], soybean curd residue [57], potato process effluents [55], potato peels [58,59], sesame peel flour [60], orange peels [61], millet flour [62] tuna fish flour [59], molasses and whey [63], tuna fish cooking residue [60] and soybean sauce [64] for lipopeptide production. Also, numerous studies reported the production of lipopeptide using cassava wastewater as substrate [65–67]. Mutant and recombinant strains: hyperproducers The genetics of the producer organism is an important factor affecting the yield of all biotechnological products, because the capacity to produce a metabolite is bestowed by the genes of the organism [1]. The bioindustrial production process is often dependent on the use of hyperproducing microbial strains, even with cheap raw materials, optimized medium, culture conditions and efficient recovery processes [1]. Generally, a production process cannot be made commercially viable and profitable until the yield of the final product by the producer organisms is naturally high. The realization of mutant and recombinant varieties present a promising strategy to enhance lipopeptide surfactants production. In this aim, a few mutant and recombinant varieties with enhanced biosurfactant production characteristics are reported in the literature. These mutant varieties were produced using various physicochemical agents. In fact, Surfactin production by B. subtilis ATCC 55033 was

enhanced four to five times by the application of N-methyl-N′-nitro-N-nitrosoguanidine treatment [68]. Similarly, using the same agent, production of Surfactin by B. licheniformis KGL11 was enhanced by about 12 times [69]. As suggested by Mulligan et al. [70] ultraviolet mutation of B. subtilis ATCC 21332 yielded a stable mutant that produced over three times more of the biosurfactant, Surfactin, than the parent strain. Also, different patents describing the use of mutant for enhanced lipopeptide production were developed [71,72]. In addition to these mutant-hyperproducing varieties, several recombinant strains producing lipopeptides in better yields and showing improved production properties have been developed in recent years. The cloning of sfp genes originating from B. subtilis RB14 in B. subtilis MI113 (pC112) permitted an enhancement of Surfactin production of about 60 % [73]. Similarly, the cloning of sfp gene from B. subtilis C9 in B. subtilis SB103 permit a great improvement of Surfactin production by the recombinant strain [74]. Leclère et al. [75] reported the generation of an over-producing recombinant strain B. subtilis BBG100 obtained from strain ATCC 6633 by replacement of the native promoter of the Mycosubtilin operon by a constitutive promoter originating from the replication gene repU of the Staphylococcus aureus plasmid pUB110. It produced up to 15-fold more Mycosubtilin than the wild type produced. Zhao et al. [76] used the genome shuffling approach for improving antimicrobial lipopeptide production by B. amyloliquefaciens permitting the generation of mutant strain F238 that exhibited 3.5- and 10.3-fold increases in Surfactin production in shake flask and fermenter, respectively. Biosynthesis of lipopeptide surfactants In the microbial community, there is a high diversity of cyclic lipopeptides (CLP) structures and producing microorganisms. The differences in chemical structures suggest that the CLP compounds may serve different and possibly multiple purposes. Among the bacterial

genera, Bacillus (Gram-positive) and Pseudomonas (Gram-negative) have received the most attention because they produce a wide range of effective lipopeptide surfactants that are potentially useful for agricultural, chemical, food and pharmaceutical industries. The biosynthetic mechanisms and gene regulation systems of lipopeptide surfactants have been extensively analyzed over the last decade. Generally, lipopeptides resulted from the condensation of peptidic and lipidic moieties synthetized independently. Peptides are generally synthesized in a ribosome-independent manner with megaenzymes called NonRibosomal Peptide Synthetases (NRPSs) [77]. They lead to a remarkable heterogeneity among the lipopeptides products generated by Bacillus with regards to the type and sequence of amino acid residues, the nature of the peptide cyclization and the nature, length and branching of the fatty acid chain. Lipopeptides are classified into three families depending on their amino acid sequence: Iturin, Fengycin and Surfactin [25,78] (Figure 1). These antibiotics are either cyclopeptide (Iturin) or macrolactones (Fengycin and Surfactin) characterized by the presence of L and D amino acids and variable hydrophobic tails [78]. The latter compounds are amphiphilic cyclic peptides composed of 7 α-amino acids (Surfactin and Iturin) or 10 α-amino acids (Fengycin) linked to one unique β-amino fatty acid (Iturin) or βhydroxy fatty acid (Surfactin and Fengycin) [78]. The length of the fatty acid chain varies from C13 to C16 for Surfactin, from C14 to C17 for Iturin and from C14 to C18 in the case of Fengycin [78]. A fourth class of lipopeptide compound called Kurstatin is also described [78]. Different homologous compounds for each lipopeptide family are thus usually coproduced [25,79]. In addition to Bacillus-derived lipopeptides, biosurfactant belonging to lipopeptide compounds can be also produced by a wide variety of gram negative isolates belonging to Pseudomonas. They are well recognized and are composed of a short oligopeptide with a linked fatty acid tail. Important determinants of the activity spectrum of cyclic lipopeptides

include the number, nature, position and configuration (L or D form) of the amino acids in the macrocyclic peptide ring as well as the type and length of the fatty acid tail [7] (Figure 1). Actinomycetes are commonly known by the production of cyclic lipopeptide antibiotics. These compounds include Friulimicin produced by Actinoplanes friuliensis [80], Amphomycin produced by Streptomyces canus [81] and Laspartomycin produced by S. viridochromogenes [82]. Structurally, they are composed of 11 amino acid residues among which are 10 residues forming a ring structure and an exocyclic aspartic acid residue linked with an acyl group [82]. All of these antibiotics have similar structures with respect to their amino acid composition and conserved residues, but they differ in the amino acid sequence, having one to three non-conservative residues and varied lipid tails (chain length, position of the double bond and configuration) [82]. Production of active-form lipopeptides requires not only transcriptional induction and translation but also post-translational modifications and assemblage [77]. Here, we will provide an overview of the biosynthetic pathways of lipopeptide surfactants in Bacillus genera including the synthesis of the peptide moiety, the synthesis of the lipid moiety and the condensation of the two parts. Biosynthesis of the peptide moiety NRPSs are multi-modular enzymes that recognize, activate, modify and link the amino acid intermediates to the produced peptide [83]. They are effective in synthesizing peptides containing unusual amino acids including D-amino acids, β-amino acids and hydroxy- or Nmethylated amino acids [77]. So, Non-Ribosomal Peptides presented a high structural diversity along with important biological activities [77]. Generally, synthetases are organized on module subdivised on domains. Each module permitted the incorporation of a specific amino-acid in the peptidic moiety. Each domain has a particular function leading to the

incorporation of the amino-acid. In fact, as described by Roongsawang et al. [77], there are four different domains. A first Adenylation domain (A) permitted the recognition of the specific amino-acid and its activation by an adenylation reaction (the amino-acid is converted to an aminoacyl adenylate). A second domain of Thiolation (T) permitted the fixation of the amino-acid on the synthetase by a covalent manner by a thioester linkage. A third domain of Condensation (C) permitted the formation of a peptidic linkage between two consecutive amino-acids. And a fourth domain of Thioesterase (Te) that permitted the liberation of neoformed peptide. These domains formed three categories of modules [77]. A first module of Initiation composed of the domain A and domain T allowed the initiation of peptide biosynthesis. A second module of Elongation composed of the domains C, A and T accomplished the elongation of the peptidic moiety. Finally, an end module of Termination composed of the four domains C, A, T and Te permitted the incorporation of the final aminoacid and the liberation of the peptide. In addition, other secondary domains allow amino-acid modifications like epimerisation (transformation of monomers from L configuration to D configuration), methylation (addition of a methyl group) and formylation (addition of a formyl group) [84]. Moreover, there are condensation domains that have epimerisation activities called dual domain of Condensation/Epimerization. They permit the epimerization of the amino-acid before the formation of a peptidic linkage with the adjacent amino-acid. Another domain is the domain of Heterocyclisation (Cy). Biosynthesis of the lipid part The biosynthesis of the lipid part of the lipopeptide is accomplished by numerous consecutive modules that permitted the coupling, the activation and the linkage of the fatty acid to the peptide moiety. According to Duitman et al. [85], five steps are required. Firstly, the domaine Acyl CoA-ligase (AL) coupled a coenzyme A to a fatty acid (an ATP-dépendant reaction). The activated fatty acid is afer that transferred to the cofactor 4-Phosphopantethein

of the first domain Acyl-carrier protein (ACP1). Similarly, a malonyl CoA is fixed to the ACP2 domain. The condensation of the malonyl and acyl thioesters catalyzed by ß-ketoacyl (KS) synthetase domain lead to the formation of a ß-ketoacyl thioester. The ß-ketoacyl thioester is converted to a ß-amino fatty acid by transamination. The ß-amino fatty acid can be transferred to the Thiolation domain by the Condensation domain. It can be coupled to the amino acid fixed on the first module of the biosynthesis of the peptidic moiety by an aminotransferase. After the biosynthesis, the lipopeptide can be submitted to modifications like glycosylation or halogenation realized by specific enzymes associated to the synthetases.

Dowstream

processing:

extraction

and

purification

of

lipopeptide

surfactants Often, incorporation of biosurfactants in an industrial process or its application in medical, pharmaceutical, agriculture and environmental field requires its retention from the fermentation supernatant and its purification. In fact, the production process still incomplete without an efficient and economical means for recovery of the products. The intense foaming produced during aerobic sparged culture is a big obstacle for the commercialization of these surface active compounds making their recovery and purification from complex fermentation broth difficult [86]. However, a great deal of monetary input is required in the purification processes [87]. Generally, the downstream processing costs account for approximately 60 % of the total production costs. During all these process, the risk of contamination with undesired compound from fermentation procedures always exist. Ionic charge (chromatography), solubility (water/organic solvents) and location (intracellular, extracellular, cell bound) ultimately determine the purification procedure for biosurfactants/bioemulsifiers to be extracted [87]. Also, they are designated in accordance to the physicochemical properties, the

potential use of the microbial derived surface active compounds and its wanted degree of purity. Several techniques were developed to extract and purify lipopeptide surfactants. Always, the aim of the extraction is to obtain a crude extract from aqueous culture medium. Among the most used techniques for lipopeptides extraction, we can present the Acid Precipitation, the Solvent Extraction, Ammonium Sulphate Precipitation and Foam Fractionation (Table 1). Generally, a combination of Acid Precipitation and Solvent Extraction was also followed [89,90]. Ammonium Sulphate Precipitation is always pursued by Dialysis to remove any small molecules that may be present [91]. For purification, various strategies were developed such as Membrane Ultrafiltration techniques, Ionic Exchange Chromatography and Adsorption-Desorption on resins or on wood activated carbon like Charcoal. Also, developed techniques like High Performance Liquid Chromatography, Hydrophobic Interaction Chromatography and Gel Filtration are well used to fractionate and purify lipopeptides compounds. Generally, HPLC is an excellent method for the separation of individual peptide/lipopeptide biosurfactants [88]. The most commonly employed technique is

Reversed

Phase

Chromatography,

which

results

in

the

separation

of each

peptide/lipopeptide structure based on polarity [88]. The separated products are detected by ultraviolet absorbance detection and each individual peak can be collected using a fraction collector for further analysis of their structure [88]. Table 2 describes the main purification techniques used along with their advantages and examples from previous studies. Often a single downstream processing technique is not enough for product recovery and purification [1]. In such cases and as suggested by Muthusamy et al. [1], a multi-step recovery strategy, using a sequence of concentration and purification steps, is more effective. In such a multi-step recovery for biosurfactants, it will be possible to obtain the product at any required degree of purity.

Pore forming capacity of lipopeptide surfactants and related application Lipopeptides are well known by their membrane permeabilization properties as they can induce pore and ion channels formation into the membrane lipid bilayer. As suggested by Bernheimer et al. [112] Surfactin, one of the most powerful biosurfactants, is known to destabilize membranes disturbing their integrity and permeability. Generally, membrane destabilization and leakage begin by the dimerization of Surfactin into bilayer [113] followed by an insertion into the lipid bilayers, chelating mono- and divalent cations and modification of membrane permeability by channel formation or membrane solubilization by a detergentlike mechanism [114]. Recently, data have shown that pore formation in membranes occurs after lipopeptide oligomer binding, some of which are Ca2+ dependent multimers [115]. These pores may cause transmembrane ion influxes, including Na+ and K+, which result in membrane disruption and cell death [115]. Heerklotz et al. [116] compared the structural effects of Surfactin on lipid membranes with those of two nonionic detergents, C12EO6 and C12EO8, by means of solid-state NMR of selectively deuterated lipids. The detergents exhibit the expected behavior of increasing the lateral pressure in the headgroup region and disordering the acyl chains [116]. In contrast, the strong activity of Surfactin to destabilize membranes is not reflected in an extreme disordering of the fatty acyl chains [116]. However, Surfactin tilts the acyl chains of the lipid and leads to a reorientation of the lipid headgroup toward the membrane interior [116]. These effects provide evidence for a rather deep insertion of the peptide moiety into the hydrophobic-hydrophilic interface of the membrane. The results are discussed in terms of the molecular parameters governing the activity of a molecule to destabilize lipid membranes and the activity of antibiotic peptides to induce unspecific leakage of membranes [116].

The detergent effect draws on Surfactin's ability to insert its fatty acid chain into the bilipidic layer causing disorganization leading to membrane permeability [117]. Insertion of several Surfactin molecules into the membrane can lead to the formation of mixed micelles by self-association and bilayer influenced by fatty chain hydrophobicity ultimately leading to bilayer solubilization [118]. Carrillo et al. [113] studied the molecular mechanism of membrane permeabilization by incorporation of Surfactin to explain Surfactin-induced ‘pore’. The Surfactin-induced vesicle contents leakage was monitored by following release of carboxyfluorescein entrapped into unilamellar vesicles made of palmitoyloleoylphosphatidylcholine (POPC) [113]. The effect of the

addition

of

cholesterol,

dipalmitoylphosphatidylcholine

(DPPC)

and

palmitoyloleoylphosphatidylethanolamine (POPE) was also checked [113]. Electron microscopy showed that Surfactin was able to induce content leakage. Cholesterol and POPE attenuated the membrane-perturbing effect of Surfactin, whereas the effect of DPPC was to promote Surfactin-induced leakage, indicating that bilayer sensitivity to Surfactin increases with the lipid tendency to form lamellar phases, which is in agreement with our previous observation that Surfactin destabilizes the inverted-hexagonal structure [113]. Fouriertransform infrared spectroscopy (FTIR) was used to specifically follow the effect of Surfactin on different parts of the phospholipid bilayer [113]. The effect on the CMO stretching mode of vibration of POPC indicated a strong dehydration induced by Surfactin [113]. On the other hand, the CUH stretching bands showed that the lipopeptide interacts with the phospholipid acyl chains, resulting in considerable membrane fluidization [113]. In a study conducted by Heerklotz and Seelig [119], the leakage and lysis of POPC vesicles induced by Surfactin was studied using calcein fluorescence de-quenching, isothermal titration calorimetry and 31P solid state NMR. Results showed that membrane leakage starts at a Surfactin-to-lipid ratio in the membrane, Rb value equal to 0.05, and an

aqueous Surfactin concentration of 2 µM Heerklotz and Seelig [119]. The transient, graded nature of leakage and the apparent coupling with surfactin translocation to the inner leaflet of the vesicles, suggests that this low-concentration effect is due to a bilayer-couple mechanism. Different permeabilization behaviour is found at Rb of 0.15 and attributed to Surfactin-rich clusters, which can induce leaks and stabilize them by covering their hydrophobic edges Heerklotz and Seelig [119]. Other microbial derived surfactants like Bacillus sp. derived Iturin [120]; Fengycin [121,122] and Lichenysin [123] are well recognized by their pore forming abilities and membrane permeabilizing properties. Also, Pseudomonas derived lipopeptides [29,124,125] and fungal derived lipopeptides [126] have similar activities. Patel et al. [121] studied the mecanism of membrane permeabilization caused by Fengycin by fluorescence lifetime-based calcein efflux measurements and cryo transmission electron microscopy. Poor miscibility of Fengycin with lipid probably promotes the formation of pores in 10% of the vesicles at only≈1 μM free fengycins and in 15% of the vesicles at 10 μM [121]. We explain why this limited, all-or-none leakage could nevertheless account for the killing of virtually all fungi whereas the same extent of graded vesicle leakage may be biologically irrelevant [121]. Then, crystallization of Agrastatin 1 and micellization of Plipastatin cause a cut-off in leakage at 15% that might regulate the biological activity of Fengycin, protecting Bacillus and plant membranes. The fact that Fengycin micelles solubilize only about 10 mol-% fluid lipid resembles the behavior of detergent resistance [121]. The mechanism of action of Enniatin; fungal cyclohexadepsipeptides derived from Fusarium sp. Verticillium and Halosarpheia; has been suggested, since the 1960's, to be an ionophore [126]. In fact, they incorporate easily into the cell membrane as a passive channel

and form cation selective pores. By forming complexes with cations like K+, Na+ and Ca2+, Enniatin evokes changes in intracellular ion concentration, disrupting cell function. Indeed, this may explain the broad range of biological activities observed for the Enniatin to know antimicrobial, cytotoxicity and anticancer activities, enzyme inhibitors and anthelmintic properties [126].

Owing to their

membrane-permeabilizing activities; the cyclic

lipodepsipeptides, Syringopeptin and Syringomycin from P. syringae pv. syringae exhibited a phytotoxic activity causing the lysis of tobacco protoplast [127]; exhibited a membranepermeabilizing activities towards human red blood cells and in bilayer lipid membranes [128] and formed ion channels in sugar beet vacuoles causing their lysis [125]. Results published by Kim et al. [129] proved that the antibiotic lipopeptide produced by B. thuringiensis CMB26 acts on the cell membrane. In fact, scanning electron and optical microscopies showed that the lipopeptide was capable of affecting the cell surface of the phytopathogenic fungus Colletotrichum gloeosporioides, E. coli O157 and Pieris rapae crucivora (larvae of the cabbage white butterfly). Regarding the lipopeptide surfactant Surfactin, the extent of perturbation of the phospholipid bilayer correlates with its concentration. At low concentrations Surfactin penetrates readily into the cell membrane, where it is completely miscible with the phospholipids and forms mixed micelles. At moderate concentrations, the lipopeptide forms domains that may contribute to the formation of ion-conducting pores in the membrane leading to membrane disruption. At high concentrations, the formed domains segregated from the phospholipid bilayer causes membrane permeabilization showing a stronger activity than that of Triton [130]. Such properties can cause structural fluctuations acting therefore on biological membrane integrity that may explain well the primary mode of the antibiotic action resulting in the important biological activities of biosurfactants including antibacterial; antifungal;

antiviral; anti-mycoplasma and hemolytic activities [114]. These activities enable the application of biosurfactants in diverse fields’ especially in agriculture as biocontrol agent, in food industry as preservative agent to control microbial invasion and in medicine and pharmaceutics as anti-pathogenic bacteria; antiviral and as inhibitors of fibrin clot formation. Other studies depicted the membrane permeabilizing activity of surfactants to increase substrate and/or enzyme diffusion. In fact, the stimulating effect of enzyme activities may be due to an effect of the surfactant on cell permeability facilitating therefore enzyme secretion [131]. In a previous work published by Goes and Sheppard [132], Surfactin; at 0.013 %; was reported to enhance α-amylase production in solid state fermentation with a higher efficiency towards synthetic surfactants. Lipopeptide surfactants as antibacterial agents In view of the increased resistance shown by pathogenic micro-organisms against the existing antimicrobial drugs, there is a high demand for new antimicrobial agents. The lipopeptide surfactant Surfactin is well known by its inhibitory activity towards pathogenic bacteria growth [56,133,134]. Other Bacillus related lipopeptides were recognized by their antibacterial activity [18,135,136]. Moreover, Pseudomonas related lipopeptides and fungal related lipopeptides were also reported to exhibit antibacterial activities [31,32,126,130]. Das et al. [137] presented a non-hemolytic lipopeptide surfactant produced by a marine microorganism possessing a pronounced antimicrobial action against a wide range of bacteria including Proteus vulgaris, Alcaligens faecalis and St. aureus. Moreover, Huang et al. [138] reported the efficiency of an antimicrobial substance produced by the strain B. subtilis fmbj, which is mainly composed of Surfactin and Fengycin, to inactivate endospores of B. cereus. Observation by transmission electron microscopy indicated that the lipopeptide could damage the surface structure of the spores [138]. Surfactin isoforms derived from B. velezensis strain

H3 were active against St. aureus, Mycobacterium, Klebsiella peneumoniae, P. aeruginosa and C. albicans [139]. Fengycin isoforms produced by a marine Bacillus strain were active against Micrococus flavus, Citrobacter fruendii, E. coli, Alcaligenes faecalis, Serratia marcescens, Proteus vulgaris and K. aerogenus [95]. An organic solvent extract of B. subtilis SSE4 culture filtrate containing a new lipopeptide antibiotic Subtulene A was toxic to Gramnegative and Gram-positive bacterial strains including human pathogens such as: Stenotrophomonas maltophilia, which has emerged as an antimicrobial resistant causative agent of serious nosocomial infections, Enterobacter cloacae, which causes nosocomial bacteremia and the plant pathogen, Xanthomonas campestris [135]. The culture filtrate of the endophytic B. amyloliquefaciens containing Fengycin and Surfactin lipopeptides strongly inhibited the growth of all Gram-positive bacteria tested except B. subtilis and all Gramnegative ones except Ochrobactrum anthropic [140]. A Mattacin (Polymyxin M) produced by Pa. kobensis M was capable of inhibiting the growth of a wide variety of Gram-positive and Gram-negative bacteria, including several human and plant pathogens with activity comparable with purified Polymyxin B [141]. Generally, Polymyxin isomers produced by Paenibacillus species are well recognized by their antibacterial activities such as Polymixin E derived from Pa. polymyxa [142], Polymixin B derived from Pa. polymyxa PKB1 [34] and Polymixin B6 derived from Pa. polymyxa strain JSa-9 [143]. Pa. elgii B69 produced two lipopeptide antibiotics, Pelgipeptins C and D with inhibitory activity against MethicillinResistant St. aureus [144]. Similarly, a new cyclic lipopeptide antibiotic from Pa. tianmuensis Battacin (Octapeptin B5) was reported active against multidrug-resistant Gram-negative bacteria [145]. As discussed by Sinnaeve et al. [31]; among the target microorganisms used (fungi, yeast and bacteria) the Gram-positive bacteria were the most sensitive to Tolassin homologues produced by P. tolaassii. Although, the antimicrobial activity is mainly correlated with the structural modification in the different analogues [31]. Enniatin B,

produced by F. tricinctum inhibited the growth of several bacterial strains that are considered normally pathogens to the intestinal tract: E. coli, Enterococcus faecium, Salmonella enterica, Shigella

dysenteriae,

Listeria

monocytogenes,

Yersinia

enterocolitica,

Clostridium

perfringens, P. aeruginosa and St. aureus [10]. Generally, the inhibition of Gram-negative bacteria is a property not typically associated with lipopeptides, whereas Gram-positive bacteria are generally more susceptible [146]. In a comparative study of the antagonistic activity of five Pseudomonas lipopeptides (Massetolide, Syringomycin, Orfamide, Arthrofactin and Entolysin), no significant inhibition of Gramnegative bacteria was observed [147]. A novel lipopeptide; from S. amritsarensis sp. nov. exhibited an antibacterial potential with a MIC values against B. subtilis, St. epidermidis, Mycobacterium smegmatis and clinical strain, Methicillin Resistant St. aureus of about 10, 15, 25 and 45 μg/ml, respectively [8]. These wide spread antibacterial activities of lipopeptide surfactants open future prospects for their use in biomedical and pharmaceutical sciences against micro-organisms responsible for diseases and infections in the urinary, vaginal and gastrointestinal tracts, as well as in the skin, making them a suitable alternative to conventional antibiotics [148, 149]. Since the bioactivity of lipopeptides is commonly attributed to membrane interactions, Reder-Christ et al. [147] analyzed the molecular interactions between the lipopeptides and bacteria-like lipid model membranes in more detail via complementary biophysical approaches. Application of the quartz crystal microbalance (QCM) showed that all lipopeptides possess a high binding affinity towards the model membranes [147]. Cyclic voltammetry (CV) experiments further demonstrated that the glycosylated lipodipeptide SB253514, produced by Pseudomonas strain SH-C52 showed no membrane permeabilization effects at inhibitory concentrations [147]. Collectively, these results suggests that the antibacterial activity of SB-253514 cannot be explained by an unspecific detergent-like

mechanism generally proposed for amphiphilic molecules but instead appears to occur via a defined structural target [147]. Biosurfactant as antifungal agent Antifungal activity can be mediated by the pore forming and permeabilizing activities of the antimicrobial compound. In this aim, it is well documented that lipopeptides, the prevailing group of biosurfactant compounds are among the most popular and powerful metabolites in combating and treating fungal diseases infection in vitro and in vivo [6]. Iturin [25,26,108] and Fengycin [15,27,55,94] were highly recognized by their antifungal activities. Moreover, other Bacillus spp. related lipopeptides exhibited antifungal activities like Plipastatin [150]; Pumilacidin [151]; Bacillomycin [16]; Kurstakin [17]; Mycosubtilin [152], Bacillomycin [16,93] and Bacitracin [153]. Also, lipopeptide antibiotics derived from Paenibacillus were described by their antifungal activities such as the fusaricidin-type compound produced by Pa. polymyxa active against Leptosphaeria maculans, the causative agent of blackleg disease of canola [154] and Polymyxin P produced by Pa. polymyxa M-1 suppressing the phytopathogenic Erwinia spp. [155]. Mohammadipour et al. [24] described the in vitro antifungal activity of Surfactin molecules towards the fungal pathogens A. flavus and Colletotrichum gloeosporioides. The effect of purified Surfactin on the growth of A. flavus was evaluated; mycelia growth was considerably reduced with increasing concentration of Surfactin, and 36 %, 54 %, 84 % and 100 % inhibitions of mycelia growth were, respectively, observed at 20, 40, 80 and 160 mg/L after 7 days of incubation [24]. Snook et al. [156] described the antagonistic activity of Surfactin produced by the endophytic bacterium B. mojavensis RRC 101 to the pathogenic and mycotoxic fungus F. verticillioides enabling its use as biocontrol agent. A B. subtilis derived lipopeptide exhibits growth inhibition of phytopathogenic fungi like Fusarium spp.,

Aspergillus spp. and Biopolaris sorokiniana enabling its use as biocontrol agent [157]. Kita et al. [158] discussed the suppressive ability of Iturin A produced by B. subtilis RB14-C against the damping-off of tomato seedlings caused by R. solani and Phomopsis root rot of cucumber. Fengycin and Surfactin produced by the endophytic B. amyloliquefaciens ES-2 isolated from Scutellaria baicalensis Georgi exhibited an antifungal activity against phytopathogenic fungi, such as Penicillium italicum (apple rot fungus), F. culmorum, llliciun verum, Botrytis cinerea Pers (tomato phytopathogenic fungus), Magnaporthe grisea (rice blast) and Erysiphe graminis hordei (rice sheath slight) [140]. Two novel bioactive linear lipopeptides compounds produced by a marine B. subtilis; gageotetrins A−C; displayed good time course motility inhibition and lytic activity against the late blight pathogen Phytophthora capsici, which causes enormous economic damages in cucumber, pepper, tomato and beans, at 0.02 μM [159]. As suggested by Chitarra et al. [89], an Iturin-like compound produced by B. subtilis YM 10-20 may permeabilize fungal spores and inhibit their germination. In fact, permeabilization and morphological changes in P. roqueforti conidia in the presence of the inhibitor were revealed by fluorescence staining and scanning electron microscopy, respectively. Comparable findings were described by Senthilkumar et al. [160] and Lin et al. [161] reporting the cell lysis of the pathogenic fungi R. bataticola when treated by antifungal metabolite produced by Paenibacillus sp. and the swelling and the deformation of fungus hyphae of Pestalotiopsis eugeniae when treated by the Iturin A of B. subtilis BS-99-H. Fluorescence microscopic analysis indicated the permeabilization and disruption of F. graminearum hyphae by a Fengycin type lipopeptide produced by B. subtilis IB [101]. Zhang et al. [102] reported the in vivo inhibition of the growth of A. flavus on peanuts was in the presence of lipopeptides compounds derived from B. subtilis B-FS06 along with an inhibition of spore germination.

Also, Pseudomonas related lipopeptides are well known by their antifungal properties. In fact, Viscosinamid and Tensin from P. fluorescens reduced fungal growth and aerial mycelium development of both Penicilluim ultimum and R. solani [162] and had antagonistic activity towards R. solani [28] respectively. Moreover, Sclerosin produced by Pseudomonas sp. DF41 was capable of suppressing Sclerotinia sclerotiorum-mediated stem rot of canola [163]. In a study conducted by Tran et al. [30], it was shown that Massetolid derived from P. fluorescens SS101 inhibited the growth of Phytophthora infestans showing therefore promising results in biological control of late blight caused by these fungi. A member of the Viscosin group of cyclic lipononadepsipeptides produced by P. putida RW10S2 was revealed to inhibit the growth of the phytopathogenic Xanthomonas species [164]. Moreover, fungal and yeast related lipopeptides were discussed for their antifungal activities. Echinocandin produced by Aspergillus fumigatus and Aureobasidin A from Aureobasidium pullulans were demonstrated active against some Aspergilli [165,166]. In a study conducted by Meca et al. [167]; bioactive compounds Enniatins derived from F. tricinctum induced the inhibition of the growth of several mycotoxigenic moulds as F. verticilloides, F. sporotrichioides, F. tricinctum, F. poae, F. oxysporum, F. proliferatum, Beauveria bassiana, Trichoderma harzianum, A. flavus, A. parasiticus, A. fumigatus, A. ochraceus and P. expansum. Also, actinomycete related lipopeptides exhibited a wide spectrum of antifungal activities [168]. Other bacterial related lipopeptide; Fusaricidin of Pa. polymyxa; was capable to inhibit the in vivo fungal growth of Leptosphaeria maculans, the causative agent of blackleg disease of canola [154]. Candida spp. are endosymbionts of animals and humans, where among these, C. albicans is a clinically significant and a well-known species. In general, Candida acts as a commensal in animal hosts and colonizes in skin, nails, mucous membranes, gastrointestinal and genitourinary tracts. The available synthetic antifungal drugs show high toxicity to host

tissues causing adverse effect. Lipopeptide surfactants are among the natural compounds reported to inhibit the growth of this pathogenic yeast. Peláez et al. [11], Miller et al. [169] and Fickers et al. [170] reported the anti-candida activity of ecomycin of P. viridiflava, mycosubtilin of B. subtilis and Pneumocandin of Glarea lozoyensis respectively. In fact, the minimum inhibitory concentration values for Ecomycin B were 4 mg/ml against Cryptococcus neoformans and 31 mg/ml against C. albicans, two human pathogenic fungi [169]. Phaeofungin, a cyclic lipodepsipeptide from a Phaeosphaeria sp. showed modest antifungal activity against C. albicans (MIC 16−32 μg/mL) and better activity against A. fumigatus (MIC 8−16 μg/mL) and Trichophyton mentagrophytes (MIC 4 μg/mL) [21]. Hemolytic activity of lipopeptide surfactants Owing to their membrane forming capacity and detergent effect, lipopeptide surfactants can induce hemolysis of human erythrocytes. The ability of erythrocytes lysis permits the use of lipopeptide surfactants as potent inhibitors of fibrin clot formation. In a study conducted by Arima et al. [171], Surfactin was discussed for the first time as inhibitor of fibrin clot formation by inhibiting the conversion of fibrin monomer to fibrin polymer. Since 1970, Bernheimer and Avigad demonstrated the inhibition of fibrin clot formation and haemolysis of erythrocyte by a B. subtilis derived Subtilysin [172]. Aranda et al. [173] discussed the hemolytic activity of the antibiotic lipopeptide Iturin A. They demonstrated its ability to cause hemolysis of human erythrocytes in a dosedependent manner at Iturin concentration below its critical micellar concentration [173]. Regarding the mechanism of action, these results indicate that Iturin A-induced hemolysis follows a colloid-osmotic mechanism, with the formation of a membrane pore of average diameter 32 A˚ and relative kinetics determinations clearly show that K+ leakage occurs prior to hemoglobin release [173]. K+ leakage was measured using K+ -selective electrode. A B.

subtilis ATCC 6633 derived lipopeptide surfactant exhibited an attenuated hemolytic effect in comparison to those of chemical surfactants such as SDS (Sodium Dodecyl Sulfate), BC (Benzalkonium Chloride), TTAB (Tetradecyl Trimethyl Ammonium Bromide) and HTAB (Hexadecyl Trimethyl Ammonium Bromide) suggesting its suitability due to low toxicity to the membrane [14]. Moreover, Pseudomonas derived lipopeptides were shown to exhibit hemolytic activities such us Syringopeptin and Syringomycin from P. syringae pv. syringae [128], Cormycin of P. corrugate [174], Entolysin of P. entomophila [175], Syringomycin of P. syringae pv. syringae [176] and Viscosin of P. putida [164]. Similarly, hemolytic activity of Nocardiopsis alba and Serratia marcescens derived lipopeptides were also discussed by Gandhimathi et al. [9] and Shanks et al. [177], respectively.

Conclusion To conclude, we can assume that nature offers a wide structural diversity of lipopeptide surfactants. Among literature studies, several bacteria, fungi, yeast and actinomycete producing strains were isolated and identified and the produced metabolites were purified and characterized and their biological activities were assessed. Great efforts were concentrated on their production optimization and extraction and purification strategies. Generally, the nature of the carbon source along with the physicochemical factors encountering the fermentation conditions can have a great effect on the production yields. A very interesting property for lipopeptide surfactants their ability to induce pore and ion channels formation in lipid bilayer membrane disturbing therefore their integrity and permeability. Their pore forming capacity is among their most popular functional properties allowing their investigation as antibacterial, antifungal, antitumor and hemolytic agents. These permitted their use in biomedical, pharmaceutical and therapeutic fields and their application as biocontrol agents in agriculture.

Conflict of interest The authors report no declaration of interest.

Acknowledegments This work has been supported by grants from ‘‘Tunisian Ministry of Higher Education, Scientific Research and Technology”.

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List of Tables

Table 1: Common techniques used to extract lipopeptide surfactants

Table 2: Methods of purification of lipopeptide surfactants

Table 1: Common techniques used to extract lipopeptide surfactants Extraction procedures

Advantages

Disadvantages

Acid precipitation

Efficient method for the recovery of crude lipopepetide preparation for large scale application Low cost ; Easy

Sulfate

ammonium

Must be followed by dialysis t

precipitation Organic

contaminants and salts solvent

extraction

Foam fractionation

Efficient

method

for

the

recovery

of

crude High

selectivity

of

solubility

biosurfactants preparation for large scale application

hydrophobicity level and the natur

Possibility of the reuse of the organic solvent

High cost and toxicity

Best utility for continuous retention process High purity of the end product

Table 2: Methods of purification of lipopeptides biosurfactants Purification procedure

Advantages

Disadvantages

Membrane ultrafiltration

Rapid process

High cost

Can be applied as a single step of retention and purification

Must be operated

High degree of purity Ionic Exchange

Fast process

Selective accord

chromatography

High purity and high quality purified lipopetides can be achieved

biomolecule

Possibility of the reuse of the resins Adsorption on resins

High purity and high quality purified lipopeptide is obtained;

Necissetitate the

Quick and can be operated in continuous system

for desorption

One-step recovery is required Possibility of the reuse of the selected resin; Results in less degradation of product. Can be operated when distillery wastewater is used as the nutrient medium for lipopetide production Adsorption

on

wood High purity

activated carbon (Charcoal)

-

Possibility of the reuse of the selected resin Can be operated in continuous system

HPLC: High Performance High purity of the end product Liquid Chromatography

Can not be op

Permit to fractionate different lipopeptides isomers for their system identification

Must be suppo

Can be qualitative and quantitative when using standard at known retention techniq concentration TLC:

Thin

Low yield of rete

Layer This can serve for the chemical characterization of the produced Very low yield o

Chromatography

biosurfactant after revelation using specific reagents.

Usually used f

Also, it can be realized directly on a colony spot to determine characterization whether or not the ability to produce biosurfactant HIC:

Hydrophobic Possibility of the reuse of the matrix

lipopeptides not

Low yield of rete

Interaction Chromatography Gel filtration

Possibility of the reuse of the matrix

Dab: Diaminobutyric acid; Dhb= Dehydroaminobutyric

Low yield of rete

Figure 1: Structures of the most important lipopeptides family and their isoforms

Pseudomonas related lipopeptide