Vol. 31, No. 3, 1968
`
`BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
`
`SURFACTIN, A CRYSTALLINE PEPTIDELIPID SURFACTANT PiIODUCED
`
`BY BACILLUS SUBTILIS
`
`:
`
`ISOLATION, CHARACTEXIZATION
`
`AND ITS INHIBITIOR OF FIBRIIJ CLOT FO:IEiATION
`
`Kei Arima, Atsushi Kakinuma, and Gakuzo Tamura
`
`Department of Agricultural
`
`Chemistry
`
`University
`
`of Tokyo, Tokyo, Japan
`
`Received
`
`April
`
`5, 1968
`
`In a study
`
`intending an approach
`
`to blood clotting
`
`and / or
`
`fibrinolytic
`
`system(s)
`
`from microbiology
`
`using microbial
`
`products, we
`
`have recently
`
`found a potent clotting
`
`inhibitor
`
`produced in
`
`the culture
`
`fluids
`
`of several strains of B. subtilis.
`
`The inhibitor
`
`was isolated as white needle crystalls
`
`and from its
`
`strong surface
`
`active
`
`nature exceeding
`
`that of sodium lauryl
`
`sulfate
`
`was named %urfactinVt.
`
`Surfactin
`
`is a peptidelipid
`
`with
`
`the molecular
`
`weight of about 1050 and is composed of L-aspartic
`
`acid, L-glutamic
`
`acid, L-valine,
`
`L-leucine,
`
`D-leucine
`
`(1:1:1:2:2)
`
`and unidentified
`
`fatty
`
`acids.
`
`This bacterial
`
`peptidelipid
`
`remarkably elongates
`
`the time
`
`necessary
`
`for
`
`fibrin
`
`clot
`
`formation by inhibiting
`
`the conversion of
`
`fibrin monomer to
`
`fibrin
`
`polymer.
`
`In
`
`this communication we wish to report on the isolation
`
`and
`
`characterization
`
`of surfactin
`
`and the determination
`
`of
`
`its
`
`inhibition
`
`site
`
`in
`
`fibrin
`
`clot
`
`formation.
`
`Further attempts carried out
`
`to know some other physiological
`
`functions
`
`of surfactin
`
`have revealed
`
`in
`
`this peptidelipid
`
`several
`
`interesting
`
`activities,
`
`for example,
`
`inhibition
`
`of protein denatur-
`
`ation,
`
`lysis of intact
`
`cells of some of both gram positive
`
`and gram
`
`488
`
`PETITIONERS
`
`EXHIBIT NO. 1021 Page 1 of 7
`
`

`

`Vol. 31, No. 3, 1968
`
`BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
`
`negative bacteria with
`
`the leakage of cell contents,
`
`etc, details
`
`of
`
`which will appear in succeeding papers.
`
`Isolation
`
`and Characterization
`
`of Surfactin
`
`Surfactin
`
`is
`
`produced
`
`in a fair amount in
`
`the culture
`
`fluids of several strains of
`
`B. subtilis.
`
`Especially,
`
`B. subtilis
`
`IF0 3039,
`
`IAN 1069,
`
`IAM 1213,
`
`IAM 1259, IAN 1260
`
`can accumulate 50 - 100 ug surfactin
`
`per ml nutrient
`
`broth
`
`in 24 h culture.
`
`Surfactin
`
`is an acidic substance soluble
`
`in alkaline water and in
`
`many kinds of organic solvents
`
`including methanol, ethanol, acetone,
`
`ethylacetate,
`
`chloroform, methylene chloride,
`
`acetic acid but insoluble
`
`in water, petroleum ester, hexane, etc.
`
`Addition
`
`of (NH4)2S04 to 50 %
`
`saturation
`
`or of divalent
`
`cations
`
`in alkaline
`
`solution makes surfactin
`
`insoluble and results
`
`in
`
`its precipitation.
`
`Using these solubility
`
`characteristics,
`
`surfactin was crystallized
`
`as follows.
`
`B. subtilis
`
`IAM 1213 was grown in a nutrient
`
`broth at 30" for 24 h
`
`on a reciprocal
`
`shaker. After
`
`the elimination
`
`of cells
`
`the reaction
`
`of
`
`filtrate
`
`was adjusted
`
`to pH 2 with HCl and the resulting
`
`precipitate
`
`was collected
`
`and dissolved
`
`in alkaline water,
`
`followed by the addition
`
`of CaCl 2' This Ca-precipitate
`HCl and newly generated acid-precipitate
`
`was collected
`
`and resuspended in 0.01 N
`
`was extracted with ether.
`
`Active material
`
`obtained by concentration was dissolved
`
`in acetone and
`
`decolorized
`
`by passing an active
`
`charcoal column pretreated with
`
`acetone.
`
`The clear acetone solution
`
`thus obtained was applied
`
`to a
`
`Sephadex LH 20 column pretreated with acetone and to
`
`the eluate contain-
`
`ing active
`
`fraction,
`
`water of about half
`
`the volume of acetone was added
`
`to make the solution
`
`slight
`
`turbid. When this solution was preserved
`
`in
`
`cold
`
`for 2 months, surfactin was obtained as crystalline
`
`white needles
`
`(Figure 11, m.p. 138 - 140°, with
`
`the yield of 40 - 50 mg per liter
`
`of
`
`culture medium.
`
`489
`
`PETITIONERS
`
`EXHIBIT NO. 1021 Page 2 of 7
`
`

`

`Vol. 31, No. 3, 1968
`
`BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
`
`Figure 1. Microuraph of crystalls
`
`of surfactin
`
`Chemical or physicochemical
`
`data of surfactin were as follows.
`
`C 59.6 %, H 9.0 %, N 9.1 %.
`
`[rx)g7 +40 (1 %, in chloroform),
`
`-39"
`
`(1 %, in methanol).
`
`By vapor pressure method,
`
`the molecular weight was
`
`calculated
`
`to be about 1050. UV absorption
`
`spectrum showed the absence
`
`of absorption maximum in
`
`the range from 230 rnp to 400 mp. On the other
`
`hand, presence of peptide bonds was cleary demonstrable
`
`from IR spectrum
`
`(Figure 2). Biuret
`
`reaction
`
`is positive,
`
`ninhydrin
`
`reaction
`
`is negative.
`
`When surfactin was subjected
`
`to acid hydrolysis
`
`in 6 N KC1 in a sealed
`
`tube at
`
`llO"
`
`for 20 h, L-aspartic
`
`acid, L-glutamic
`
`acid., L-valine,
`
`L-
`
`leucine, D-leucine were detected by an amino acid analyzer and by
`
`microbioassay
`
`in
`
`the molecular
`
`ratio of 1:1:1:2:2,
`
`always accompanied
`
`490
`
`PETITIONERS
`
`EXHIBIT NO. 1021 Page 3 of 7
`
`

`

`Vol. 3 1, No. 3, 1968
`
`BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
`
`I
`
`0
`
`I
`40003ooo
`
`2ocO
`
`1oooBoo800
`1!500
`WAVE NUMBER (cm-‘)
`
`I
`
`700
`
`Figure 2.
`
`IR spectrum of surfactin
`
`(KBr)
`
`by a slight amount of L-isoleucine.
`
`And simultaneously,
`
`yellowish
`
`brown-colored HCl-insoluble
`
`oily material was liberated with
`
`the re-
`
`covery of 20 - 25 % original
`
`surfactin.
`
`This oily
`
`material,
`
`after
`
`purification
`
`by repeated
`
`transfer
`
`between ether and alkaline water, did
`
`not contain N and showed in IR spectrum typical
`
`features
`
`characteristic
`
`to
`
`fatty
`
`acids.
`
`From these data, surfactin
`
`is confirmed
`
`to be a peptidelipid,
`
`with
`
`the molecular weight aound 1050,composed of amino acids and fatty
`
`acids.
`
`The identification
`
`of
`
`fatty
`
`acids are now in progress and will
`
`be reported
`
`in a forthcoming paper.
`
`Surfactin was proved
`
`to have a potent surface activity
`
`by far
`
`stronger
`
`than that of sodium lauryl
`
`sulfate
`
`(TABLE 1).
`
`It might be
`
`the first
`
`time
`
`that such a strong surfactant was isolated
`
`from
`
`microbial
`
`origins.
`
`The role
`
`that surfactin may play
`
`in
`
`the physiology
`
`of parent bacteria has not yet been sufficiently
`
`clarified
`
`and remains
`
`for us as a tempting problem in
`
`the
`
`future.
`
`Inhibition
`
`of Fibrin Clot Formation When surfactin was added to
`
`the thrombin-fibrinogen
`
`system,
`
`the formation of
`
`fibrin
`
`clot was dra-
`
`matically
`
`inhibited
`
`and a remarkable elongation of clotting
`
`time
`
`and
`
`491
`
`PETITIONERS
`
`EXHIBIT NO. 1021 Page 4 of 7
`
`

`

`Vol. 31, No. 3, 1968
`
`BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
`
`TABLE
`
`1
`
`Surface
`
`activity
`
`of surfactin
`
`water
`Distilled
`0.1 14 NaHCO
`3
`
`containing
`
`0.005 % SLS(b)
`0.05
`% SLS
`0.005 %
`surfactin
`0.05
`%
`surfactin
`
`Tension
`Surface
`(dyne/cm)
`
`(a)
`
`71.98
`
`71.57
`56.56
`31.25
`27.90
`27.00
`
`(a) Determined
`(b)
`Sodium
`lauryl
`
`by drop weight
`sulfate.
`
`method
`
`using
`
`a stalagometer.
`
`0
`
`2
`
`6
`4
`TIME (min.)
`
`8
`
`10
`
`3
`
`Inhibition
`Figure
`of clotting
`A. Elongation
`Fibrinogen
`(Armour
`Bovine
`was used
`clottable
`protein
`(dissolved
`and neutralized
`0.01 M tris-0.073
`M NaCl
`B. Retardation
`of Opacity
`0.15 %, surfactin
`0 ug/ml
`2.5 U./ml,
`0.02 M
`tris-0.145
`development
`after
`thrombin
`absorbance
`at 600 mu
`in
`
`with
`
`492
`
`by surfactin
`formation
`clot
`fibrin
`of
`fibrinogen
`:
`system
`Clotting
`time
`approximately
`I containing
`Fraction
`experiments),
`these
`throughout
`with
`NaOH) as
`indicated,
`thrombin
`(pH 7.4).
`37".
`:
`system
`Clotting
`Development
`(cl,
`500 ug/ml
`(b),
`(a),
`50 ug/ml
`1964).
`(Yachnin,
`M NaCl
`(pH 7.4)
`addition
`was
`followed
`at 20° by
`cuvettes
`1 cm light
`path.
`
`0.15 %,
`30 %
`surfactin
`5 u./ml,
`
`fibrinogen
`thrombin
`Opacity
`reading
`
`PETITIONERS
`
`EXHIBIT NO. 1021 Page 5 of 7
`
`

`

`Vol. 31, No. 3, 1968
`
`BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
`
`a retardation
`
`of opacity
`
`development
`
`were
`
`observed
`
`(Figure
`
`3A, Figure
`
`3B).
`
`Calculated
`
`from
`
`the
`
`relationship
`
`between
`
`thrombin
`
`concentration
`
`and
`
`clotting
`
`time,
`
`the
`
`concentration
`
`of surfactin
`
`needed
`
`to bring
`
`about
`
`50 % inhibition
`
`of
`
`thrombin
`
`activity
`
`was about
`
`30 ug/ml.
`
`The
`
`inhibition
`
`site
`
`of surfactin
`
`was determined
`
`by several
`
`methods
`
`including
`
`sedimentation
`
`analyses
`
`with
`
`an ultracentrifuge.
`
`In Figure
`
`4,
`
`which
`
`is a sedimentation
`
`diagram
`
`of surfactin-inhibited
`
`clotting
`
`system
`
`containing
`
`fibrinogen,
`
`surfactin
`
`and
`
`thrombin,
`
`nearly
`
`complete
`
`dis-
`
`fibrinogen
`
`and appearance
`
`of a much heavier
`
`component
`
`are
`
`appearance
`
`of
`
`cleary
`
`indicated.
`
`This
`
`new
`
`component
`
`is
`
`comparable
`
`with
`
`those
`
`inter-
`
`mediary
`
`polymers
`
`which
`
`appeared
`
`in
`
`thrombin-fibrinogen
`
`systems
`
`inhibited
`
`by hexamethylene
`
`glycol
`
`(Ferry
`
`and Shulman,
`
`19491,
`
`by urea
`
`(Ehrlich
`
`et al,
`
`19521,
`
`and by 1 M NaBr
`
`at pH 6 (Donnelly
`
`et al,
`
`1955).
`
`The
`
`fact
`
`that
`
`no clotting
`
`occurred
`
`even after
`
`the
`
`proteolytic
`
`action
`
`of
`
`thrombin
`
`has
`
`completed
`
`with
`
`the
`
`generation
`
`of polymerization
`
`inter-
`
`mediates
`
`shows
`
`that
`
`the
`
`inhibition
`
`site
`
`of surfactin
`
`in
`
`this
`
`clotting
`
`of
`diagram
`4 Sedimantation
`Figure
`clotting
`system
`surfactin-inhibited
`fibrinogen
`0.3 %,
`:
`Clotting
`system
`thrombin
`0.5 U./ml,
`surfactin
`1.0 %,
`M NaCl
`(pH 7.4).
`0.01 M tris-0.073
`30 min.
`Sedimented
`Reaction
`: 20°,
`left
`in a Hitachi
`from
`right
`to
`at
`Type WA-1A
`Ultra-centrifuge
`a
`ll“,
`using
`55,430
`rpm and at
`cell.
`synthetic
`boundary
`Photograph
`attaining
`top
`after
`at 15 min.
`speed.
`70".
`Abbrivations
`Bar angle
`in photograph
`: S, surfactin;
`F,
`fibrinogen;
`(F),,
`intermediates
`polymerization
`from
`fibrin
`to
`fibrin
`polymer.
`
`in
`monomer
`
`493
`
`PETITIONERS
`
`EXHIBIT NO. 1021 Page 6 of 7
`
`

`

`Vol. 31, No. 3, 1968
`
`BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
`
`system is
`
`the polymerization
`
`step from fibrin monomer to fibrin
`
`polymer.
`
`This conclusion was strongly
`
`supported by both
`
`the facts
`
`that esterase
`
`activity
`
`of
`
`thrombin against
`
`tosyl arginine methyl ester was not
`
`inhibited
`
`at all by surfactin
`
`of the concentration
`
`used in experiments
`
`of Figure 3 and that
`
`fibrin monomer prepared
`
`in 1 M NaBr, pH 5.3
`
`(Donnelly et al,
`
`1955)
`
`did not clot
`
`in
`
`the presense of surfactin
`
`even
`
`after dilution with
`
`tris-NaCl,
`
`pH 7.4.
`
`Several surfactants
`
`like sodium lauryl
`
`sulfate are known to dis-
`
`aggregate and solubilize
`
`the
`
`fibrin
`
`polymer (Loewy et al, 19611.
`
`The
`
`ability
`
`of surfactin
`
`to
`
`inhibit
`
`clot
`
`formation may derive
`
`from
`
`its
`
`strong surface active nature.
`
`Acknowledffements We are indebted
`
`to Drs. M. Isono and T. Takahashi
`
`of Takeda Research Laboratories
`
`for preparation
`
`and supply of surfactin
`
`from tank cultures. We are also
`
`indebted
`
`to Dr. J. Kirimura of
`
`Ajinomoto Co. for microbioassay and Dr. K. Hattori
`
`of Kao Soap Co. for
`
`determination
`
`of surface
`
`tension.
`
`References
`
`J.A. and Dunathan, K.
`
`(1961).
`
`J. Biol. Chem. a,
`
`Loewy, A.G., Gallant,
`2648.
`Ehrlich,
`2258.
`J.D. and Shulman, S.
`(1949).
`Ferry,
`Yachnin, S. (19641,
`Blood. 24, 553.
`Donnelly, T.H., Laskowski, M., Jr., Notley, N. and Scheraga, H.A.
`Arch. Biochem. Biophs. z,
`369.
`
`P., Shulman, S. and Ferry,
`
`J.D.
`
`(1952). J. Amer. Chem. Sot. 2,
`
`J. Amer. Chem. Sot. &
`
`3198.
`
`(1955).
`
`494
`
`PETITIONERS
`
`EXHIBIT NO. 1021 Page 7 of 7
`
`