APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1994, p. 31-38
`0099-2240/94/$04.00+0
`Copyright X 1994, American Society for Microbiology
`
`Vol. 60, No. 1
`
`Structural and Immunological Characterization of a
`Biosurfactant Produced by Bacillus licheniformis JF-2
`SUNG-CHYR LIN,' MARK A. MINTON,2 MUKUL M. SHARMA,3 AND GEORGE GEORGIOUl*
`Departments of Chemical Engineering,' Chemistry,2 and Petroleum Engineering,3
`The University of Texas at Austin, Austin, Te-xas 78712
`
`Received 30 August 1993/Accepted 28 October 1993
`
`BaciUlus licheniformis JF-2 produces a very active biosurfactant under both aerobic and anaerobic
`conditions. We purified the surface-active compound to homogeneity by reverse-phase C18 high-performance
`liquid chromatography and showed that it is a lipopeptide with a molecular weight of 1,035. Amino acid
`analysis, fast atom mass and infrared spectroscopy, and, finally, 'H, '3C, and two-dimensional nuclear
`magnetic resonance demonstrated that the biosurfactant consists of a heterogeneous Cl5 fatty acid tail linked
`to a peptide moiety very similar to that of surfactin, a lipopeptide produced by BaciUus subtilis. Polyclonal
`antibodies were raised against surfactin and shown to exhibit identical reactivity towards purified JF-2
`lipopeptide in competition enzyme-linked immunosorbent assays, thus providing further evidence for the
`structural similarity of these two compounds. Under optimal conditions, the B. licheniformis JF-2 biosurfactant
`exhibits a critical micelle concentration of 10 mg/liter and reduces the interfacial teinsion against decane to 6 x
`lo-3 dyne/cm, which is one of the lowest interfacial tensions ever reported for a microbial surfactant.
`
`Microbial compounds which exhibit pronounced surface
`activity are classified as biosurfactants. Microbial biosurfac-
`tants include a wide variety of surface- and interfacially
`active compounds, such as glycolipids, lipopeptides, po-
`lysaccharide-protein complexes, phospholipids, fatty acids,
`and neutral lipids (6). Biosurfactants consist of distinct
`hydrophilic and hydrophobic moieties. The former can be
`either ionic or nonionic and consist of mono-, di-, or polysac-
`charides; carboxylic acids; amino acids; or peptides. The
`hydrophobic moieties are usually saturated, unsaturated, or
`hydroxylated fatty acids. Biosurfactants are easily biode-
`gradable and thus are particularly suited for environmental
`applications such as bioremediation and the dispersion of oil
`spills (8, 20-22).
`Among the many classes of biosurfactants, lipopeptides
`are particularly interesting because of their high surface
`activities and therapeutic potential. For example, surfactin,
`a well-studied lipopeptide antibiotic produced by Bacillus
`subtilis, is not only a very effective biosurfactant (5) but is
`also an inhibitor of fibrin clotting (1, 3) and cyclic AMP
`phosphodiesterase (10).
`Bacillus lichenifornis JF-2, isolated from oil-field injection
`brine (13), has been shown to be able to grow and produce a
`very effective biosurfactant under both aerobic and anaero-
`bic conditions at a very wide range of temperatures and in
`the presence of high concentrations of salts (12, 17). We
`have studied the effects of various environmental parameters
`on the production of the biosurfactant, the formation of
`fermentation end products, and the growth of B. licheni-
`formis JF-2 in batch cultures (17, 18). Although it has been
`speculated that the JF-2 biosurfactant is similar to surfactin
`from B. subtilis (7, 19), the chemical structure of the JF-2
`biosurfactant had not been characterized. Recently, two
`other Bacillus isolates, B. licheniformis 86 (9) and another B.
`licheniformis strain isolated by Jenny et al. (14), have been
`
`* Corresponding author. Mailing address: Department of Chemi-
`cal Engineering, The University of Texas at Austin, Austin, Texas
`78712. Phone: (512) 471-6975. Fax: (512) 471-7963. Electronic mail
`address: gg@mail.che.utexas.edu.
`
`shown to produce lipopeptides with peptide moieties con-
`taining C-terminal amino acid residues different from those
`of surfactin.
`In this study, we purified the surface-active compound
`from B. licheniformis JF-2 to apparent homogeneity. The
`structure of this compound was characterized by amino acid
`analysis and various spectroscopic techniques. As part of
`the characterization studies, we raised polyclonal antibodies
`against surfactin and showed that they exhibit the same
`reactivity for the JF-2 surfactant. This result together with
`amino acid analysis and two-dimensional nuclear magnetic
`resonance (NMR) data indicated that the peptide sequence
`of the JF-2 surfactant is identical to that of surfactin.
`However, the two compounds differ with respect to the
`composition of their fatty acid tails. In addition to the
`chemical characterization of the JF-2 lipopeptide, we studied
`its interfacial properties and found that under optimal con-
`ditions the JF-2 lipopeptide is one of the most effective
`bacterial lipopeptide surfactants known.
`
`MATERUILS AND METHODS
`Microorganisms and growth conditions. B. lichenifonnis
`JF-2 (ATCC 3097) was obtained from the American Type
`Culture Collection (Rockville, Md.). The bacteria were
`grown aerobically in a mineral salt medium (13), containing
`0.1% (NH4)2SO4, 0.025% MgSO4, 1% (wt/vol) glucose, 0.5%
`NaCl in 100 mM phosphate buffer (pH 7.0), and 1.0%
`(vol/vol) trace metals solution, in 2-liter Erlenmeyer flasks
`with a working volume of 1 liter at 42°C for 15 h. The trace
`metals solution contained 0.1% (wt/vol) EDTA, 0.3%
`MnSO4, 0.001% FeSO4, 0.01% CaCl2, 0.01% CoCl2, 0.01%
`ZnS04, 0.001% CuS04, 0.001% AlK(S04)2, 0.001% H3B04,
`and 0.001% Na2MoO4 (4).
`Isolation and purification. The cells were removed from
`the culture by centrifugation at 8,000 x g for 15 min.
`Surface-active compounds were then isolated from the clear
`broth either by acid precipitation with concentrated HCl at
`pH 2.0 (7) or by XAD-2 (Sigma, St. Louis, Mo.) adsorption
`chromatography. For the former, the surfactant-containing
`
`31
`
`PETITIONERS
`
`EXHIBIT NO. 1031 Page 1 of 8
`
`

`

`32
`
`LIN ET AL.
`
`APPL. ENvIRON. MICROBIOL.
`
`c
`
`B
`
`A
`
`o
`1-
`
`0Q
`
`) I
`
`4i
`9c
`CZ
`
`C4
`
`precipitate was collected by centrifugation and resuspended
`in 15 ml of water adjusted to pH 6.0 and subsequently
`lyophilized. The lyophilized material was then extracted
`with 5 ml of a mixture of chloroform and methanol (1:2
`[vol/vol]). For XAD-2 adsorption chromatography, the fer-
`mentation broth supernatant was loaded onto a column (16
`by 500 mm) at a flow rate of 1 ml/min. The column was eluted
`with 1.5 bed volume of methanol following a 1.5 bed volume
`of water wash. Nonvolatile material in the eluent was
`concentrated by evaporation in a rotary evaporator at 45°C.
`Samples obtained by either procedure were designated as
`the crude biosurfactant preparation. The crude biosurfactant
`preparation was dissolved in 3 ml of a mixture of chloroform
`and methanol (1:1) and further separated into five fractions
`by liquid chromatography on a silica gel (no. 62; Mallinck-
`rodt, Paris, Ky.) column (28 by 500 mm) eluted with 5%
`methanol in chloroform at a flow rate of 2 ml/min. The
`fraction containing the surface-active component was iden-
`tified by interfacial tension measurements, as described
`below. The active compound was purified to homogeneity by
`preparative reverse-phase liquid chromatography at room
`temperature with a Waters high-performance liquid chroma-
`tography (HPLC) system (Milford, Mass.) equipped with a
`Waters C18 ,uBondapak column (19 by 300 mm). The solvent
`system consisted of mobile phase A (10 mM KH2PO4 buffer
`at pH 6.0) and mobile phase B (20% tetrahydrofuran in
`acetonitrile [HPLC grade; Fisher Scientific, Fair Lawn,
`N.J.]). The column was developed with 53% B isocratically
`at a flow rate of 2 ml/min. Biosurfactant-containing fractions
`were lyophilized and extracted with methanol to remove
`salt. For analytical reverse-phase C18 HPLC analysis, a
`pBondapak C18 column (7.8 by 300 mm) was used at a flow
`rate of 0.5 ml/min. The A210 of the eluent was monitored.
`Characterization. Infrared spectroscopy was performed on
`a Nicolet 60SXR FT-IR spectrometer. The spectrum was
`measured in a sample compartment purged for at least half
`an hour with dry nitrogen before acquiring data, which were
`measured at a resolution of 4 cm-' and were averaged over
`500 scans. Baselines were electronically adjusted to zero
`absorbance for the measurement of spectral intensities.
`For amino acid analysis, the purified biosurfactant was
`hydrolyzed in 6 M HCl at 105°C for 24 h. The hydrolysate
`was subjected to an Applied Biosystems 420H Derivatizer/
`Analyzer with on-line 130A Separation System and 920A
`Data Analysis Module (Foster City, Calif.). Norleucine was
`added in the samples to a final concentration of 500 pM as an
`internal standard.
`Fast atom bombardment-mass spectroscopy (FAB-MS)
`analysis was performed on a Finnigan TSQ 70 mass spec-
`trometer with an NBA matrix. Mass spectra were collected
`from 100 to 1,200 AMU. Positive ions were detected.
`NMR analysis (1H, '3C, COSY, TOCSY, and ROESY
`NMR) of JF-2 biosurfactant was performed in a Bruker
`high-field (11.9-T) NMR spectroscope, with 1,2-dideuterio-
`tetrachloroethane (CDCl2CDCl2) (Norell, Landisville, N.J.)
`as solvent at 348 K.
`Production of polyclonal antibodies against surfactin. A
`total of 2 mg of surfactin (Calbiochem, San Diego, Calif.),
`dissolved in a 400-,ul mixture of phosphate-buffered saline
`(PBS) and dimethylformamide (PBS-dimethylformamide
`[3:1]), was mixed with 300 RI of Pierce Imject keyhole limpet
`hemocyanin (KLH)-100 pl of 1 M [1-ethyl-3-(dimethylami-
`no)propyl]-carbodiimide-50 ,ul of 0.1 M N-hydroxyl-sulfo-
`succinimide (Pierce, Rockford, Ill.) at room temperature.
`The conjugation reaction was allowed to proceed for 20 min
`and subsequently quenched by the addition of 3.0 ml of 50
`
`I
`
`30
`
`1
`J1
`20
`10
`Retention Time
`(minute)
`FIG. 1. Analytical reverse-phase C18 HPLC chromatogram of
`surface-active material obtained from silica gel liquid chromatogra-
`phy. Fraction C was identified as the most active component by
`interfacial tension measurements.
`
`I
`
`40
`
`mM glycine (11). The conjugate was dialyzed against PBS
`overnight and used to immunize rabbits by subcutaneous
`injection with incomplete Freund's adjuvant (Sigma) on the
`basis of a standard schedule (2). Blood samples were col-
`lected from the ear vein 10 weeks after immunization (2
`weeks after the booster injection). Sera were prepared by
`centrifuging the blood samples at 5,000 x g for 10 min.
`ELISA. Microtiter plates were coated by an overnight
`incubation at 37°C with a surfactin-ovalbumin conjugate
`which was prepared in the same procedure as the surfactin-
`KLH conjugate used for immunization. The plate was then
`blocked with 3% bovine serum albumin (BSA) in PBS by
`incubation at 37°C for 3 h. For competition enzyme-linked
`immunosorbent assays (ELISAs), 50 ,ul of diluted rabbit
`surfactin-specific serum (1:1,000 in 3% BSA in PBS) and 100
`pA of sample were preincubated at 37°C for 2 h. Subse-
`quently, the mixture was transferred to microtiter plates
`precoated with the surfactin-ovalbumin conjugate and incu-
`
`PETITIONERS
`
`EXHIBIT NO. 1031 Page 2 of 8
`
`

`

`VOL. 60, 1994
`
`96.6
`
`B. LICHENIFORMIS JF-2 SURFACTANT
`
`33
`
`uJ
`
`Oz§60
`
`'0-
`
`zcn
`m 50-
`
`40-
`
`27.5
`
`._
`
`4000
`4000.9
`
`V l
`
`_
`
`_
`3500
`
`3000
`
`IMBERS
`WAVEM
`trum of the JF-2 biosurfactant.
`FIG. 2. The infrared spect
`
`bated for an additional 2 h. A 3% BSA solution in PBS and
`surfactin at a concentration of 25 mg/liter also in PBS
`were used as negative and positive controls, respectively.
`Plates were washed with deionized water 10 times; washing
`was followed by the addition of 100 ,ul of diluted goat
`anti-rabbit immunoglobulin G-horseradish peroxidase conju-
`gate diluted 1:1,000 in 3% BSA in PBS (Bio-Rad, Richmond,
`Calif.). The plates were incubated for 2 h at 37°C and washed
`again as described above. A total of 100 ,ul of substrate,
`1-Step ABTS (Pierce), was then added to each well. The
`
`enzyme reaction was allowed to proceed for 10 min and then
`stopped by the addition of 50 ,ul of 1% sodium dodecyl
`sulfate (SDS). The A405 of the solution in each well was
`measured with a Dynatech MR300 microtiter plate reader
`(Chantilly, Va.).
`To quantify the binding affinities of the serum towards
`surfactin and the JF-2 biosurfactant, the percent inhibition of
`antibody-conjugate binding by free antigens is defined as
`follows (23): percent inhibition = {1 - [(AEX - AN )I(Ap0
`-ANeg)]} x 100, where AEXP is the A405 of the sampfe, APos
`
`154
`
`100 -
`
`80
`
`60
`
`40
`
`20
`
`0
`
`1036
`
`329
`
`460
`1
`
`613
`
`685
`
`1018
`
`200
`
`800
`600
`400
`FIG. 3. The FAB-MS spectrum of the JF-2 biosurfactant.
`
`1000
`
`- 1.2
`
`1.0
`
`0.8
`
`0.6
`
`- 0.4
`
`0.2
`
`ti - 0.0
`1200
`
`PETITIONERS
`
`EXHIBIT NO. 1031 Page 3 of 8
`
`

`

`34
`
`LIN ET AL.
`
`APPL. ENVIRON. MICROBIOL.
`
`A
`
`I..
`PPM
`
`7
`
`6
`
`5
`
`4
`
`3
`
`FIG. 4. Proton NMR spectrum of the JF-2 biosurfactant.
`
`0
`
`2
`
`I
`
`0
`
`is the A405 of the positive control (colorless), and ANeg is the
`A405 of the negative control (green).
`Interfacial properties. Interfacial tension measurements
`against decane were performed in a spinning drop interfacial
`tensiometer (Model 300; University of Texas, Austin). The
`critical micelle concentration (CMC) was determined by
`measuring the interfacial tension of the biosurfactant solu-
`tion following serial dilution. All interfacial tensions were
`measured against decane.
`
`RESULTS
`Isolation and purification. Interfacial tension measure-
`ments indicated that the biosurfactant can be effectively
`isolated from the cell-free culture supernatant by either acid
`precipitation or XAD-2 adsorption chromatography. The
`interfacial tension increased from 0.085 dyne/cm for the
`cell-free culture to more than 25 dyne/cm for the reneutral-
`ized acid precipitation supernatant of the culture. When acid
`precipitation was used as the first purification step, 250 mg of
`acid precipitate was obtained per liter of culture after lyoph-
`ilization. Subsequently, 110 mg of water-soluble material
`remained after organic extraction. The crude biosurfactant
`preparation was further separated into five fractions by silica
`
`gel chromatography. Only one fraction, which was eluted
`after 1.8 bed volumes, contained surface active material.
`Analytical reverse-phase C18 HPLC analysis showed the
`presence of three major peaks in this material (Fig. 1).
`Preparative reverse-phase C18 HPLC was used to isolate
`three fractions corresponding to the material in each of the
`three peaks. Interfacial tension measurements indicated that
`fraction C contained the most surface active compound. At
`a concentration of 50 mg/liter in fresh medium with 5% NaCl
`and adjusted to pH 6.0, the material obtained from fractions
`A, B, and C gave interfacial tensions against decane of 1.317,
`1.873, and 0.060 mN/cm, respectively. Material from frac-
`tion C was extracted with an equal volume mixture of
`chloroform and methanol to remove the salt from the HPLC
`mobile phase. Approximately 25 mg of the highly active
`compound per liter of culture was obtained. Since the
`concentration of the surfactant in the fermentation broth is
`34 mg/liter, the overall yield is approximately 70%. The
`purified material gave a single peak in analytical reverse-
`phase C18 HPLC (data not shown). Lack of anomalous peaks
`in proton and carbon NMRs, as discussed below, provides
`further confirmation of the purity of the biosurfactant. The
`same yield and final purity were obtained with either acid
`precipitation or XAD-2 chromatography as the first separa-
`
`PETITIONERS
`
`EXHIBIT NO. 1031 Page 4 of 8
`
`

`

`VOL. 60, 1994
`
`B. LICHENIFORMIS JF-2 SURFACTANT
`
`35
`
`Glu
`Leu
`
`Leu
`
`Asp
`Fatty Acid
`A1_
`
`Fatty
`Acid
`
`Asp
`
`Glu
`
`I
`
`Leu
`
`Leu
`
`Leu
`
`Fatty Acid-Glu
`
`I
`
`Ii
`
`v
`
`I,l
`
`Glu-Leu
`
`V
`
`Leu-Asp
`
`Leu-Asp
`
`V
`
`II
`Leu-Leu
`
`l
`
`I
`
`-7.0
`
`-7.2
`
`7.4
`
`-7.6
`
`PPM
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`I
`
`.
`
`.
`
`4.5
`3.5
`5.0
`4.0
`ppM
`FIG. 5. The ROESY spectrum of the JF-2 biosurfactant obtained
`proton-proton relationships are indicated by filled arrowheads and labe
`
`.
`
`.
`
`I
`
`.
`
`.
`
`.
`
`.
`
`I
`
`.
`
`.
`
`.
`
`.0.
`I
`.5
`1.0
`1.5
`2.5
`2.0
`3.0
`in CDCl2CDC12 at 348 K. Signals corresponding to nongerminal
`led.
`
`tion step. XAD-2 chromatography is likely to be better
`suited for the continuous removal of the surfactant from the
`fermentation broth and for scale-up purposes.
`Chemical structure of the surfactant. Figure 2 shows the
`infrared spectrum of the JF-2 biosurfactant. Bands charac-
`teristic of peptides (wave number 3,430:NH, wave number
`1,655:CO, and wave number 1,534:CN) and aliphatic chains
`(wave number 3,000 to 2,800, CH2 and CH3) were observed,
`indicating that this compound is a lipopeptide. Also ob-
`served was a band corresponding to an ester carbonyl group
`(wave number 1,730:CO) (17). Amino acid analysis indicated
`the presence of four different amino acid residues in the
`peptide moiety of the biosurfactant. The composition was
`determined to be glutamic acid:aspartic acid:valine:leucine
`= 1:1:1:4. The FAB-MS spectrum (Fig. 3) indicated that the
`biosurfactant has a molecular mass of 1,035 Da.
`Proton NMR (Fig. 4) showed seven NH signals (8 7.0 to
`7.7) and seven corresponding CH signals (8 3.9 to 4.9) for the
`a-amino acids of the peptide. These were readily correlated
`with one another as well as with the signals of the corre-
`sponding alkyl residues via two-dimensional COSY and
`TOCSY spectra (data not shown). Since there were no
`
`signals for free CONH2, eliminating asparagine and glu-
`tamine as possibilities, the identities of the amino acids were
`confirmed as aspartic acid, glutamic acid, leucine, and
`valine. An additional low-field signal at 8 5.3 consistent with
`CHO of the alcohol moiety of an ester (or lactone) was also
`observed.
`Attempts to obtain the sequence of the amino acids by
`two-dimensional NOE (ROESY) NMR were moderately
`successful (Fig. 5). Germinal as well as more distant proton-
`proton relationships were observed in the ROSEY spectrum.
`These germinal proton-proton relationships, which can be
`considered as noises in peptide sequencing, were also ob-
`served in the TOCSY spectrum (data not shown) and,
`therefore, can be eliminated from the ROESY spectrum,
`leaving signals resulting from distant proton-proton relation-
`ships. These remaining proton-proton relationships yield the
`following partial sequences that are the same as those found
`in surfactin (15): fatty acid-Glu-Leu, Asp-Leu, and Leu-Leu.
`On the bases of the composition of the peptide moiety and
`the molecular weight of the molecule, the lipid chain was
`determined to be a C15 fatty acid amidated to the N-terminal
`amine of the peptide. It was obvious that a mixture of
`
`PETITIONERS
`
`EXHIBIT NO. 1031 Page 5 of 8
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`

`

`36
`
`LIN ET AL.
`
`APPL. ENVIRON. MICROBIOL.
`
`CH3-CH
`(1)
`
`2-CH R
`1
`CH3
`(3)
`
`Ante
`biso-
`
`CH3 -CH -CH2-R
`1
`(4)
`CH;
`(4)
`Hso-
`
`CH3 -CH2-CH2-CH2 -R
`(2)
`
`Normal-
`
`3 1
`
`2
`
`wm
`
`z22
`
`20
`16
`t
`14
`FIG. 6. Part of carbon NMR spectrum of the JF-2 biosurfactant.
`
`12
`
`washed plates are incubated with the secondary antibodies.
`The results of the competition ELISA are shown in Fig. 7.
`The absence of any color response, resulting from the
`complete blocking of the antigen binding sites on antibodies
`by free surfactin molecules in the preincubation mixture in
`row D, indicated that the polyclonal antibodies specifically
`recognize free surfactin. The absence of a decrease in color
`response in control with SDS (50mg/liter in PBS) in row B
`indicated that (i) the serum does not react nonspecifically
`with other surface active compounds and (ii) the lack of
`color response when the sera were preincubated with sur-
`factin was not due to the detergent character of the molecule
`causing the desorption of the bound antigen from the wells.
`We found that the cyclic conformation of the peptide moiety
`is important for antibody binding since no competition was
`observed upon addition of a synthetic peptide with the same
`amino acid sequence as surfactin (L-Glu-L-Leu-D - Leu-L -
`
`A B
`
`FIG. 7. The competition ELISA of surfactin and the JF-2 bio-
`surfactant. (A) Control; (B) SDS; (C) the JF-2 biosurfactant; (D)
`surfactin. The lack of color response indicates that the binding of
`antibodies to the coated surfactin-ovalbumin conjugate is inhibited.
`Fifty percent serial dilutions of the samples in each row were
`performed from left to right.
`
`normal, anteiso, and isobranched forms were present (CH3
`at 8 13.4, 10.7, and 18.7, and ca. 22, respectively) as
`observed in the carbon spectrum (Fig. 6).
`Immunoreactivity with surfactin-specific antibodies. In or-
`der to further assess the structural similarity in peptide
`moiety between surfactin and the JF-2 biosurfactant, we
`determined the immunoreactivities of the JF-2 biosurfactant
`against antibodies specific to surfactin. Polyclonal antibodies
`were raised against a surfactin-KLH conjugate prepared by
`[1-ethyl-3-(dimethylamino)propyl]-carbodiimide (EDC) cou-
`pling. Conjugation of the surfactin to KLH was necessary
`because surfactin (molecular weight, 1,035) alone is proba-
`bly too small to elicit an immune response in rabbits. With
`the surfactin-KLH conjugate, high antibody titers were
`obtained. Sera were collected and used to develop an ELISA
`for surfactin. The ELISA analysis showed that the poly-
`clonal antibodies bound to the surfactin-ovalbumin conju-
`gate but not to ovalbumin alone, indicating that they are
`specific for the surfactin moiety of the surfactin-KLH con-
`jugate (data not shown).
`A competition ELISA was developed to demonstrate that
`the polyclonal antibodies can bind free surfactin molecules.
`Detection by direct ELISA was not possible because free
`surfactin molecules could not be applied effectively as a
`coating to microtiter plate wells, presumably because of the
`amphiphilic nature of the surfactin. In competition ELISA, a
`surfactin-containing sample is first preincubated with the
`polyclonal antibodies and then the mixture is transferred to
`microtiter wells coated with a surfactin-ovalbumin conju-
`gate. The formation of antigen-antibody complexes during
`preincubation decreases the amount of free antibodies that
`can bind to surfactin-ovalbumin. The decreased amount of
`bound antibodies reduces the signal resulting when the
`
`PETITIONERS
`
`EXHIBIT NO. 1031 Page 6 of 8
`
`

`

`VOL. 60, 1994
`
`B. LICHENIFORMIS JF-2 SURFACTANT
`
`37
`
`100
`
`90
`
`.
`
`80
`~70
`|60
`
`50
`
`40
`
`-2
`
`-1
`
`0
`Log [concentration]
`FIG. 8. Inhibition of binding of antibodies to coated surfactin-
`ovalbumin conjugate by surfactin (-) and the JF-2 biosurfactant
`(0).
`
`1
`
`2
`
`Val - L - Asp - D - Leu - L - Leu) (Bio-Synthesis, Inc.,
`Lewisville, Tex.) (data not shown).
`Preincubation of the serum with a solution of the JF-2
`surfactant serially diluted twofold from an initial concentra-
`tion of 25 mg/liter gave an inhibition pattern identical to that
`obtained with surfactin (Fig. 7, row C). The percent inhibi-
`tion by surfactin and the JF-2 biosurfactant at different
`concentrations is shown in Fig. 8. It is evident that the
`percent inhibition is identical, indicating that the surfactin-
`specific antibodies recognize and exhibit the same affinity for
`the JF-2 lipopeptide.
`Interfacial activity. The effect of pH on the interfacial
`activity of the JF-2 biosurfactant against decane was inves-
`tigated. As shown in Fig. 9, the biosurfactant preparation
`exhibited optimal interfacial activity at pH 6.0. The interfa-
`cial tensions of the solutions at pH 6.0 (0.006 and 0.023
`dyne/cm in the presence of 5 and 0.5% of NaCl, respectively)
`were 10 times lower than those at pH 7.0 with the same ionic
`strength (0.060 and 0.024 dyne/cm in the presence of 5 and
`0.5% of NaCl, respectively). At pH 6.0, the interfacial
`tension in the presence of 5% NaCl (0.006 dyne/cm) was four
`
`pH
`FIG. 9. The effect of pH on the interfacial activity of the JF-2
`biosurfactant. Purified biosurfactant was dissolved in buffer solu-
`tions with 0.5% (squares) or 5% (diamonds) NaCl.
`
`C
`B
`A
`D
`FIG. 10. The effects of microbial metabolites and NaCl concen-
`tration on the interfacial activity of the biosurfactant produced by B.
`licheniformis JF-2. The interfacial tension against decane for solu-
`tions of purified biosurfactant resuspended at a concentration of 50
`mg/liter in fresh medium with 0.5% NaCl at pH 6.0 (A), fresh
`medium with 5% NaCl at pH 6.0 (B), the supernatant of acid
`precipitation with 0.5% NaCl at pH 6.0 (C), and the supernatant of
`acid precipitation with 5% NaCl at pH 6.0 (D) were measured.
`
`times lower than that in the presence of 0.5% NaCl (0.023
`dyne/cm). A similar profile of pH effect on surfactin interfa-
`cial activity was also observed (data not shown). Interfacial
`tension measurements at each dilution of biosurfactant in
`freshly prepared medium with 0.5% NaCl at pH 6.0 indicated
`that the biosurfactant has a CMC of 10 mg/liter.
`The effects of various additives on the interfacial activity
`of the biosurfactant are shown in Fig. 10. For these experi-
`ments, the interfacial activity of the biosurfactant was arbi-
`trarily defined as the reciprocal of interfacial tension against
`decane. The activity of the JF-2 biosurfactant resuspended
`in the supernatant of acid-precipitated fermentation broth
`containing 5% NaCl was almost 20 times higher than that of
`the biosurfactant in freshly prepared growth medium with
`0.5% NaCl. This result indicates that both NaCl and acid-
`soluble components in the fermentation broth function as
`cosurfactants to augment the interfacial activity of the JF-2
`lipopeptide. The interfacial tension of solution containing the
`JF-2 biosurfactant in the supernatant of acid-precipitated
`fermentation broth with 5% NaCl at pH 6.0 at a concentra-
`tion of 50 mg/liter was as low as 0.006 dyne/cm.
`
`DISCUSSION
`
`We have purified the B. licheniformis JF-2 lipopeptide by
`either acid precipitation or XAD-2 adsorption chromatogra-
`phy as the first step followed by silica gel chromatography
`and preparative-scale C18 reverse-phase HPLC. These pro-
`cedures gave about 25 mg of homogeneous material, as
`determined by analytical HPLC and NMR, with an overall
`yield of 70%.
`The results of chemical analysis, immunological reactiv-
`ity, and NMR spectra indicate that the chemical structure of
`the peptide moiety of the JF-2 surfactant is very similar to
`that of surfactin from B. subtilis. However, some differences
`between these two biosurfactants were detected. First of all,
`the FAB-MS spectrum of the JF-2 biosurfactant indicated
`that the purified surfactant is a homogeneous preparation.
`The presence of a family of lipopeptides with the same
`peptide domains but different chain lengths of lipid tails,
`
`PETITIONERS
`
`EXHIBIT NO. 1031 Page 7 of 8
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`

`38
`
`LIN ET AL.
`
`APPL. ENVIRON. MICROBIOL.
`
`which has been frequently observed for lipopeptides pro-
`duced by other microorganisms (9), was not observed for the
`biosurfactant produced by B. licheniformis JF-2. In addition,
`13C NMR analysis indicated that the lipid tail of JF-2
`biosurfactant is present in three different configurations,
`namely, n-, iso-, and anteisoforms. The anteisoform of fatty
`acids has not been observed in surfactin or other lipopeptide
`surfactants. Nevertheless, the difference in lipid moieties
`between surfactin and the JF-2 biosurfactant does not affect
`their interfacial activities.
`The effect of pH on the interfacial activity of surfactin and
`the JF-2 biosurfactant cannot be easily explained by proto-
`nation of the carboxylic side chains of glutamic and aspartic
`acids in the peptide moiety, since the pK. values are 4.25
`and 3.86, respectively. We suspected that the pH depen-
`dence may be a consequence of a metal chelation effect
`which alters the electrostatic properties of the molecule.
`However, molecular modeling by energy minimization of the
`cyclic peptide conformation revealed that the interatomic
`distances of the carboxylic groups are inconsistent with a
`metal binding site either in a monomer or as a dimer (1Sa).
`Therefore, the pH dependence of the biosurfactant is most
`likely the result of complex electrostatic interactions at the
`water/decane interface.
`Results of competition ELISA with the surfactin-specific
`antibodies further confirmed that surfactin and the JF-2
`biosurfactant have exactly the same amino acid composition
`and sequence. The competition ELISA can also be used for
`the quantification of biosurfactant concentration in the fer-
`mentation culture supernatant. Detection of the biosurfac-
`tant by competition ELISA allows high specificity and
`sensitivity (at least as low as 0.01 mg/liter) as well as the
`ability to analyze a large number of samples simultaneously.
`The immunoassay can also be used as a screening assay for
`detecting lipopeptide-overproducing mutants of B. subtilis or
`B. licheniformis JF-2 (data not shown).
`The CMC of the JF-2 biosurfactant was determined to be
`10 mg/liter, significantly lower than that of other biosurfac-
`tants as well as many synthetic surfactants. The CMC can be
`interpreted as the solubility of surfactant in water or the
`minimum concentration required to reach the maximum
`interfacial or surface activity. Therefore, in terms of CMC,
`the JF-2 biosurfactant satisfies an important criterion for
`commercial detergency applications.
`Furthermore, we
`showed that sodium chloride and metabolites in the culture
`supernatant work synergistically with the JF-2 biosurfactant
`in reaching the maximum interfacial activity. Under optimal
`conditions, JF-2 biosurfactant at a concentration of 50 mg/
`liter reduced the interfacial tension of aqueous phase against
`decane to 0.006 dyne/cm, a 500-fold reduction from the
`interfacial tension obtained in the absence of surfactant (32
`dyne/cm). This is the lowest value that has been reported for
`microbial surfactant so far (6).
`
`ACKNOWLEDGMENTS
`Support for this work was provided by the Department of Energy,
`the Texas Advanced Research and Technology Program, and the
`Center for Enhanced Oil and Gas Recovery Research at the Uni-
`versity of Texas.
`We are grateful to Brent Iverson for advice on producing the
`surfactin conjugates and for help with computer simulations and to
`Sandies Smith for help with amino acid analysis and Peter Zuber for
`providing us with B. subtilis strains.
`
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