Surface and self-assembling properties of cyclic
lipopeptide surfactin produced by Bacillus subtilis
Toshiaki Taira*Research Institute for Chemical Process Technology, National Institute of Advanced Industrial Scienceand Technology (AIST), Central 5-2, 1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan.
t-taira@aist.go.jp
Peptides produced from amino acids are versatile oligomers. When amino acids are organized
into oligomers with specific sequences, they provide folded and flexible structures, resulting inwide-ranging functionalities including molecular recognition, self-assembly, catalysis, andmore. This review describes surface and self-assembling properties of cyclic lipopeptide (CLP)of surfactin (SF) produced by Bacillus subtilis (B. subtilis). The sodium salt of SF (SFNa) isregarded as one of the promising surface-active CLPs and exhibits remarkable self-assembly
behavior under considerably low concentration. SFNa also have several advantages overconventional
petroleum-derived◆ Low CAC
surfactants such as high◆ Remarkable self-assembly behavior
biodegradability.
With
regard to its numerous◆ Excellent surface tension-lowering ability
attractive characters, it is◆ Encapsulation of cations
presumed that these areowing to the hydrogen◆ High biodegradability
bondinteractions◆ Low toxicity
between the cyclic peptide
moieties
toform
secondary structures.
Keyword: cyclic lipopeptide, surfactin, biosurfactant, self-assembly, micelles
Toshiaki Taira is a senior researcher at National Institute of Advanced Industrial
Science and Technology (AIST). He got his Ph.D. degree in 2010 at Tokyo institute ofTechnology under the supervision of Prof. Kohtaro Osakada. Then he worked with
Prof. Julius Rebek, Jr. at The Scripps Research Institute (TSRI) as a postdoctoral
research fellow from 2010-2012. Since 2012, he has been a member of AIST. His
research interest lies in colloid and interface science, particularly self-assembling
system of bio- and metallo-surfactants. He got several awards including JOCS Young
Scientist Award (The Japan Oil Chemists’ Society, 2020).

Acc. Mater. Surf. Res. 2023, Vol.8 No.1, 37-45.

37

Surface and self-assembling properties of cyclic
lipopeptide surfactin produced by Bacillus subtilis
Toshiaki Taira*Research Institute for Innovation in Sustainable Chemistry, National Institute of Advanced
Industrial Science and Technology (AIST), Central 5-2, 1-1 Higashi, Tsukuba, Ibaraki
305-8565, Japan.
three types of CLPs7). High-performance

1. Introduction
Peptides produced from amino acids are

liquid chromatography (HPLC) analysis

versatile oligomers1). When amino acids

suggested that these CLPs were thought toare organized into oligomers with specificbe SF, iturin A, and fengycin. They aresequences, they provide folded and flexible

usually

structures,

non-ribosomal

resultingsynthetizedthroughainwide-ranging

includingmolecular(NRPSs). Among them, SF is one of therecognition, self-assembly, catalysis, andpromising surface-active CLPs (Figure

more2,3).

1)8-11).

functionalities
Recentprogressin

peptidepeptidesynthetases

SF consists of a peptide loop,

libraries encouraged by both genetic andwhich contains both L- and D-amino acids

synthetic methods enabled their application(L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-L

as drugs as well as supramoleculareu), and a fatty acid side chain. There is a

structures in material sciences

3,4). In thiscommercially available sodium salt of SF

review, recent research findings of surface(sodiumand self-assembling properties of cyclic

produced by Kaneka Corporation.

lipopeptide

(CLP)

of

surfactin

SFNa)

which

is

(SF)

produced by Bacillus subtilis (B. subtilis)
are presented.

3. Surface activities of SFNa
The surface-active CLP of SFNa isregarded2.

surfactin,

Cyclic lipopeptides produced by

Bacillus subtilisasbiosurfactants
advantages

one
8-11)

.

of
SFNa

over

the

promisinghas

severalconventional

It is well known that B. subtilis produces
cyclic lipopeptides (CLPs) as secondary
metabolites5). The series of CLPs generally
consists of the cyclic peptides with different
amino acid sequences together with a longalkyl chain. They are produced as a
mixture of analogues, and the composition
varies depending on conditions, such as
strain species and medium compositions6).
We investigated sixty B. subtilis strains fortheir ability to produce CLPs and found that

Figure 1. Structure of surfactin (SF).

B. subtilis NBRC 109107 produced at least
Acc. Mater. Surf. Res. 2023, Vol.8 No.1, 37-45.

38

petroleum-derived counterparts such as
high biodegradability. We isolated one
strain of

Pseudomonas putida as a

SFNa-degrading bacterium12). This strain
degraded 1 g/L SFNa below the detectablelevel of HPLC analysis within 14 days. The
high biodegradability of SFNa hold promisefor the potential application in microbial
enhancedrecovery13

oil

）

or

14)

bioremediation .
Moreover, owing to its unique molecular
structure including a bulky head group andchiral centers, they show remarkableself-assembly.
tensionWe

lowering

measuredabilityofsurface

SFNa

byFigure 2. Relationship between surface
tension and concentration of SFNa andWilhelmy plate method15). The critical

SDS at 25 oC.

aggregation concentration (CAC) of SFNa

[ Arial : 10pt ]was estimated to be 2.7 × 10−5 M (Figure 2),
whose value is significantly lower than thatof sodium dodecyl sulfate (SDS). SFNa
can reduce surface tension of water from
72

to

27

mN/m,

tension-loweringwhose

abilityissurfacesignificantly

higher than that of SDS. Its numerous
attractive characters are presumably owing
to the hydrogen bond interactions betweenthecyclicpeptidesecondary structures.

moieties

toform

When the cyclic

peptide of SFNa adopts a horse saddle
shape via intramolecular hydrogen bonds,
two carboxy groups of L-Glu and L-Asp on
the same side of the ring form “claw”
configuration16).
secondaryWe

structures

found

that

the

of

SFNa

are

significantly enhanced through increased
dissociation of L-Asp and L-Glu as revealed
by a titration experiment of SF solution toestimate apparent pK1 and pK2 values
together with circular dichroism
spectroscopy17).

(CD)

The apparent pK1 andAcc. Mater. Surf. Res. 2023, Vol.8 No.1, 37-45.

pK2 of SF were estimated to be 5.8 and 6.8,
respectively. Additionally, we conducted atitration in water; complete dissolution ofSF

wasobservedwhen

1.5–2.0

equivalents of NaOH were added (pH >6.9). The apparent pKa values were higher
than those of related amino acids (pKa for
Asp and Glu are expected to be around 3.4
and 4.2, respectively) as well as -peptides(pKa for Asp and Glu are expected to bearound

4.3

and

4.5,

respectively)18);

however, they are similar to that of SF at anair–water interface (pKa around 6)19). These
results indicate that the dissociation of thetwo acidic amino acids of SF is suppressed
because of their incorporation into thecyclic peptide and/or location at the air–solvent interface. The secondary structureformation enhanced with increasing pH aswell as counter cations, obviously becauseofenhancementofintermolecularhydrogen bond interactions.
The critical aggregation concentrations(CACs) of SF with different counter cations
39

such

as

potassium(SFK),

monoethanolamine
diethanolamine(SFMEA),(SFDEA),and

4. Self-assembling properties of SFNa
Dynamiclightscattering(DLS)

measurement of an aqueous solution oftriethanolamine (SFTEA) were 2.8–4 times

SFNahigher than that of SFNa20). Furthermore,

spontaneously

the CACs increased as the organic

Basedcounter-cations became less hydrophobic

distribution of SFNa micelles shows a peak

(SFTEA (8.7 × 10−5 M) < SFDEA (9.8 × 10−5

top around 300–500 nm. The giant micelles−4M) < SFMEA (1.1 × 10

(1

mM)

on

revealed

forms

the

that

giant

histogram,

SFNa

micelles15).
the

size

M)) as shown inof SFNa can be separated by ultrafiltration

Figure 3(a) and (b). In addition, CD

technique. The amount of SFNa in bothmeasurements revealed that a negative

retentates and permeates were measured

band corresponding to -sheets increasedusing HPLC. The giant micelles of SFNa

with increasing organic counter cation

were recovered quantitatively and detectedhydrophobicity

mostly in the retentate (> 99%). Knoblich et(SFMEA

<

SFDEA

<

SFTEA) (Figure 3(c)). These results clearlyal.

reported

indicate that intermolecular hydrogen bondsurfactin micelles is significantly affectedinteraction promote self-assembly of SF in

by external conditions such as pH, metal

aqueous solution.

ions, and temperature by cryo-transmission
electron

that

the

microscopy

morphologyof(cryo-TEM)21).

Ishigami et al. reported the formation of

Figure 3. (a) Structures of SF salts with inorganic and organic counter-cations. (b) CAC values of
SFK, SFMEA, SFDEA, and SFTEA. (c) CD spectra of SF-MEA, SF-DEA, and SF-TEA in water(0.25 mM, 25 °C).
[ Arial : 10pt ]Acc. Mater. Surf. Res. 2023, Vol.8 No.1, 37-45.

40

largemicelles22).

rod-shaped

small-angle

x-ray

scatteringThe(SAXS)

measurement revealed that presence ofsmall micelles in water23). Consequently,
morphologies of SF micelles change
depending on the external conditions,which are different from those of synthetic
surfactants.
Another unique feature of SFNa is toform various lyotropic liquid crystals24).
Such

self-assembling

aqueoussolutionwithbehavior
relatively

in
high

concentrations is also crucial not only forthe application of SFNa in various fields,
but also to understand its role in abiological system. Bellow 50 wt%, SFNa
providedtransparent

andless-viscousaqueous solutions, indicating the formationof micellar phases (Figure 4). By polarized
light microscopy, the aqueous solutions of
SFNa at the concentrations above 50 wt%
were optically anisotropic and gave mosaic
textures(InsetofFigure4).

SAXS

diffractograms of the SFNa solution above
50 wt% clearly gave the three peaks whose

Figure 4. (top) Visual observation of
aqueous solutions of SFNa at different
concentrations under cross polarized light.
(bottom) SAXS patterns of lyotropic liquid
crystal of SFNa. Inset represents a mosaic
texture of 80 wt% of SFNa.

spacing ratio of 1:2:3, indicating the

SF allows the molecules to specificallypresence of the lamellar phase.

recognize each other.

We reported the molecular interactionsof monolayers composed of cyclic andlinear forms of SF, which have the same
peptide sequence and alkyl chain, butdifferent

topology25).

microscopic(AFM)monolayers

revealed

Atomicimages

force
of

the

phase-separated

microdomain structures, indicating that thecyclic and linear SFs are nearly immiscible.
These results indicated that topologicalfeatures of SF are crucially important for
self-assembling systems: the restriction ofconformational freedom of cyclic forms

5. Encapsulation properties of SFNa
Cyclic peptides are known to act as
ionophores. SF is expected to be a usefulas a novel sorbent for large monovalentCs+ because the heptapeptide moiety ofSF provides an ideal coordination number
for Cs+ (between 6 and 8). The cavity
diameter (4–7 Å) is larger than that of theion (3.38 Å), making it suitable for Cs+
encapsulation.

SF

also

has

two

−

carboxylate groups (R-COO ) that bind Cs+
to give a neutral salt (RCOOCs) through aof
41
Acc. Mater. Surf. Res. 2023, Vol.8 No.1, 37-45.

as Na+ and K+. Thus, the use of the giant
micellar system of SF with its high Cs+
affinity

anddistinctive

assemblingproperties would be a new approach for thetreatment of contaminated soil and water.
Mixing SFNa with alkylammonium
bromides with different alkyl chain lengths
leads to form vesicles and provides stable
colloidal

suspensions20).

When

the

approximate effective ionic radii of thecyclic peptide moiety of SF and theammonium moiety are considered, SFNa
and didodecyldimethylammonium bromide(C12DMA-Br) can be regarded as having
cone and inverse truncated cone shapes,
Figure 5. Cartoon representation of Cs+

respectively. In the case of SFNa alone,

removal from water using the micellar

which is a single-chain anionic surfactant

system of SF.

with a large head group (cone shape), a
polyethertransparent micellar solution was formed.

ionophores. Moreover, SF forms giant

In contrast, as an inverse truncatedmicelles in aqueous solution, with thecone-shaped surfactant with a small headsimilarmechanism

asheptapeptide moieties aligned on thesurface. High binding affinity and effective
removal of Cs+ can thus be achieved
throughmicellar-enhanced

ultrafiltration(MEUF), a low-energy separation process
combing entrapment by surfactant micelles
and ultrafiltration (Figure 5)15). We foundthat SF micelles selectively encapsulate
Cs+,

which

was

suggestedbymatrix-assisted laser desorption/ionization
time-of-flightmass

spectrometry(MALDI-TOFMS), together with nuclear
magnetic resonance (NMR) and Fouriertransform infrared(FT-IR) spectroscopy.

A highly effective separation of

Cs+

Figure 6. Photograph of the colloidal

immobilized on the surface of the SF

suspension of the mixture of SF-Na andmicelles was also achieved through facile

C12DMA-Br. freeze-fracture transmission

centrifugal ultrafiltration in 91% even inelectron microscopy (FF-TEM) images ofcoexisting with other alkali metal ions suchthe colloidal solution are also shown.

Acc. Mater. Surf. Res. 2023, Vol.8 No.1, 37-45.

42

group and a double chain, C12DMA-Br

SFNa

also

shows

remarkabkegave a colloidal suspension. Generally,emulsifying activities against cyclosiloxane

anionic surfactants with steric bulky organic

oils as octamethylcyclotetrasiloxane (D4)counter-cations exhibit decreased waterand decamethylcyclopentasiloxane (D5)solubility and form precipitates, which canwhich are regarded as causative agents for

hamper industrial applications. In contrast,

pollution from silicon sealants26). Generally,

SFNa, which has a bulky cyclic peptide

removing cyclosiloxanes is troublesome

moiety, provides open space at thedue to their super-hydrophobic characters.

air/water interface, which can mitigate theThus, SFNa would contribute to application

negative impact of organic counter-cations

for the environmentally friendly surfactant

includingfor various wastes in industry.

bulky

double-tailed

cationic

surfactants that give vesicles in water
without any precipitation. The combinationof SFNa as an environmentally friendly
anionic

surfactant

counter-cationsorganicIn

this

review,

self-assemblingthesurfaceandproperties

of

SFNa

provideideal

produced by B. subtilis are described.

cosmetic

andSFNa having a cyclic heptapeptide moiety

pharmaceutical applications with reduced

allows the molecules to recognize each

irritant potential and enhanced mildnessotherand comfort.

interactions.

formulations

may

with

7. Conclusionsfor

specifically,

via

Thus,

hydrogen
SFNa

bond

exhibits

remarkable self-assembly behavior under
6. Applications

considerably

lowconcentration.

SFNa

As described above, changes in the

self-assembled to form giant micelles

secondary structure of SFNa induced bywhich can be separated by ultrafiltration

increased pH. We found that the secondary

technique. Another unique feature of SFNa

structure formation of SF is significantly

is formation of lamellar liquid crystals at

enhanced through increased dissociationhigh concentrations. Together with theof L-Asp and L-Glu. We also studied thenumerous

activity of the common detergent enzyme

space of the cyclic peptide can be

subtilisin in SFNa solution at above its pK2

applicable for encapsulation of cations(pH 7.6) to understand the role of thesuch as Cs+.

dissociation degree in the interaction with17)

attractive

characters,

inner

SFNa has also several advantages overthe protein . The mixing of SFNa withtypically used synthetic surfactants, such

subtilisin suppressed the decrease in

as low toxicity and high biodegradability.

protease activity observed in the presence

Because of the environmental compatibility,of synthetic surfactants such as SDS and

SFNa can be applicable for various

polyoxyethylene alkyl ether. Thus, SF has

industrial applications, e.g., in cosmetics,

great potential for use in laundry detergent

food additives, enhancement of oil recovery,

formulations, to improve the stability andand detergent formulations.

reliability of detergent enzymes.
Acc. Mater. Surf. Res. 2023, Vol.8 No.1, 37-45.

43

8. Acknowledgement
I

would

like

to

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supervision and support by the late10) N. S. Shaligram, R. S. Singhal, Food

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thank

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Oleo Sci., 2021, 70, 581–587.
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