PEPTIDE HAVING ANTIMICROBIAL AND/OR BIOSURF ACTANT PROPERTY

Field of the Invention

The present invention relates to a peptide. In particular, the present invention relates to a peptide with antimicrobial and/or biosurfactant properties. The peptide may be useful in preparations for human skin and keratinous surfaces such as hair and nails, including cosmetic and transdermal formulations.

Background of the Invention

Antimicrobial Peptides and Biosurfactants

Antimicrobial peptides (AMPs) are small cationic molecules composed of positively charged amino acids, which are widely distributed in microorganisms, plants, and animals as a part of the innate immune response. AMPS have been found which may have activity against Gram positive bacteria, Gram negative bacteria, fungi and protozoa.

Currently, there are two hypotheses for the action mode of AMPS: peptide-lipid interactions, and receptor-mediated recognition processes. Peptide-lipid interactions include a barrel-stave mode and a carpet mode. Receptor-mediated recognition processes may be related to association of AMPS with DNA, autolysins and permeability of cell structures.

In general, a surfactant is composed of a non-polar lipophilic portion and a polar hydrophilic portion. After reaching a certain concentration, surfactants form aggregates and dissolve in water. However, because of the balance between the polar and non-polar portions, surfactants can exhibit surface activities. That is, surfactants enhance the dissolution of a lipid in water, and reduce the surface tension of the interface of two phases.

Biosurfactants are molecules produced by organisms which reduce the surface tension of aqueous solutions and hydrocarbons and the critical concentration and surface tension of micelles. In the formation of micelles, solutions can be microemulsified by dissolving hydrocarbons in water or vice versa. Biosurfactants contain a hydrophilic moiety, such as that derived from amino acids, peptides, cations, anions, monocarbohydrates, dicarbohydrates or polycarbohydrates, and a hydrophobic, or lipophilic, moiety, such as that derived from saturated or unsaturated or hydroxy fatty acids, or hydrophobic peptides.

It has been reported that microorganisms such as Bacillus subtilis, Candida tropicalis, Brevibacterium casei, Flavobacterium aquatile, Pseudomonas aeruginosa, Pseudomonas fluorescens, Torulopsis bombicola, Candida sp., and Echinacea purpurea produce biosurfactants. The rhamno lipid produced by Pseudomonas aeruginosa and the sophoro lipid produced by Torulopsis bombicola may eliminate heavy metal contamination in soil, and may have a bactericidal effect on Gram positive bacteria or some enterobacteria. Pseudomonas fluorescens may emulsify and decompose aliphatic and aromatic petroleum hydrocarbons in soil. The peptidoglycolipid produced by Pseudomonas aeruginosa may emulsify crude oil and kerosene; and the glyco lipid produced by Candida sp. may decrease the surface tension between water and kerosene. Phenolic acid derivatives extracted from Echinacea purpurea, such as cichoric acid, can be formulated into tablets and used as surfactants. In addition, iturin produced by Bacillus subtilis may have a fungicidal effect.

Various AMPS or biosurfactants have been described in the literature. For example, WO 99/62482 reports surfactants for use in external preparations for skin, the reference which is hereby incorporated by reference in its entirety. Alternatively, US Patent 5,554,507 reports a sequence from Bacillus subtilis, and proposes that the sequence has at least one siderophore biosynthetic gene. EP 0 576 050 reports a similar sequence from Bacillus subtilis, but indicates that an allele with this sequence is not functional (page 8, lines 6-7).

Surfactin is a known AMP and biosurfactant. It was reported in 1968 in a study on blood clotting inhibitors (Arima et al, Biochemical and Biophysical Research Communications, vol. 31, no. 3, pages 488-494, 1968). The primary structure of surfactin has been determined by Kakinuma et al. (Agr. Biol. Chem., vol. 33, no. 6, pages 971-972, 1969). It is a macrolide containing a heptapeptide sequence (L)Glu-(L)Leu-(D)Leu-(L)Val-(L)Asp- (D)Leu-(L)Leu linked to a beta-hydroxy fatty acid with a distribution of several different lengths of carbon atoms and various branched or unbranched configurations at the fatty acid terminus. The carbonyl group of the fatty acid is connected to the GIu moiety, while the beta- hydroxy group connects to the terminal Leu to form a large ring. While the (L) designations are included above, it is also conventional to omit the (L) designation for amino acids. Cyclic peptides are also known as depsipeptides.

In view of the environmentally hazardous nature of synthetic microbicides and synthetic surfactants, additional biosurfactants and bioemulsifϊers which could substitute for synthetic surfactants or emulsifϊers would be commercially useful. Thus, there remains a need for novel AMPS or biosurfactants that have antimicrobial activity and/or surface active activity as replacements for synthetic microbicides or synthetic surfactants.

Summary of the Invention

In order to meet this need, this invention provides for a surfactin variant having antimicrobial activity and surface active properties. The surfactin variant according to the invention is a seven amino acid macrolide with a particular distribution of fatty acid acyl groups.

In another embodiment, this invention provides for a combination of four specific surfactin analogs that in combination possess antimicrobial activity and surface active properties.

The surfactin compositions of the invention possess both antibacterial and antiviral activity and are useful for inhibiting the activity of both bacterial and viral microorganisms.

In another embodiment, the invention provides for compositions of either of the above components. The above components may preferably replace synthetic surfactants or antimicrobial agents in compositions according to the invention.

In another embodiment, the invention provides for methods of providing transdermal delivery enhancement by administering either of the above in combination with an active agent intended to penetrate one or more layers of the skin.

In another embodiment, the present invention provides for a novel sfp peptide comprising SEQ ID NO:1 and provides for methods for producing surfactin variants using the novel sfp. In another aspect, the present invention provides a nucleic acid molecule comprising SEQ ID NO:2 which encodes the sfp peptide of the invention. The invention also provides a vector comprising the nucleic acid molecule, and a transformed microorganism comprising said nucleic acid molecule or vector. In a further aspect, the present invention provides a method for preparing the sfp peptide of the invention, which comprises culturing a microorganism capable of producing the sfp peptide.

The antimicrobial surfactins of this invention have application as additives in compositions in which providing antibacterial or antiviral activity is useful to maintain the integrity of the composition. Thus, the compositions of the invention are useful as preservatives. Such compositions may be useful in compositions including, for example, cosmetics, pharmaceutical formulations, particularly topical formulations, and others.

Brief Description of the Drawings

FIG. 1 depicts the full-length cDNA sequence of the gene encoding the sfp peptide, which is composed of 1177 nucleotides, including the 5' UTR composed of 148 nucleotides, the coding region composed of 438 nucleotides and the 3' UTR composed of 591 nucleotides. The start codon is in bold, and the stop codon is marked with the symbol *. It is predicted that the coding region encodes a sequence of 145 amino acids.

FIG. 2 shows the HPLC analysis results of the fermentation broth of Bacillus subtilis.

FIG. 3 shows the bacterial inhibition of transformed Bacillus subtilis cells.

FIG. 4 shows the growth curves of Bacillus subtilis cultured in various media.

FIG. 5 shows the growth curves of Bacillus subtilis cultured under various temperatures.

FIG. 6 shows the inhibitory activity of the peptide of the invention produced in Bacillus subtilis in the formation of membrane.

FIG. 7 shows a gas chromatogram of the methyl esters of the beta-hydroxy fatty acids of the lipopeptide according to the invention. (1) is isoC13; (2) is n-C13; (3) is isoC14; (4) is anteiso C14; and (5) is isoC15. Percentages are (1) 11%; (2) 26%; (3) 37%; (4) 24%; and (5) 2%. The major form is isoC14.

FIG. 8 shows mass spectra of the lipopeptide according to the invention in the region of molecular masses. (A) Mass spectrum of lipopeptide according to the invention with [M+H]+ at m/z 994, 1008 and [MSNa]' at m/z 1016, 1022. (B) Mass spectrum of control (standard surfactin) with [M+H]+ at m/z 1022, 1036 and [M+Na]+ at m/z 1044, 1058.

FIG. 9 shows mass spectra of the lipopeptide according to the invention. The sample is from a methanol extract of Bacillus sp Th. The parent ion at mlz 931.7 corresponds to the Na

adduct containing a fatty acid residue with 14 carbon atoms. Fragments representing the calculated masses derived from the stepwise cleavage of the amino acids L-leucine (818.9), D-leucine (705.4), L-aspartic acid (590.4), L-valine (491.4), D-leucine (378.5) and L- glutamic acid (249.4) from the lipopeptide as marked.

FIG. 10 shows various applications for biosurfactants.

FIG. 11 shows a comparison of moisturizing effects of biosurfactants on cultured human skin.

FIG. 12 shows that increased concentration of micelles may promote skin penetration.

FIG. 13 shows the anti-virus activity of antimicrobial peptides and surfactins. Effect of various AMPs and surfactins on the infectivity of NNV, Nerve Necrosis Virus, a common virus found in fish. NNV was incubated in the presence of the indicated concentrations of other AMPs , surfactin-1 (a standard surfactin sample available from , for example, Sigma Chemical Company) and sufractin-2 (a surfactin composition according to the invention) at 26 ⁰ C for 60 min and assayed for residual infectivity by plaque assay. The data depict the means of two to three experiments.

Detailed Description of the Invention

In one embodiment, the product according to the invention is a surfactin variant. By "surfactin variant" is meant a surfactin having a heptapeptide sequence (L)Glu-(L)Leu- (D)Leu-(L)Val-(L)Asp-(D)Leu-(L)Leu and a distribution of fatty acid chains, or isoforms, having an increased ratio of isoC13, nC13 and isoC14 and lower ratios of nC14 and iC15 compared to that present in the surfactin published by Yakimov et al., supra. Preferably, the surfactins of the present invention have a percent of fatty acids as follows: isoC13, greater than 3% , and more preferably greater than 10%; nC13, greater than 0.65% and more

preferably greater than 25%; isoC14, greater than 17% and more preferably greater than 35%; nC14, less than 41%, and more preferably less than 25%; and isoC15, less than 11% and more preferably less than 3%. For purposes of this invention, the term "surfactin variant" can be used interchangeably with "surfactin isoform" and both terms will be used to indicate variations in the fatty acid chain, while the term "surfactin analog" will be used to indicate variations in the amino acid sequence compared to surfactin. The particular surfactin variant or distribution of fatty acid chains according to the present invention may also be referred to as UMO-Biosurfactin or alternatively "Cyclic Depsipeptide C14".

The surfactin variant according to the present invention can be seen in Figure 7 with the beta-hydroxy fatty acids of the lipopeptide according to the invention in the distribution as follows: (1) is isoC13, 11%; (2) is n-C13, 26%; (3) is isoC14, 37%; (4) is anteiso C14, 24%; and (5) is isoC15, 2%. The major form is isoC14. For comparison, Yakimov et al. (Applied and Environmental Microbiology, vol. 61, no.5, page 1706-1713, Table 1, 1995) reports that surfactin has the following distribution: 1) isoC13=3%, 2) nC13=0.65%, 3) isoC14=16.8%, 4) nC14=40.8%, and 5) isoC15=l l.l% , as well as several other moieties. Therefore, the surfactin variant of the present invention has higher percentages of isoC13 (11% versus 3%), nC13 (26% versus 0.65%), and isoC14 (37% versus 16.8%), while also having a lower percentage of nC14 (24% versus 40.8%) and isoC15 (2% versus 11.1%).

The surfactin variant has improved properties compared to known surfactin. For example, the higher proportion of iso C 14 beta-hydroxy fatty acid results in higher surfactant activity and higher bactericidal capacity compared to known surfactin. In addition, the surfactin variant of the present invention has better emulsification capability that can lower water surface tension from 72 mN/m to 27mN/m at lOμM. Known surfactin has a critical micellar concentration (CMC) of 9.4 μM in 200 mM NaHCO ₃ at pH 8.7 (Ishigami et al., 1995). A preferred critical micelle concentration (CMC) of the surfactin variant according to

the invention is about lμM. Bactericidal activity of the surfactin variant according to the invention is presented below in Tables 1 and 2. For purposes of comparison, surfactin (derived from ATCC 21332) can be purchased from Sigma Co. (USA) and used as a standard to show a reduction in water surface tension from 72 mN/m to ~30 mN/m at concentrations of -10 μM.

In another embodiment, the product according to the invention is a surfactin analog. In other words, the invention also provides for a combination of lipopeptides with improved properties compared to known lipopeptides. There are many surfactin- like peptides produced by Bacillus that differ from surfactin in the amino acid sequence, which will be referred to in the present specification as surfactin analogs. One such surfactin analog is lichenisin produced by B. licheniformis JF-2. In one embodiment according to the invention, one combination of surfactin analogs according to the present invention is the following combination of four surfactin analogs:

1) ELLVDLL (GIu- Leu-(D)Leu- VaI- Asp-(D)Leu-Leu)

2) LLDLLDL (Leu-Leu- Asp-Leu-Leu- Asp-Leu) (D or L not available)

3) ELLIDLI (GIu- Leu-(D)Leu- lie- Asp-(D)Leu-Ile) or EILIDLI (GIu- He-

(D)Leu- lie- Asp-(D)Leu-Ile)

4) ELLVDLL (GIu- Leu-(D)Leu-Val- Asp-(D)-Leu-Val).

The amount of each surfactin analog l)-4) is between about 1% to about 97% such that the total amount of l)-4) is less than or equal to 100%. The fatty acid portions can be distributed according to the natural fatty acid distribution in surfactin according to the surfactin published by Yakimov, or in one embodiment, the fatty acid distribution can have higher ratios of isoC13, nC13 and isoC14 and lower ratios of nC14 and isoC15 compared to the surfactin published by Yakimov. A single amino acid substitution in the heptapeptide moiety of surfactins may strongly modify the properties. Modifications, for example, may include the substitution of the L-valine residue at the fourth position by a more hydrophobic residue,

i.e., leucine or isoleucine. These [Leu ⁴ ]- and [lie ⁴ ] surfactins may have a higher affinity for hydrophobic solvents and an improved surfactant power. Structure-property correlations can be modeled by analysis of the hydrophobic residue distribution in the three-dimensional model of the structure of surfactin in solution. The surfactin analogs may be produced by bacteria or produced synthetically, i.e. not by bacteria.

In another embodiment, the product according to the invention is an sfp peptide. The invention provides for an sfp peptide comprising a sequence as shown in SEQ ID NO: 1, or a functional fragment thereof.

Phosphopantetheinyl transferase (PPTase) transfers the 4'-phophopantetheinyl group of Coenzyme A to the side hydroxy group in a serine residue preserved in carrier proteins and thereby activates the carrier proteins from the inactive apo-form, rendering them in fully activated holo-form. The activated carrier proteins transfer an acyl group and play an important role in the synthesis of substances such as fatty acids, polyketone and non- nucleotide polypeptides. PPTase can be classified into the AcpS type, the sfp type, and the domain type. PPTase has been used in genetic engineering research on polyketone and non- nucleotide polypeptides. The sfp type is also involved in nonribosomal peptide synthesis.

The term "functional fragment" used herein refers to the fragment that has a partial sequence of the sequence depicted in SEQ ID NO: 1 which retains the sfp activity of the full length sequence. The sfp peptide of the invention was isolated from Bacillus subtilis isolated from soil near a pond in Thailand. The sequence of the sfp peptide has been determined and compared with those of known sfp peptides. The results show that the sfp peptide of the invention is a novel protein of the PPTase family. The sfp peptide according to the invention is useful for the production of a surfactin variant or analog composition according to the invention.

Microorganisms which may be able to use the sfp peptide according to the invention can be naturally occurring microorganisms which express the sfp peptide, or transformed microorganisms. In an embodiment of the invention, the microorganism is selected from the group consisting of Bacillus sp., Escherichia coli, yeast, Candida tropicalis, Brevibacterium casei, Flavobacterium aquatile, Pseudomonas aeruginosa and pseudomonas fluorescens. Preferably, the microorganism is B. subtilis, B. amyloliquefaciens or B. circulars. Most preferably, the microorganism is Bacillus subtilis isolated from soil.

In another aspect, the invention provides a nucleic acid molecule which encodes the sfp of the invention. Preferably, the nucleic acid molecule has a sequence as depicted in SEQ ID NO: 2. The nucleic acid according to the invention can be used to produce the sfp peptide of the invention. Preferably, the nucleic acid is in a vector. The vector can be used to preserve and produce the nucleic acid molecule, or introduce the nucleic acid molecule into a host cell. Preferably, the vector comprises a selectable mark. The vector may also preferably comprise the origin for reproduction in a prokaryotic cell and restriction sites for gene manipulations. Preferably, the nucleic acid molecule of the invention is regulated by a promoter. In an embodiment of the invention, the promoter is an inducible promoter.

This invention also provides a transformed microorganism, which comprises the nucleic acid molecule of the invention or a vector comprising the nucleic acid molecule. The term "transformed microorganism" used herein refers to a microorganism whose genetic materials have been altered via the introduction of a nucleic acid molecule. The transformation can be performed by persons of ordinary skill in the art underlying the invention on the basis of the teachings of the invention and the general knowledge of molecular biology. For instance, heat shock or electroporation can be used to transform a bacterium with a vector.

As an example, E. coli is used with a suitable vector and restriction cites in the vector are selected. The vector and nucleic acid molecule comprising the sequence encoding the sfp peptide of the invention are treated with relevant restriction enzymes. The treated vector and the nucleic acid molecule are thus ligated. Transformation of E. coli cells with the vector comprising the nucleotide sequence encoding the sfp peptide of the invention may thus be accomplished using heat shock or electroporation, for example. The sfp peptide in the transformed E. coli cells is then expressed.

As an alternate example, yeast is used with a suitable vector and restriction cites in the vector are selected. The vector and nucleic acid molecule comprising the sequence encoding the sfp peptide of the invention are treated with relevant restriction enzymes. The treated vector and the nucleic acid molecule are thus ligated. Transformation of yeast cells with the vector comprising a nucleotide sequence encoding the sfp peptide of the invention may thus be accomplished using heat shock or electroporation, for example. Prior to the transformation, the yeast cell walls can be removed to form a spheroplast. Alternatively, the transformation can be performed via heat shock in the presence of basic anions, e.g., LiCl or RbCl. After the transformation, the yeast cells are induced to express the sfp peptide with a suitable inducer.

The expression of the sfp peptide of the invention can be detected by known methods such as methods for protein detection, e.g., agar electrophoresis, Western Blotting and immunoreaction analysis, or methods for mRNA detection, e.g., Northern Blotting.

When an sfp peptide according to the invention is used to produce the surfactin variant according to the invention, the yield of the surfactin variant can be increased by adjusting the culture conditions. For instance, the yield can be improved by optimizing the culture medium. In accordance with the invention, different culture media and salts result in different growth curves of transformed cells. Preferably, the culture medium comprises

divalent cations or activated carbon. The number of cells increase along with cultivation time. In addition, matrices such as potatoes, corn starch or sweet potatoes can be added to the culture medium. Furthermore, the yield of surfactin variant can also be promoted by addition of a foaming agent. Preferably, the cultivation is conducted at 42°C to 48 ⁰ C. Preferably, the oxygen content in the medium is maintained at 30% for the first 48 hours of cultivation, and at 60% in the subsequent 48 hours.

The invention also provides for methods of using the above variant of surfactin or combination of surfactin analogs as an antimicrobial peptide (AMP) or surfactant in a variety of compositions. In an even further aspect, the invention provides the use of the components according to the invention in any composition for which an antimicrobial would be useful to prevent the growth of microorganisms including bacteria and viruses. Non- limiting examples of such compositions include, pharmaceutical compositions, food compositions, cosmetic compositions, agrochemical compositions, preservative compositions, surfactant compositions, detergent compositions, emulsifier compositions, humectant compositions, dispersion compositions, dissolution compositions, antistatic compositions, anti-clouding compositions and lubricant compositions. In a further aspect, the present invention provides a pharmaceutical composition, which comprises the surfactin variant of the invention. Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In an embodiment of the invention, the pharmaceutical composition further comprises a humectant or adjuvant. The pharmaceutical composition according to the invention can be administered to an animal in need of treatment by various routes. Preferably, the pharmaceutical composition is in the form of an oral composition or an injectable composition. The latter form is preferably an injectable composition for intravenous administration. The surfactin variant of the invention exhibits biosurfactant property, and thus can achieve an effect of a detergent, emulsifier or humectant. The composition of the

invention can exist in various forms to suit different needs. For instance, the composition can be in the form of a solution, gel, emulsion, emugel, cream, ointment, lotion, transdermal system, injectable fluid, suspension or patch.

The amount of surfactin variant or analog to be included in the composition may vary from about 0.01% by weight to about 30% by weight. For example, the amount of surfactin variant or analog may be about 0.05% by weight, about 0.1 % by weight, about 0.5% by weight, about 1% by weight, about 2% by weight, about 3% by weight, about 4% by weight, about 5% by weight, about 10% by weight, about 20% by weight, about 30% by weight, or any amount in between the listed amounts.

The surfactin variant or analog of the invention is effective in killing Gram positive bacteria, Gram negative bacteria, fungi, protozoa, viruses with an envelope and tumors. In an embodiment of the invention, the surfactin variant or analog of the invention is effective in killing E. coli, V. harveyi, V. alginolyticus, V. anguillarum, V. salmonicida, A. hydrophila, S. epidermidis, Iridovirus, Herpes simplex virus, suid herpes virus-1, Vesicular stomatitis virus and Simian Immunodeficiency virus. In an even further aspect, the invention provides the use of the surfactin variant or analog of the invention in the preparation of an antimicrobial composition. The antimicrobial composition is preferably used against microorganisms selected from the group consisting of Gram positive bacteria, Gram negative bacteria, fungi, protozoa and viruses with an envelope. More preferably, the microorganisms are selected from the group consisting E. coli, V. harveyi, V. alginolyticus, V. anguillarum, V. salmonicida, A. hydrophila, S. epidermidis, Iridovirus, Herpes simplex virus, suid herpes virus-1, Vesicular stomatitis virus and Simian Immunodeficiency virus. About 80% of viruses have a lipid envelope composed of a membrane and glycoproteins, which facilitates host infection. It is believed that the surfactin variant or analog of the invention can destroy the

lipid envelope of viruses, rendering it unable to adsorb onto and then invade a host cell (which can be an animal, plant or bacterial cell).

The present invention also provides for a transdermal delivery enhancer. In other words, the present invention also provides for methods of improving transport across one or more dermal layers comprising administering the above variant of surfactin or combination of surfactin analogs in combination with an active agent such as a drug to be transported. According to the present invention, the surfactin variant is a skin penetration enhancer, i.e. the surfactin variant serves as a microemulsion to increase transdermal drug delivery. By "transdermal delivery enhancer" is also meant a "skin penetration enhancer" or a "natural cellular transduction vector", all three phrases being equivalent for purposes of this disclosure.

Although transdermal drug delivery is more attractive than injection, it has not been applied to macromolecules because of low skin permeability. Agents released from a transdermal delivery system must be capable of penetrating each layer of skin. In order to increase the rate of permeation of an agent, a transdermal drug delivery system must be able in particular to increase the permeability of the outermost layer of skin, the stratum corneum, which provides the most resistance to the penetration of molecules. Surfactin variants and analogs according to the invention serve as transdermal enhancers.

We have studied the effects of extrinsic factors on the conformation of surfactin, a heptapeptide biosurfactant from a Bacillus strain, in aqueous solutions. It has been made clear that temperature, pH, Ca ²⁺ ions affect the conformation of surfactin in aqueous solutions. The P -sheet formation reached a maximum at 4O ⁰ C. Ca ²⁺ induced the formation of α-helices and caused this transition at 0.3 mm with surfactin monomers or at 0.5 mm with surfactin micelles, but above these transition concentrations of Ca ²⁺ β-sheets were observed. In

micellar solution the β-sheet structure was stabilized at pH values below 7 or upon addition Of Ca ²⁺ in concentrations above 0.5 mm. Our results indicated that the bioactive conformation of surfactin is most likely the β-sheets when the molecules are assembled in micelles. The β- sheet structure in micelles could be retained by tuning the micelles. Surfactin micelles could be tuned in the bioactive conformation by manipulating pH, temperature, Ca ²⁺ concentrations in surfactin solutions. Our results indicated that Ca ²⁺ may function as directing templates in the assembly and conformation of surfactin in micelles. Thus, environmental manipulation and template-aided micellation (TAM) are provided as a new approach for preparing predesigned micelles, microemulsions or micro-spheres for specific application purposes.

Our studies indicate that surfactin can create pores on the epithelium to prolong the life -time of the aqueous pathways through the epithelium, for the consideration that surfactant can reduce the surface energy of the pore edge. Without being bound by theory, the biological activity of surfactin may be related to its ability to destabilize and permeabilize membranes at concentrations far below the onset of micellization. The preference of surfactin for micelles is RT In(AT ^• CMC) 5 21.1 kcal/mol compared to ~ 20.5 kcal/mol for strong non- ionic detergents (at 0.1 M salt). This is surprising because micelle formation of surfactin is opposed by a strong electrostatic repulsion between the peptidic head groups of ~3 kcal/mol, which does not occur for non-ionic detergents. Despite this electrostatic effect, surfactin prefers to form micelles rather than to insert into a lamellar structure.

While not wishing to be bound by theory, one can suppose that surfactin acts first by penetrating readily into the cell membrane, where it is completely miscible with the lipid components. Surfactin penetrates spontaneously into lipid membranes by means of hydrophobic interactions. The insertion in the lipid membrane is accompanied by a conformation change of the peptide cycle. Furthermore, the ion-conducting pores induced by surfactin are not due to the formation of lipid/surfactin structures in the lipid bilayer but,

rather, to the presence of surfactin dimers. The insertion of surfactin into the lipid membrane may be the first step in the formation of pores. Subsequently, surfactin may aggregate in the lipid membrane to form pores.

The ability of certain biosurfactants to accelerate the transport of genetic material or drugs through biological membranes is widely sought in biotechnology and pharmacy. The most important applications for surfactants, however, are related to their self-organization in solution. Self-organization leads to the formation of micelles, liposomes, and microemulsions. These self-organized structures are used for solubilization, transport, and separation processes, and as templates for nanoparticles. Biosurfactants may affect membrane permeability to increase drug absorption. Alternately, surfactin variants or analogs according to the invention can decrease the critical micelle concentration (CMC) of a drug. Through this effect, the biosurfactant act as an " drug delivery enhancer " to help the penetration of the drug across the cytoplasmic membrane of the skin cell.

By the term "about" is meant within +10% of the stated amount, or within experimental error of the measuring technique.

The compositions and methods of the present invention will be described in more detail in the following non-limiting examples.

EXAMPLE 1 : Preparation of sfp peptide and transformed cells.

The total RNA of B. subtilis was obtained, and the cDNA thereof was synthesized with a IK reverse transcription buffer containing lOμg total RNA, 200 U Molony murine leukemia virus reverse transcriptase, ImM dNTP, 160U RNAse inhibitor and 1.6μg random primer, followed by transcription at 42°C for 30 mins. The sfp gene was amplified with the polymerase chain reaction with 80μl of IX PCR buffer containing 20μ g reverse transcription product, 0.5U Taq DNA polymerase, lμg forward 5 primer and 1 μg reverse primer. The

amplification was performed in a cycle of 94°C for 1 min., 55°C for 1 min. 30 sec, and 72°C for 1 min. 30 sec, for 35 cycles.

The purified PCR products were cloned to the Sma I position of the plasmid M13mpl8 or pBluescript, and sequenced. The sequence is depicted in SEQ ID NO: 2. The analysis of the sequence is shown in Fig. 1.

In addition, the purified PCR products were cloned into the expression vector pMK4 containing the promoter fenC that can regulate the expression of the downstream sfp gene. After ligation, the host cell E. coli JM83, or B. subtilis 168 was transformed with resultant pMK4 vector comprising the PCR products, and cultivated at 200rpm at 37°C.

After centrifugation at 800Og for 10 min. to remove the bacterial cells, the supernatant was filtered through a 20 ultra filtration membrane with a pore size of 0.2 mm (30 kDa MWCO) to obtain micelles having biosurfactant property, which was then broken with 50% (v/v) methanol. The product collected was then subject to chromatography of an HPLC reverse phase column (Techsphere 5mm ODS C 18, Merck, German) under the following conditions: temperature: 30 ⁰ C ; mobile phase: 3.8 MM 5 trifluoroacetic acid: acetonitrile, 1 :4 (v/v); flow rate: 0.5 ml/min.; wavelength: 210 nm; and the amount of sample: 20 μl. Standards were purchased from Sigma, USA. The results are shown in Fig. 2. The molecular weight of the product expressed in B. subtilis 168 or pKM4 vector is 1.1 kDa.

The inhibitory activity of naturally occurring and transformed B. subtilis is shown in Fig. 3.

EXAMPLE 2: Antimicrobial Activity

Surfactin variant was isolated with ultrafiltration, and the purity determined with HPLC was over 90%. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the surfactin variant were determined as follows.

Preparation of Bacterial Solutions

E. coli., V. harveyi, V. alginolyticus, V. anguillarum, 20 V. salmonicida, A. hydrophila and S. epidermidis were cultivated with LBA (E. coli DH5α) or TSA (plus 1.5% NaCl) at 37 ⁰ C for 16 hours. The colonies were added to suitable culture media. A bacterial solution (500μL) where OD540 is 1, with a concentration around 1 x 109 cells, was added to 500μL LB or TSB (plus 1.5% NaCl) to reach the concentration of 1 x 10 ^4'5 cells per cc.

Susceptibility Test

The bacterial solutions (130μL) with concentration of 1 x 10 ^4'5 cells per cc were added to a 96-well microplate, followed by the addition of 20μL of surfactin variant produced by B. subtilis at various concentrations. The microplate was cultivated at 37°C for 16 hours. The MIC value was determined as the lowest concentration of the surfactin variant where the culture medium remained clear without the growth of tested bacteria. The bacterial solutions which remained clear without the growth of tested bacteria were applied to the culture medium LBA or TSA. After cultivation at 37 ⁰ C for 16 hours, the colonies were observed and the MBC values were determined. Each test was repeated three times and compared to a control group.

The MIC and MBC of the surfactin variant produced by B. subtilis against E. coli., V. harveyi, V. alginolyticus, V. anguillarum, V. salmonicida, A. hydrophila and S. epidermidis were detected at various concentrations (0.075 μM to 0.675 μM) The results are shown in following Table 1.

Table 1 : Antimicrobial Susceptibility to UMO Bio-surfactin

MIC= minimum inhibitory concentration MBC= minimum bactericidal concentration

The surfactin variant killed Gram positive and negative bacteria. Accordingly, the surfactin variant has a killing and suppressing effect on various microorganisms.

EXAMPLE 3 : Preparation of sfp

In this example, conditions for cultivating sfp peptide were tested.

Culture Media

Culture medium E containing the following was used: 20g/L of L-glutamine, 12g/L of citric acid, 80g/L of glycerol, 7g/L Of NH ₄ Cl, 0.5g/L Of K ₂ HPO ₄ , 0.5g/L of MgSO4, 0.04g/L OfFeCl ₃ 6H ₂ O, 0.15g/L of CaCl ₂ 2H ₂ O, and 0.1 g/L Of MnSO ₄ H ₂ O, pH=6.5.

Culture medium F containing the following was used: 65g/L of L-glutamine, 22g/L of citric acid, 170g/L of glycerol, 5 7g/L of NH ₄ Cl, 0.5g/L of K ₂ HPO ₄ , 0.5g/L of MgSO4, 0.04g/L Of FeCl ₃ 6H ₂ O and 0.15 g/L of CaCl ₂ 2H ₂ O, pH=6.5.

The culture medium LB was also used.

The results of bacterial growth are shown in Fig. 4. As shown, the growth curves varied with the culture media and salts used. The best conditions were observed with culture medium E where the number of cells increased along with cultivation time. The number of cells decreased after 24 hours when LB was used. The cell growth with culture medium F was slow.

Non-traditional carbon sources such as agrochemical products were also tested. It was found that 180mglL, 1715mg/L and 2200mg/L of sfp were produced with the addition of 80% potato, 12% corn starch and 16% sweet potato, respectively. When 8% corn starch of technical grade was used, 2205mg/L of sfp was produced.

Culture Temperature

The culture medium containing the following was used to cultivate B. subtilis at 45°C and 37°C: 5.OgIL of peptone, 3.OgIL of meat extract, and lOmg of MgSO4, FeSO4, ZnSO4 and MnSO4, pH=7.5. The results in Fig. 5 show that cultivation at 45°C achieves better results.

Oxygen Content

The oxygen content was adjusted at different times of cultivation. It was found that 2730mg/L of sfp peptide were obtained if the oxygen content was maintained at 30% for the first 48 hours and 60% in the subsequent 48 hours.

Increasing the scale of Fermentation

The fermentation of product was increased to 5L and 2OL fermentation, and 3g/L and 4g/L of peptide was obtained, respectively. The fermentation in fermentation tanks of different sizes remained good stability.

Addition of Anti-Foaming Agent and Foaming Agent

Addition of an anti-foaming agent was found to reduce the production time and did not affect the recovery rate of acid precipitation. While the yield at 48 hours was 0.9g/L of lipopeptide where no anti-foaming agent was added, the yield at 12 to 24 hours was 0.6g/L if an anti-foaming agent was added. In addition, the addition of a foaming agent also increased the yield of the lipopeptide.

EXAMPLE 4: Purification of lipopeptide

The above fermentation broth of B. subtilis was extracted with methanol and precipitated with 6 mol/L of HCl to obtain crude lipopeptide, which was then purified by gel filtration with the Sephadex LH-20 column.

The fermentation broth of B. subtilis was also ultrafiltered with the 30 kDa MWCO ultramembrane to obtain micelles with the biosurfactant property, which were then broken with 50% (v/v) methanol. The lipopeptide was collected and the recovery rate was about 95%.

EXAMPLE 5 : Antiviral effect of lipopeptide

Fin cell line derived from the fin tissue of a grouper was used as the host cell for cultivating viruses.

Cell Culture

Fin cell line was cultivated in the culture medium L- 15 (containing 10% serum). It was observed that cells grew to the full scale after 2 to 3 days' cultivation. The culture was then passaged.

Viral Infection

After passages, the culture medium was discarded. After washing with PBS, cells were inoculated in the 96-well microplate, with 5 x 10 ⁴ cells in each well. After addition of viral solutions, the microplate was shaken for an hour, followed by addition of fresh culture medium without serum. The cell morphology was observed.

Viral Titer Test

Fin cell line was infected with viral solutions with concentrations from 10 ^"1 to 10 ^~10 following the TCID50 protocol, with 8 repeats for each concentration. The cell line was observed for 3 to 7 days. The viral titer was calculated (PFU ImI). The titer was adjusted to be over 10 ^~8 PFU/ml by filtrating cell debris and repeating the TCID50 protocol.

Amplification of Viruses

Fin cell line was infected with the viruses at M.O.I = 0.1 to 0.01 (PFU/cell). After the cytopathology reached over 90%, cells were subject to a freezing (-8O ⁰ C) and defrosting (25 ⁰ C ) cycle three times to release the virus, which was then recovered.

Purification of Virus

The virus was collected by centrifugation of 8000rpm at 4 ⁰ C for 10 min. 2.2% NaCl and 7% PEGβoo were added to the supernatant and reacted for 2 to 4 hours. After centrifugation 20 with lOKrpm at 4°C for an hour, the reticular structure and virus were collected. The debris was dissolved by TNE buffer. The product was then subject to ultra centrifugation of 35 Krpm at 4°C for 17 hours with CsCl gradients. The portion with density of 1.15 to 1.35 g/cm ³ was collected. The virus was dissolved in 5OmM Tris-HCl buffer, pH 8.0, and stored at -70 ⁰ C.

The addition of 120μm sfp peptide of the invention was found to reduce the titer of the virus Iridovirus, >4.4 loglO CCID ₅₀ /ml. The lipopeptide of the invention did not activate the virus Iridovirus that has the envelope.

EXAMPLE 6: Inhibition of formation of cell membrane

The inhibitory effect of the lipopeptide of the invention on the formation of cell membrane of Salmonella enterica on a PVC matrix was tested in this example.

S. enterica was added into PVC tubes plated with the sfp peptide and cultured at 30 ⁰ C overnight. After wetting, the PVC tubes were dyed with crystal violet. The results in Fig. 6 show that cell membrane was observed in the interface of air and liquid (see the arrows).

EXAMPLE 7

Lipid Analysis Method: The lipid moiety contained P-hydroxy fatty acids. The fatty acid methyl esters were obtained by the action of diazomethane. They were analyzed by gas chromatography on a fused-capillary column SP 2100 (SO m X 0.32 mm) from 190 ⁰ C to 250 ⁰ C with a temperature programming of 1 "C/min. They were identified by their retention times in comparison with standard /3 -hydroxy fatty acids and their structures were confirmed by combined gas chromatography/mass spectrometry. Gas chromatography was performed on a DB 5 capillary column (0.32 mm X 30 m) with temperature programming from 60 ⁰ C to 180 ⁰ C at 30"/min and then isotherm at 180 ⁰ C for 20 min. Electron impact mass spectrometry was performed at 200 ⁰ C and 70 eV.

Mass spectrometry: Mass spectra were recorded on a Kratos (Kratos Analytical Ltd, Manchester, UK) MS80 RF instrument which was operated in the liquid secondary-ion mass spectrometry (LSIMS) mode using a cesium ion gun (Phrasor Scientific, Duarte, CA, USA) at 20 kV. Samples (2-5 pg) were introduced on a copper probe tip using thioglycerol (1 pi) as matrix. Data were acquired with a DS-90 data system.

It will be apparent to those skilled in the art that various modifications and variations can be made in the methods of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention include modifications and variations that are within the scope of the appended claims and their equivalents. All references referred to herein are hereby incorporated by reference in their entirety.