Biocatalytic synthesis, antimicrobial properties and toxicity studies of arginine derivative surfactants

Article abstract of DOI:10.1007/s00726-015-1979-0

Abstract Two novel arginine-based cationic surfactants were synthesized using as biocatalyst papain, an endopeptidase from Carica papaya latex, adsorbed onto polyamide. The classical substrate N ^α-benzoyl-arginine ethyl ester hydrochloride for the determination of cysteine and serine proteases activity was used as the arginine donor, whereas decyl- and dodecylamine were used as nucleophiles for the condensation reaction. Yields higher than 90 and 80 % were achieved for the synthesis of N ^α-benzoyl-arginine decyl amide (Bz-Arg-NHC₁₀) and N ^α-benzoyl-arginine dodecyl amide (Bz-Arg-NHC₁₂), respectively. The purification process was developed in order to make it more sustainable, by using water and ethanol as the main separation solvents in a single cationic exchange chromatographic separation step. Bz-Arg-NHC₁₀ and Bz-Arg-NHC₁₂ proved antimicrobial activity against both Gram-positive and Gram-negative bacteria, revealing their potential use as effective disinfectants as they reduced 99 % the initial bacterial population after only 1 h of contact. The cytotoxic effect towards different cell types of both arginine derivatives was also measured. Bz-Arg-NHC_n demonstrated lower haemolytic activity and were less eye-irritating than the commercial cationic surfactant cetrimide. A similar trend could also be observed when cytotoxicity was tested on hepatocytes and fibroblast cell lines: both arginine derivatives were less toxic than cetrimide. All these properties would make the two novel arginine compounds a promising alternative to commercial cationic surfactants, especially for their use as additives in topical formulations.

Full text of DOI:10.1007/s00726-015-1979-0

Amino Acids
DOI 10.1007/s00726-015-1979-0
ORIGINAL ARTICLE
Biocatalytic synthesis, antimicrobial properties and toxicity
studies of arginine derivative surfactants
M. Elisa Fait1 · Graciela L. Garrote2 · Pere Clapés3 · Sebastian Tanco4,5,6
Julia Lorenzo4 · Susana R. Morcelle1
·
Received: 7 November 2014 / Accepted: 30 March 2015
© Springer-Verlag Wien 2015
Abstract Two novel arginine-based cationic surfactants
were synthesized using as biocatalyst papain, an endopepti-
dase from Carica papaya latex, adsorbed onto polyam-
ide. The classical substrate Nα-benzoyl-arginine ethyl ester
hydrochloride for the determination of cysteine and serine
proteases activity was used as the arginine donor, whereas
decyl- and dodecylamine were used as nucleophiles for the
condensation reaction. Yields higher than 90 and 80 % were
achieved for the synthesis of Nα-benzoyl-arginine decyl
amide (Bz-Arg-NHC10) and Nα-benzoyl-arginine dode-
cyl amide (Bz-Arg-NHC12), respectively. The purification
process was developed in order to make it more sustainable,
by using water and ethanol as the main separation solvents
in a single cationic exchange chromatographic separation
step. Bz-Arg-NHC10 and Bz-Arg-NHC12 proved antimicro-
bial activity against both Gram-positive and Gram-negative
bacteria, revealing their potential use as effective disinfect-
ants as they reduced 99 % the initial bacterial population
after only 1 h of contact. The cytotoxic effect towards dif-
ferent cell types of both arginine derivatives was also meas-
ured. Bz-Arg-NHCn demonstrated lower haemolytic activity
and were less eye-irritating than the commercial cationic
surfactant cetrimide. A similar trend could also be observed
when cytotoxicity was tested on hepatocytes and fibroblast
cell lines: both arginine derivatives were less toxic than
cetrimide. All these properties would make the two novel
arginine compounds a promising alternative to commercial
cationic surfactants, especially for their use as additives in
topical formulations.
Handling Editor: P. Meffre.
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-015-1979-0) contains supplementary
material, which is available to authorized users.
* Susana R. Morcelle
morcelle@biol.unlp.edu.ar
Keywords Arginine-based surfactants · Papain ·
1
Biocatalysis · Antimicrobial activity · Cytotoxic effect
Centro de Investigación de Proteínas Vegetales (CIPROVE),
Departamento de Ciencias Biológicas, Facultad de Ciencias
Exactas, Universidad Nacional de La Plata (UNLP), La Plata,
Argentina
Introduction
2
Centro de Investigación y Desarrollo en Criotecnología de
Alimentos (CIDCA-CONICET-UNLP), La Plata, Argentina
Antiseptics are biocides or products that destroy or inhibit
the growth of microorganisms in or on living tissue (e.g.,
health care personnel hand washes and surgical scrubs),
whereas disinfectants are similar but generally are products
or biocides that are used on inanimate objects or surfaces.
Infection control practices and prevention of nosocomial
infections, as well as the potential microbial contamina-
tion and infection risks in food and other consumer mar-
kets have increased the use of antiseptics and disinfect-
ants in general. These kind of compounds are also used as
3
Biotransformation and Biomolecules Group, Catalonia
Institute of Advanced Chemistry (IQAC-CSIC), Barcelona,
Spain
4
Institut de Biotecnologia i Biomedicina, Departament de
Bioquímica i de Biologia Molecular, Universitat Autònoma
de Barcelona, Barcelona, Spain
5
Present Address: Department of Medical Protein Research,
VIB, Ghent, Belgium
6
Department of Biochemistry, Ghent University, Ghent,
Belgium
1 3

M. E. Fait et al.
preservatives in many pharmaceutical formulations (Car-
mona-Ribeiro and de Melo Carrasco 2013). In this context,
quaternary ammonium compounds, such as cetyl and hexa-
decyl trimethylammonium bromide (CTAB and HTAB,
respectively), as well as the cetrimide (a mixture consisting
mainly of tetradecyl trimethylammonium bromide together
with smaller amounts of dodecyl and hexadecyl trimethyl-
ammonium bromides) are the most useful antiseptics and
disinfectants. Solutions containing cetrimide 0.1–1 % are
used for cleaning skin, wounds and burns, as well as con-
taminated catheters and sterilized surgical material storage
(Florence and Attwood 2006). Many of these compounds
are amphiphilic, i.e., they can reduce surface and interfacial
tensions by accumulating at the interface of immiscible flu-
ids and increase the solubility and mobility of hydrophobic
or insoluble organic compounds. This kind of amphiphilic
compounds are considered as surfactants (surface active
agents) (Singh et al. 2007). Bio-based surfactants are syn-
thetic amphipatic structures based on natural structures of
biosurfactants (glycolipids, lipopeptides, phopholipids and
fatty acids). Lipoamino acids and glycerolipids have been
exhaustively studied, demonstrating their good surface
activity, aggregation properties, broad biological activity
and low toxicity profile (Pérez et al. 2002). In particular,
amino acid-based surfactants constitute a class of tensio-
active compounds with excellent adsorption and aggrega-
tion properties, high biodegradability, environmentally
friendly and broad antimicrobial activity. Among them,
arginine-based surfactants are an important group of cati-
onic surfactants with antimicrobial activity against a broad
spectrum of bacteria, biodegradability and low toxicity. All
these properties turn them into interesting additives to be
included as food preservatives and pharmaceutical formula-
tions as well as active ingredients in dermatology and per-
sonal care products. In this sense, arginine-based cationic
surfactants have been proposed as alternatives to quater-
nary ammonium halides, which have an important intrinsic
toxicity as well as a questioned biodegradability (Castillo
et al. 2004).
out that the use of this approach would not meet the stand-
ards of green chemistry, since this methodology of produc-
tion could result in the presence of hazardous by-products
coming from the starting materials that could be toxic in
the final formulations. These drawbacks could be circum-
vented by the use of enzymes as biocatalysts, due to their
selectivity and their ability to act under mild reaction con-
ditions. In particular, for the synthesis of amino acid-based
surfactants, hydrolases (lipases and proteases) were exten-
sively studied (Valivety et al. 1997; Clapés et al. 1999;
Morán et al. 2001a; Morcelle et al. 2009). It is important
to point out that during the past decades, striking advances
concerning the use of enzymes in the field of biocatalysis
have impacted upon a wide range of industries ranging
from pharmaceuticals and fine chemicals to different mate-
rials, polymers and food. Enzymatic strategies for diverse
steps in chemical production are in agreement with the
development of sustainable manufacturing processes.
Papain (E.C. 3.4.22.2) is a plant cysteine endopeptidase
from Carica papaya and is mainly used in food, brewer-
ies, pharmaceuticals, cosmetics, leather and textile indus-
tries. It is less expensive than microbial enzymes and has a
good thermal stability when compared to other proteases.
Papain preferably cleaves peptide bonds involving basic
amino acids in P1 position and aromatic or bulky aliphatic
moieties in P2 position (Schechter and Berger nomen-
clature) (Schechter and Berger 1967). On the other hand,
papain also displays esterase activity (Chamani and Hesh-
mati 2008). As a biocatalyst in organic chemistry, papain is
one of the most used peptidases for miscellaneous peptide
and peptide derivative synthesis, including polymers of one
single amino acid (Narai-Kanayama et al. 2008), peptide
sweeteners (Nakaoka et al. 1998), peptide hormones (Klein
and Cerovský 1996; Fité et al. 2002) and peptide isosteres
(Lang et al. 2007), due to its broad specificity and robust-
ness. Papain has also proved to be a suitable catalyst for the
synthesis of arginine-N-alkylamides and arginine-O-esters
derivatives, even at temperatures as high as 65 °C (Clapés
et al. 1999). All these properties, including its easy prepara-
tion from the natural source, turn papain into a very ver-
satile biocatalytic tool for condensation reactions involving
the synthesis of amide and ester bonds between a broad
range of amino acids and a plethora of simple nucleophiles
bearing alcohol or amine moieties. In this context, papain
adsorbed onto polyamide has demonstrated to be the opti-
mum catalyst for the synthesis of many arginine-based sur-
factants. In this context, the aims of the present work were
to obtain arginine alkylamide derivatives (Bz-Arg-NHC10
and Bz-Arg-NHC12) using papain adsorbed onto polyamide
as biocatalyst and to evaluate some biological activities of
these compounds, such as their antimicrobial properties, as
well as their cytotoxicity and haemolytic activity. As start-
ing materials, the synthetic substrate Nα-benzoyl arginine
Arginine-based surfactants can be synthesized from
raw materials as amino acids and fatty acids of amines
(Castillo-Expósito 2006). Traditional chemical synthe-
sis and purification methods of this kind of compounds
involve steps of protection and deprotection reactions, use
of dangerous organic solvents and other harmful chemi-
cals, including BF3, strong organic bases and chlorinated
solvents among others. Drastic conditions, such as high
temperatures and pressures, as well as hydrogenation reac-
tions could also be required depending on the desired final
product (Pérez et al. 1996; Mitin et al. 1997; Piera et al.
1998; Castro et al. 2004). On the other hand, global yields
for these syntheses were 45–60 % in the best case (Piera
et al. 2000; Pérez et al. 2014). It is also important to point
1 3

Biocatalytic synthesis, antimicrobial properties and toxicity…
ethyl ester (Bz-Arg-OEt, also known as BAEE), which is
commonly employed to determine enzymatic hydrolytic
activity of proteases such as papain and trypsin (Walsh and
Wilcox 1970; Arnon 1970), and two fatty amines were used
as acyl donor and nucleophiles, respectively.
enzymatic activity was performed before and after immobi-
lization using BAPNA as substrate (Ota et al. 1964; Mor-
celle et al. 2009), and it was expressed in terms of interna-
tional units (IU), defined as the amount of p-nitroaniline
(μmoles) released per min at 37 °C and pH 7.4. For this pur-
pose, a calibration curve of p-nitroaniline was performed.
Materials and methods
Chemicals
Microscale Nα‑benzoyl‑arginine dodecylamide
(Bz‑Arg‑NHC12) enzymatic synthesis: optimization
of the product extraction process
Papain (E.C. 3.4.22.2) from Carica papaya latex, crude
powder (3.11 units/mg solid, one unit hydrolyzes 1.0 μmol
of Nα-benzoyl-arginine ethyl ester hydrochloride per min at
pH 6.2 and 25 °C), dodecylamine and molecular sieves (4
Å) were obtained from Fluka. Bovine seroalbumin (BSA),
Coomassie Brilliant Blue G-250, Nα-benzoyl-arginine ethyl
ester hydrochloride (Bz-Arg-OEt or BAEE), decylamine
and Nα-benzoyl-d,l-arginine p-nitroanilide hydrochloride
(BAPNA) were from Sigma. Polyamide-6 (EP-700, parti-
cle size <800 μm, mean pore diameter 50–300 nm, specific
surface area BET method 8.4 m2/g) was an Akzo (Obern-
burg, Germany) contribution. 1,4-Dithio-d,l-threitol (DTT)
was purchased to Invitrogen (Carlsbad, CA, USA). Cet-
rimide was from Dishman Pharmaceuticals and Chemicals
(India). Nutrient broth was obtained from Biokar Diagnos-
tics (Beauvais, France). The rest of the chemicals and sol-
vents used in this work were of analytical grade.
Bz-Arg-OEt·HCl (0.02 mmol) and dodecylamine
(0.03 mmol) were dissolved in anhydrous acetonitrile (1 ml)
containing 0.25 % v/v water. Papain adsorbed onto polyam-
ide (100 mg) was added as biocatalyst. Reactions were car-
ried out under nitrogen atmosphere at 37 °C in closed flasks
vessels placed on an orbital shaker (150 rpm) for 72 h. They
were stopped by addition of a methanol/acetic acid (MeOH/
AcOH) 4:1 mixture, followed by a thorough wash of the
biocatalyst (3 × 1 ml) with the same solvent mixture (Cla-
pés et al. 1999). Solvent extraction mixture containing water
instead of AcOH was also tested for extraction of Bz-Arg-
NHC12 following the same procedure.
All the extracts were analysed by HPLC (Agilent
1100), after filtration through a 0.22-μm nylon membrane
(Osmonics) using a LiChrosorb propyl cyano column
(250 × 4 mm, 5 μm, Hibar). Chromatographic conditions
were as follows: solvent A, 0.1 % (v/v) TFA (trifluoroacetic
acid) in H2O; solvent B, 0.1 % (v/v) TFA in CH3CN/H2O
80:20; flow rate 1 ml/min; detection at 215 and 254 nm.
For identification purposes, standards of Bz-Arg-OEt and
Bz-Arg-OH were also analysed. Quantification was made
from peak areas according to the external standard method.
Retention times for each compound were as follows: Bz-
Arg-OH, 4.7 min; Bz-Arg-OEt, 5.3 min; Bz-Arg-NHC10,
13.6 min; Bz-Arg-NHC12, 15.6 min.
Microorganisms
Escherichia coli ATCC 11229, Bacillus cereus ATCC
10876, Staphylococcus aureus ATCC 6538, Pseudomonas
aeruginosa ATCC 15442, Kocuria rhizophila ATCC 9341,
Enterococcus faecalis ATCC 51299, Serratia marcescens
ATCC 14756, Bacillus subtilis subsp. spizizenii ATCC
6633, Shigella flexneri ATCC 9199 and Enterococcus fae‑
cium ATCC 6569 from American Type Culture Collec-
tion (ATCC, Manassas, USA) were employed as indicator
microorganisms.
Bz-Arg-OH standard was obtained by hydrolysis of
Bz-Arg-OEt (30 mM in 0.1 M phosphate buffer pH 7.0
containing 5 mM EDTA and 5 mM cysteine) with papain
(5 mg) after 30 min incubation (37 °C). Reaction was
stopped by precipitation of papain with MeOH, and the
precipitated protein was eliminated by centrifugation
(10 min at 9000×g). The supernatant containing Bz-Arg-
OH was evaporated and the remaining solid was redis-
solved in MeOH for further analysis.
Biocatalyst preparation
Protein content of crude papain was determined by means
of the Bradford’s method (Bradford 1976) using a standard
curve of BSA. The enzyme was immobilized by deposition
onto polyamide according to the following procedure: First,
the enzyme (100 mg) was dissolved in boric acid-sodium
borate buffer 0.1 M pH 8.5 with EDTA.
Semipreparative Bz‑Arg‑NHC10 and Bz‑Arg‑NHC12
synthesis. Purification by cationic exchange
chromatography
1 mM (1 ml) in presence of DTT (150 mg); then, the
solid support (1 g) was added and shaken vigorously with
the enzymatic solution, and the mixture was finally lyo-
philized for 24 h (Morcelle et al. 2009). Determination of
Bz-Arg-OEt (0.2 mmol, 68.4 mg) and decylamine
(0.3 mmol, 47.2 mg) or dodecylamine (0.3 mmol, 55.7 mg)
were dissolved in anhydrous acetonitrile (10 ml) containing
1 3

M. E. Fait et al.
0.25 % v/v water. Papain adsorbed onto polyamide (1 g) was
added as biocatalyst. Individual reactions were performed
under nitrogen atmosphere in Erlenmeyer flasks (100 ml)
placed on an orbital shaker (250 rpm) at 37 °C for 72 h.
After incubation, the biocatalyst was removed by filtration
and washed with acetonitrile (3 × 10 ml) and diethyl ether
(3 × 10 ml), to eliminate the excess of fatty amine. Finally,
reaction products were extracted from the biocatalyst sur-
face using MeOH/H2O 4:1 (3 × 10 ml) and filtered through
a nylon membrane filter (0.22 μm, Osmonics). Purification
of each surfactant was achieved in an Äkta Purifier 10 (GE
Healthcare) by cation exchange chromatography as follows:
aliquots of the filtrate (1 ml) were loaded onto a SP Sepha-
rose Fast Flow column (10 ml bed volume, GE Healthcare)
preequilibrated with boric acid-sodium borate buffer 0.01 M
pH 8.5/ethanol 1:1 (solvent A). Unbound material was
eluted after washing the column with 2 bed volumes (BV)
of the same solvent. Elution of each condensation product
was accomplished by a step of 31 % of solvent B, consisting
in boric acid-sodium borate 0.01 M pH 8.5 buffer/ethanol
(1:1) with 1 M NaCl (2 BV). The column was washed with
100 % of solvent B (2 BV) and reequilibrated to the start-
ing conditions. Flow rate was kept at 1 ml/min throughout
the whole process. Eluted peaks were detected at 215 and
254 nm. Collected fractions containing the purified products
were pooled and the solvent was evaporated. The resulting
solid was desalted by precipitation with absolute ethanol
and centrifugation. Supernatant was evaporated under vac-
uum. The purity of the products obtained was analysed by
HPLC under the above conditions. Identities of both com-
Bz-Arg-NHC10 1H NMR (400 MHz, CD3OH): δ 7.80–7.76
(m, 2H, Ph, H-2′, H-6′), 7.48–7.43 (m, 1H, Ph, H-4′), 7.40–
7.34 (m, 2H, Ph, H-3′, H-5′), 4.74 (s, 6H, NH, COCHNH),
4.46 (dd, J = 8.9, 5.5 Hz, 1H, (NH)2CHNH), 3.18–3.12
(m, 2H, CH3(CH2)8CH2NHCO), 3.10 (t, J = 7.0 Hz, 2 H),
2.84–2.74 (m, 1H), 1.91–1.80 (m, 1H), 1.79–1.69 (m, 1H),
1.68–1.48 (m, 2H), 1.48–1.34 (m, 2 H), 1.33–1.11 (m, 18
H), 1.10–1.03 (m, 1H), 0.79 (m, 4H, CH3, NH). 13C NMR
(101 MHz, CD3OD) δ 172.45 (NHCHCONH), 168.79
(PhCO), 157.17 (Ph, C-1), 133.63 (NHC(NH)(NH2), 131.55
(Ph, C-4), 128.14 (Ph, C-3, C-5), 127.11 (Ph, C-2, C-6),
53.47, 40.56, 39.06, 31.60, 29.26, 29.24, 29.01, 28.98, 28.93,
28.89, 26.52, 25.13, 22.28, 12.98.
Antimicrobial assays
Determination of minimum inhibitory and bactericidal
concentrations
Antimicrobial activity of Bz-Arg-NHC10 and Bz-Arg-
NHC12 against several bacterial strains was determined by
the microdilution assay in 96-well flat-bottom plastic tis-
sue culture plates (TCP-96T-SI, Axygen, USA). In each
case, 125 µl of sterile nutritive broth (Biokar Diagnostics,
Beauvais, France) was placed into second column’s wells
of the microplate. Subsequently, 125 µl of the correspond-
ing surfactant solution in sterile nutritive broth (800 µg/
ml for Bz-Arg-NHC10 and 200 μg/ml for Bz-Arg-NHC12)
was added to the first and second columns’ wells of the
microplate. From the second well, 125 µl was transferred
serially to the subsequent wells, discarding 125 µl of the
mixture in the tenth column, so that the final volume con-
tained in each well was 125 µl. This process resulted in
twofold serial dilutions of the surfactant solution within the
first ten columns. Columns 11 and 12 did not contain sur-
factant and served as negative and positive growth controls,
respectively. All wells (except for the 11th column) were
inoculated with 2.5 µl of a bacterial suspension adjusted to
0.5 McFarland (1.5 × 108 CFU/ml) in physiological solu-
tion. Microplates were covered and incubated for 24 h at
37 °C. Optical density at 600 nm (OD600) was determined
with a spectrophotometer (ELISA Plate Reader SLT Lab
instruments Rainbow Reader, Vienna, Austria). The mini-
mum inhibitory concentration (MIC) was determined for
each strain as the lowest concentration of surfactant that
completely inhibits measurable growth (OD600 = 0 and
GI % >95 %, where GI % means growth inhibition per-
centage). Samples of those wells that did not show visible
growth were transferred to nutrient agar plates. Presence
of colonies was analysed for after incubation at 37 °C for
24 h. Minimum bactericidal concentration (MBC) was
defined as the lowest concentration of surfactant that com-
pletely inhibits bacterial growth (absence of colonies).
1
pounds were confirmed by H-NMR and 13C-NMR, using
bidimensional techniques (HSQC and COSY, see Supple-
mentary Material), as well as ESI MS. Theoretical exact
masses were obtained using the ChemCalc online service
(Patiny and Borel 2013) and compared with the experimen-
tal values obtained by ESI MS. Bz-Arg-NHC10, ESI (+)-
TOF–MS [M + 1] calculated for C23H40N5O2: 418.31820;
found: 418.31889. Bz-Arg-NHC12, ESI (+)-TOF–MS
[M + 1] calculated for C25H44N5O2: 446.34950; found:
446.34924.
Bz-Arg-NHC12 1H NMR (400 MHz, CH3OD) δ 7.88–
7.85 (m, 2 H, Ph, H-2′, H-6′), 7.58–7.50 (m, 1 H, Ph, H-4′),
7.46 (ddt, J = 8.2, 6.7, 1.3 Hz, 2 H, Ph, H-3′, H-5′), 4.83
(s, 6 H, NH, COCHNH), 4.54 (dd, J = 8.9, 5.7 Hz, 1 H,
(NH)2CHNH), 3.28–3.10 (m, 2 H), 2.89 (t, J = 7.0 Hz,
1H), 2.02–1.56 (m, 6 H), 1.49 (dd, J = 13.8, 6.9 Hz, 2
H), 1.40–1.22 (m, 20 H), 1.16 (t, J = 7.1 Hz, 1 H), 0.87
(t, J = 6.8 Hz, 3 H, CH3). 13C NMR (101 MHz, CH3OD)
δ 172.47 (NHCHCONH), 168.79 (PhCO), 157.17(Ph,
C-1), 133.62 (Ph, C-4), 128.13 (Ph, C-3, C-5), 127.12 (Ph,
C-2, C-6), 53.50, 40.56, 39.36, 39.06, 31.62, 29.34, 29.29,
29.27, 29.26, 29.03, 28.92, 27.20, 26.52, 26.02, 25.14,
22.28, 12.99.
1 3

Biocatalytic synthesis, antimicrobial properties and toxicity…
Effect of surfactants on pathogens survival
sodium lauryl sulphate (SDS) as a positive control. The
denaturation index (DI) was calculated using the following
formula: DI (%) = [(R1 − R3)/(R1 − R2)] × 100, where
R1, R2 and R3 are the ratios between the absorbance read-
ings of the haemoglobin released from the erythrocytes
when in contact with (1) MilliQ® nanopure water, (2) SDS
at 1 mg/ml and (3) the product under analysis, respectively.
Irritation index was determined according to the lysis/
denaturation ratio (L/D) obtained by dividing the HC50 by
the DI. Surfactants can be classified according to this L/D
ratio as follows: non-irritant, >100; slightly irritant, >10;
moderately irritant, >1; irritant, >0.1 or very irritant, <0.1
(Sanchez et al. 2006).
Pathogen survival in nutrient broth added with Bz-Arg-
NHC10 or Bz-Arg-NHC12 at different concentrations was
studied. For both compounds, the corresponding MIC
and a concentration 50 % lower than that (1/2 MIC) were
tested, using E. coli and S. aureus as indicator microor-
ganisms. Overnight cultures of pathogenic bacteria were
diluted in nutrient broth to reach a final concentration of
approximately 1 × 106 CFU/ml. Surfactant solutions were
prepared in sterile nutrient broth (4.5 ml) and inoculated
with 500 µl of the bacterial suspension. At predetermined
intervals (0, 30, 60 min, 3 and 6 h), aliquots were removed,
serially diluted in 0.1 % v/v tryptone and plated on nutrient
agar plates. Colony counts were performed after incubation
at 37 °C for 24 h.
Cytotoxicity evaluation
Cell cultures
Haemolytic activity
The cytotoxicity of Bz-ArgNHC10, Bz-ArgNHC12 and cet-
rimide was tested in human HepG2 and human fibroblast
1BR.3.G cells. Human hepatocellular carcinoma HepG2
cell line ATCC HB-8065, derived from human hepatocytes,
was maintained in a minimum essential medium (MEM)
alpha supplemented with 10 % v/v heat inactivated foetal
bovine serum (FBS). Human fibroblast 1BR.3.G cell line
(catalog number EC90020507, European Collection of Cell
Cultures, Salisbury, UK) was cultured in Dulbecco’s Modi-
fied Eagle’s Medium (DMEM) containing 4500 mg/ml
glucose, GlutaMAX-I and 10 % v/v heat inactivated FBS.
Both cell lines were cultured in a highly humidified atmos-
phere of 95 % air with 5 % CO2 at 37 °C.
Preparation of the erythrocyte suspension
Human red blood cells (RBCs) were obtained from the
blood of healthy volunteers from our laboratory staff
(CIPROVE, La Plata, Argentina) and EDTA was used to
prevent the blood clotting. RBCs were separated by cen-
trifugation at 1302×g for 15 min at room temperature
and washed three times with a phosphate buffer solution
(PBS) containing 123.3 mM NaCl, 22.2 mM Na2HPO4
and 5.6 mM KH2PO4 in MilliQ® nanopure water (pH 7.4;
300 mosmol/l). Cells were finally suspended in PBS at a
density of 8 × 109 cells/ml (Pape et al. 1987).
Haemolysis assay
Experimental treatments
Haemolytic activity of Bz-ArgNHC10 and Bz-ArgNHC12
was contrasted to cetrimide, a commercial surfactant,
according to Sanchez et al. (2006). Aliquots of the eryth-
rocyte suspension (25 μl) were exposed to different con-
centrations of the surfactants (ranging from 1 to 1000 μg/
ml) dissolved in PBS to a final volume of 1 ml. Following
incubation at 37 °C for 10 min and centrifugation (1302×g,
10 min), the extent of haemolysis was quantified spectro-
photometrically at 540 nm and contrasted to that of Mil-
liQ® nanopure water (100 % haemolysis). The concentra-
tion inducing 50 % haemolysis (HC50) was calculated from
the dosis–response curves determined for each compound.
Surfactants were dissolved in dimethyl sulfoxide (DMSO)
at a stock concentration of 50–100 mM, sterilized by filtra-
tion and stored at −20 °C. Dilutions of these stock solu-
tions were prepared in a range of concentrations from 2 to
400 μM using sterile culture medium suited for each cell
line. Cells were seeded in 96-well plate at a cell density
of 6 × 103 cell/well for HepG2 and 3 × 103 cell/well for
1BR.3.G and incubated for 24 h before 100 μl of the sur-
factant solutions was added. Controls, containing culture
medium only, were included in each plate and they were
independent for each of the different surfactants tested.
Plates were incubated at 37 °C, 5 % CO2 and the growth
inhibitory effect was evaluated after 24 or 72 h by the XTT
assay.
Haemoglobin denaturation assay: evaluation of potential
ocular irritation
XTT Assay
The haemoglobin denaturation for the HC50 was assessed
by monitoring the absorbance at 575 and 540 nm, against
a blank containing only the sample diluted in PBS, using
A modified version of the classical MTT assay (Mosmann
1983; Scudiero et al. 1988) was performed using the EZ4U
1 3

M. E. Fait et al.
reduction assay kit from Biomedica Corp. (Vienna, Aus-
tria). After treatment with the surfactants, 20 μl of the XTT
solution was added to each well. Following 3 h incuba-
tion at 37 °C and 5 % CO2, the coloured formazan product
formed was spectrophotometrically quantified at 490 nm in
a microplate reader (Perkin Elmer Victor3 V).
strategy for using enzymes in organic solvents because it
consists in an easy and low cost procedure (Miletić et al.
2012). Polyamide EP-700 is an organic synthetic polymer
of slight hydrophobicity which demonstrated to be an ade-
quate support for enzymatic peptide and peptide derivative
syntheses (Clapés et al. 1999; Morcelle et al. 2009). Poly-
amide has the property of absorbing water in such a propor-
tion that reassures a good enzyme flexibility in high organic
solvent-containing media with low concomitant hydrolysis
reactions of the synthesis products (Adlercreutz 1991).
Bearing all this information in mind, we chose polyamide
EP-700 to immobilize papain to obtain a suitable catalyst
for the synthesis of the Arg derivatives.
Characterization of the free and adsorbed papain in
terms of enzymatic (hydrolytic) activity is shown in
Table 1. BAPNA was chosen as specific substrate to deter-
mine hydrolytic activity. BAPNA and Bz-Arg-OEt (the
acyl donor substrate in the synthetic reaction catalyzed
by papain) have the same acyl group (Bz-Arg). This fact
is of utmost importance, since enzymatic units should be
expressed according to the particular reaction a certain
enzyme will catalyse.
As can be seen in Table 1 from the recovered amidolytic
activity and amidolytic specific activity of the adsorbed
enzyme, papain lost about 70 % of its initial activity after
the immobilization process. According to Lartigue (1975),
possible explanations for this behaviour include protein
denaturation during the lyophilization step, occlusion of the
enzyme active site and/or that the adsorption of the enzyme
through residues required for the catalytic activity. In this
particular case, the main advantage of enzyme immobiliza-
tion is the ability to stop the reaction rapidly by removing
the biocatalyst from the medium, as well as the simplifi-
cation of the product purification process, as proposed by
Messing (1975).
Statistical analysis
Antimicrobial activity was expressed as a percentage of
growth inhibition (GI %) compared with control wells (the
mean optical density of untreated cells was set to 100 %
viability). Both antimicrobial and XTT experiments were
performed at least three times using four wells for each
surfactant concentration tested. For the haemolysis assay,
three replicate samples were tested. For the cytotoxicity
calculation, we assumed that cell viability is proportional
to the production of the red formazan derivative that is
measured spectrophotometrically and, consequently, the
percentage of cell viability was calculated by dividing the
average absorbance of the cells treated with the surfactant
by that of the control. Cytotoxicity of each surfactant was
expressed in terms of its IC50 (concentration of surfactant
causing 50 % death of the cell population or, similarly, at
which 50 % of the cells are viable relative to the control),
calculated from the concentration–response curves by lin-
ear regression analysis using the GraphPad Prism® soft-
ware. Haemolytic activity was expressed as a percentage of
haemolysis compared with control completely haemolyzed
(the mean absorbance of RBCs treated with MilliQ® nan-
opure water was set to 100 % haemolysis) in terms of its
HC50 (concentration of surfactant causing 50 % haemoly-
sis), calculated from the concentration–response curves by
linear regression analysis using the OriginPro 8® software.
All data were compared by one-way analysis of variance
(ANOVA) and Student’s t test using the GraphPad Prism®
software. P < 0.05 was considered to denote significance.
Table 1 Biocatalyst characterization
Results and discussion
Free papain
Protein content
Biocatalyst characterization
(μg of protein/mg of crude powder)
Amidolytic activity
254.0
0.049
0.176
Enzyme immobilization is a very useful strategy in biocata-
lytic reactions occurring in organic media, since enzymatic
dispersion is optimized, whereas there is an improved
accessibility for the substrates and the aggregation of the
hydrophilic protein particles is avoided (Hanefeld et al.
2009). On the other hand, immobilization allows an easy
separation of the biocatalyst from the reaction mixture,
facilitating product recuperation (Tischer and Wedekind
1999). Physical adsorption is considered as the best suited
(IU/mg of crude powder)
Amidolytic specific activity
(IU/mg of protein)
Papain adsorbed onto polyamide
Amidolytic activity
(IU/mg of adsorbed crude powder)
Amidolytic specific activity
(IU/mg of adsorbed protein)
0.013
0.051
1 3

Biocatalytic synthesis, antimicrobial properties and toxicity…
Fig. 1 Papain-catalysed syntheses of Nα-benzoyl-arginine decyla-
mide (Bz-Arg-NHC10) and Nα-benzoyl-arginine dodecylamide (Bz-
Arg-NHC12)
Microscale Bz‑Arg‑NHC12 enzymatic synthesis.
Optimization of the extraction process of the product
Fig. 2 Synthesis of Bz-Arg-NHC12 catalysed by papain adsorbed
onto polyamide at 72 h reaction. Yields for the condensation and the
hydrolysis products, as well as the remaining substrate, are shown for
both extraction mixtures assayed (MeOH/AcOH 4:1 and MeOH/H2O
4:1)
Syntheses of Bz-Arg-NHC10 and Bz-Arg-NHC12 was car-
ried out using papain adsorbed onto polyamide as bio-
catalyst as described in “Materials and methods” section
(Fig. 1). At the end of the reaction, the use of the solvent
mixture MeOH/AcOH 4:1 has two aims: to stop the reac-
tion and to extract the reaction products (Clapés et al.
1999). In this context, the low pH of the resulting mixture
that had to be loaded onto the column could negatively
affect the separation process of the analytes. In order to
avoid this undesired effect, the extraction of the arginine
derivatives was tested using a MeOH/H2O 4:1 mixture for
the microscale synthesis of Bz-Arg-NHC12. All compounds
present in the extract were analysed by HPLC, comparing
the yields to those obtained when the extraction mixture
was MeOH/AcOH.
As depicted in Fig. 2, there was no significant variation
in Bz-Arg-NHC12 content for both extraction mixtures, as
well as for the other arginine-containing analytes, indi-
cating that the solvent mixture MeOH/H2O 4:1 could be
used to remove the desired product from the biocatalyst
surface and for further purification by cation exchange
chromatography.
Fig. 3 Semipreparative synthesis of Bz-Arg-NHC10 and Bz-Arg-
NHC12 at 72 h of reaction using papain adsorbed onto polyamide as
biocatalyst. Hydrolysis product (Bz-Arg-OH) and remaining substrate
(Bz-Arg-OEt) yields are also shown
chromatography, using as stationary phase a column con-
sisting in an hydrophilic matrix based on chains of agarose
with different degrees of intra-chain cross-linking function-
alized with sulfopropyl groups. Based on previous inves-
tigations (Torres et al. 2001), the mobile phase tested was
boric acid-sodium borate buffer 0.01 M pH 8.5/ethanol
1:1. At the selected pH the hydrolysis product (Bz-Arg-
OH) would be in its zwitterionic state, thus allowing for its
separation as it would elute with the unbound material and
washing fractions.
Semipreparative Bz‑Arg‑NHC10 and Bz‑Arg‑NHC12
synthesis. Purification by cationic exchange
chromatography
Semipreparative synthesis of surfactants was accomplished
as described in the “Materials and methods” section, using
for the extraction step the mixture consisting on MeOH/
H2O 4:1. In this case, the yield obtained for the conden-
sation product was 86.17
8.14 % for Bz-Arg-NHC12,
almost 25 % higher than conversion observed for the micro-
scale synthesis (69.62 3.48 %), and 98.04 4.90 % for
Bz-Arg-NHC10 (Fig. 3). The increase in Bz-Arg-NHC12
yield could be attributed to the change in the reactor
geometry.
Elution of each condensation product (Bz-Arg-NHCn)
was accomplished by a 31 % of solvent B step, as described
in “Materials and methods” section. The use of ethanol as
co-solvent for the product purification had two main goals:
to keep the solutes in solution and to avoid hydrophobic
interactions with the stationary phase. On the other hand,
ethanol has low toxic potential and low cost, characteristics
Purification of the synthesized arginine-based sur-
factants was achieved by extraction and cationic exchange
1 3

M. E. Fait et al.
Table 2 Minimum inhibitory and bactericidal concentrations (μg/ml) for Bz-Arg-NHC10 and Bz-Arg-NHC12
Microorganism
MIC μg/ml (μM)
MBC μg/ml (μM)
Bz-Arg-NHC10
Bz-Arg-NHC12
Cetrimide
Bz-Arg-NHC10
Bz-Arg-NHC12
Cetrimide
Gram-positives
B. cereus
50 (119.53)
50 (119.53)
100 (239.05)
50 (119.53)
100 (239.05)
62,5 (149.41)
200 (478.10)
100 (239.05)
100 (239.05)
50 (119.53)
50 (112.02)
50 (112.02)
25 (56.01)
25 (56.01)
25 (56.01)
100 ()
1.95 (5.80)
ND
>800 (1912.42)
>800 (1912.42)
400 (956.21)
100 (239.05)
100 (239.05)
>800 (1912.42)
200 (478.10)
200 (478.10)
200 (478.10)
200 (478.10)
>200 (448.09)
100 (224.04)
200 (448.09)
200 (448.09)
>200 (448.09)
50 (112.02)
3.91 (11.62)
ND
B. subtilis
E. faecalis
E. faecium
K. rhizophila
S. aureus
ND
ND
ND
ND
ND
ND
1.95 (5.80)
31.25 (92.89)
125 (371.55)
ND
3.91 (11.62)
31.25 (92.89)
250 (743.10)
ND
Gram-negatives
E. coli
100 (224.04)
50 (112.02)
50 (112.02)
50 (112.02)
100 (224.04)
>200 (448.09)
200 (448.09)
100 (224.04)
P. aeruginosa
S. flexneri
S. marcescens
ND
ND
ND not determined
which turn it into one of the most used co-solvents for envi-
ronmentally friendly synthesis methods.
The antimicrobial activity of arginine-based surfactants
with a certain structural similarity to the two synthesized
Bz-Arg-NHCn has been reported, such as ACA (Arg-
N-decylamide), CAM (Nα-decyl-Arg-methyl ester), ALA
(Arg-N-dodecylamide) and LAM (Nα-dodecyl-Arg-methyl
ester) (Morán et al. 2001b; Pérez et al. 2002). In this con-
text, both Bz-Arg-NHCn demonstrated to be effective
against almost all the microorganisms tested. Bz-Arg-
NHC10 against E. coli showed a MIC value of 200 µg/
ml, whereas ACA and CAM proved to be ineffective. Bz-
Arg-NHC12 revealed a MIC value of 100 µg/ml, half of the
inhibitory concentration of LAM (253 µg/ml) against E.
coli. In this sense, the other Arg derivative having a 12 car-
bon alkyl chain, ALA, had no antimicrobial activity against
this microorganism. Similarly, the MIC values for both Bz-
Arg-NHCn registered against P. aeruginosa (see Table 2)
were higher than those of LAM and ALA (32 and 128 µg/
ml, respectively) (Morán et al. 2001b). The observed dif-
ferences among the Arg-based compounds could be due to
the some differences in their chemical structures: whereas
ALA has two positive charges (lacking of the α-amino
arginine moiety protecting group), LAM has the hydro-
phobic tail attached to the arginine α-amino group and its
α-carboxyl function is involved in a methyl ester bond. The
structural differences between these compounds and the
Bz-Arg-NHCn could result in different interactions with
bacterial cell membranes. Furthermore, these interactions
could be influenced not only by the chemical structure of
the surfactant molecule, but also by the composition of bac-
terial cell walls. Remarkably, Bz-Arg-NHCn showed higher
inhibitory effect on Ps. aeruginosa, than cetrimide, an anti-
microbial widely used as disinfectant in hospitals.
Antimicrobial activity
Inhibitory effect of Bz‑Arg‑NHCn
Bacterial susceptibility tests for the arginine-derived sur-
factants were performed by measuring the growth inhibi-
tion percentages for several human pathogenic bacteria.
From those experiments, MIC for each microorganism
was determined and whenever possible, the MBC was
also determined (Table 2). To compare more precisely
the antimicrobial activity of both compounds, and taking
into account their different molecular weight, MIC and
MBC values were also expressed in µM. Both surfactants
inhibit the growth of all the microorganisms tested, albeit
to different degrees. Overall, the lowest MIC values (most
potent antimicrobial activity) against both Gram-positive
and Gram-negative bacteria were detected with Bz-Arg-
NHC12. It has been observed for this kind of compounds
that the shorter alkyl chains of a homologous series dis-
play a lower antimicrobial activity than those having longer
alkyl chains. The combination of different physicochemical
parameters, such as hydrophobicity, adsorption, aqueous
solubility and transport in the test medium, depend on the
alkyl chain length and determine the different antimicrobial
efficiency of this kind of compounds, solubility being the
most limiting step for the transport (Cornellas et al. 2011).
In the case of Bz-Arg-NHC12, as well as for other homolo-
gous surfactants of 12 carbon atoms (Morán et al. 2001b),
the cooperative interaction of these parameters enhances
the surfactant’s tendency to be adsorbed at the bacterial/
water interface and therefore exert its antimicrobial action
at lower concentrations.
It is also noticeable that Bz-Arg-NHCn showed antimi-
crobial activity towards Gram-positive and Gram-negative
strains in more or less the same extent, a fact that reveals a
1 3

Biocatalytic synthesis, antimicrobial properties and toxicity…
Fig. 4 Survival curves of Escherichia coli (a, b) and Staphylococcus aureus (c, d) in nutrient broth added with Bz-Arg-NHC10 (a, c) or Bz-Arg-
NHC12 (b, d). Values are plotted as the mean SD
contrast with other Arg-based surfactants previously stud-
ied, which were in general more effective against Gram-
positive bacteria. (Morán et al. 2001b; Pérez et al. 2014).
The performance of the killing kinetics of E. coli
reported in literature using other related bromide halides
(Vieira et al. 2008) was compared with that shown by
Bz-Arg-NHCn.
Effect of surfactants on pathogens survival
Bz-Arg-NHC10 0.45 mM (MIC) caused a 2 log reduc-
tion (i.e., 99.0 % decrease) in bacteria population after
60 min of interaction, whereas decyl trimethylammonium
bromide (C10TAB) 0.6 M caused a 70 % decrease of the
initial inoculum for the same interaction time. In the case
of Bz-Arg-NHC12, ½ MIC (0.1 mM) caused a 4 log reduc-
tion (99.99 % decrease) of the initial bacteria population in
60 min of interaction, whereas when dodecyl trimethylam-
monium bromide (C12TAB) 0.45 mM was tested in similar
conditions, a pathogen survival of 11 % was achieved. A
continuous decrease in S. aureus population could be also
observed when both Bz-Arg-NHCn were tested: reductions
up to 2 log and 4 log in the initial inoculums were achieved
after 6 h of interaction with Bz-Arg-NHC10 and Bz-Arg-
NHC12, respectively.
E. coli and S. aureus were used as indicator microorgan-
isms for the study of pathogen survival in nutrient broth
added with Bz-Arg-NHC10 or Bz-Arg-NHC12. To study the
effect of the antimicrobial agents on the bacterial popula-
tion, two concentrations were selected on an MIC basis: the
first was the corresponding MIC and the second was 50 %
of the MIC value (½ MIC). In each case, survival curves
were constructed (Fig. 4).
Bz-Arg-NHC12 and Bz-Arg-NHC10 proved bactericidal
effect for both microorganisms tested, even at concentra-
tions equal to half the MIC, showing a remarkable decrease
of the number of viable microorganism after 6 h of incuba-
tion when compared to that of the control in absence of the
surfactant. In this sense, Bz-Arg-NHC10 showed a bacterio-
static effect at a concentration of ½ MIC up to 3 h. After
that time, an increase of microbial growth was observed
(Fig. 4a).
E. coli and S. aureus are microorganisms usually used
for testing antimicrobial hand washing formulations, dis-
infectants, sanitizers and pharmaceutical and personal care
formulations. Bearing this in mind and considering the
1 3

M. E. Fait et al.
fact that the synthesized compounds produce the elimi-
nation of microorganisms at MIC, it can be concluded
that they could have a potential application as effective
disinfectants.
Haemolytic activity
When the compounds were added to the erythrocyte sus-
pension in aqueous medium, they could first distribute
between the erythrocyte membrane and the solution by
adsorption until equilibrium is reached. The interac-
tion between the compound and erythrocyte membrane
at sublytic concentration might be governed by the parti-
tion of the compound between the aqueous medium and
the membrane. This partitioning is closely related to both
the hydrophobicity of the compound and the ionic interac-
tions present. Haemolysis probably begins when the eryth-
rocyte membranes are saturated with the given compound
(Castillo et al. 2006). In this context, antimicrobial agents
that kill or inhibit the growth of microbial cells can also be
cytotoxic to others such as red blood cells. The determina-
tion of haemolytic action is an accurate way to discrimi-
nate cytotoxic from non-cytotoxic compounds and also
to assess the potential for acute eye irritation (Pape et al.
1987). All the surfactants showed haemolytic activity and
the extent of haemolysis obtained at different concentra-
tions were presented in a dose–response curve (Fig. 5). The
values of HC50 (concentration of surfactant causing 50 %
haemolysis) determined are presented in Table 3 along with
the denaturation index (DI) and the lysis/denaturation ratio
(L/D). Results showed that the haemolytic effect of.Bz-
Arg-NHC10 was almost 50 % lower than that of Bz-Arg-
NHC12. According to their L/D ratio, both compounds can
be considered as non-irritant to eyes (Sanchez et al. 2006).
In contrast, cetrimide showed a more toxic effect, since the
value obtained for its L/D ratio allowed us to classify this
disinfectant as a moderately irritant compound. Finally, all
values obtained for the two novel surfactants demonstrated
that they are less toxic than LAM, which is also, in con-
trast, moderately irritant (Martinez et al. 2006).
Fig. 5 Haemolysis induced by Bz-Arg-NHC10, Bz-Arg-NHC12 and
cetrimide. Values are plotted as the mean SD of three independent
experiments
the characteristic of a normal hepatocyte (Kelly and Dar-
lington 1989), providing a good model for studying the
toxic effect of exogenous compounds, since hepatic cells
are specialized in metabolizing this kind of compounds.
The human skin fibroblast 1BR.3.G cell line was selected
for this assay since it constitutes an appropriate in vitro
model for skin or transcutaneal irritation (Sanchez et al.
2004; Chimutengwende-Gordon et al. 2014).
The mentioned cell lines were exposed to the surfactants
for 24 and 72 h and the cytotoxicity was tested by measur-
ing the ability of viable cells to metabolize a tetrazolium
salt to a soluble, brightly coloured orange formazan deriva-
tive product (XTT assay) as indirect measurements of cell
viability. All the surfactants revealed cytotoxic effects, evi-
denced by the decrease in the reduction of XTT salt com-
pared to the control in absence of the compounds. They
also induced clear dose–response relationships (see Fig. 6)
that allowed us to determine the half-maximal inhibitory
concentrations or IC50 (Table 4). The lowest IC50 value
(higher cytotoxic activity) for both cell lines and both incu-
bation times tested was achieved by cetrimide, whereas Bz-
Arg-NHC10 showed the least potent cytotoxicity effect of
all three surfactants assayed. In this context, the IC50 of cet-
rimide is three to four times higher than those of Bz-Arg-
NHC12, whereas it is five to six times higher than the IC50
values shown by Bz-Arg-NHC10. All the tested compounds
revealed to be slightly more toxic towards the fibroblasts
than towards the hepatocytes.
Cytotoxicity evaluation
The effect of Bz-Arg-NHCn on the viability of two cell
lines was assessed using the XTT (2,3-bis-(2-methoxy-
4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide)
method. Cetrimide was also proved as the reference com-
pound. The chosen cell lines for this assay were of differ-
ent origins for a more reliable evaluation of the potential
cytotoxicity of these compounds (Nogueira et al. 2011):
HepG2 (human hepatocellular carcinoma cell line, ATCC
HB-8065) and 1BR.3.G (human skin fibroblast cell line).
The human hepatoblastoma line HepG2 exhibits many of
Amino acid-based surfactants are, in general, less cyto-
toxic than quaternary ammonium halides. Lysine-based
surfactants are about one hundred times less toxic than
the commercial cationic tensioactive HTAB (hexade-
cyl trimethylammonium bromide) when tested using the
mouse fibroblast 3T6 cell line (Sanchez et al. 2006). A
similar behaviour was observed for a family of dicationic
1 3

Biocatalytic synthesis, antimicrobial properties and toxicity…
Table 3 Haemolytic activity of surfactants in terms of HC50 (μg/ml), denaturation index (DI), lysis/denaturation ratio (L/D) and classification of
the surfactants by their potential ocular irritation according to their L/D ratio
Surfactant (MW)
HCa50 μg/ml (mM)
DI
L/D
Classification
Bz-Arg-NHC10 (418.32)
Bz-Arg-NHC12 (446.34)
Cetrimide (336.42)
1100.40 24.20 (2.63 0.06)
630.58 7.04 (1.41 0.02)
52.21 4.67 (0.16 0.01)
7.98
6.19
8.58
101.82
137.81
6.06
Non-irritant
Non-irritant
Moderately irritant
a
Mean SD. The figures in brackets give the concentrations in mM. Concentration values that cause 50 % haemolysis (HC50)
Fig. 6 Viability of cell lines vs. concentration of each surfactant measured by the XTT assay. a, c 1BR.3.G, after 24 and 72 h of incubation in
presence of the surfactants, respectively. b, d HepG2, after 24 and 72 h of incubation in presence of the surfactants, respectively
Table 4 Cytotoxicity of surfactants in 1BR.3.G and HepG2 cells in terms of IC50 (μM)
Surfactant (MW)
ICa50 μg/ml (μM)
HepG2
1BR.3.G
24 h
24 h
72 h
72 h
Bz-Arg-NHC10 (418.32)
Bz-Arg-NHC12 (446.34)
Cetrimide (336.42)
32.1 2.4 (76.7 5.7)
25.2 2.5 (56.4 5.5)
4.2 0.5 (12.4 1.5)
43.6 3.0 (104.2 7.2)
27.1 2.9 (60.8 6.4)
6.8 1.0 (20.1 3.1)
21.8 2.6 (52.1 6.2)
14.2 2.0 (31.9 4.4)
2.4 0.2 (7.2 0.5)
26.1 2.5 (62.3 5.9)
20.0 2.3 (44.9 5.1)
3.9 0.3 (11.7 1.0)
a
Mean SD. Concentration values that cause 50 % death (IC50) of the cell population after 24 or 72 h of exposure to the surfactants
arginine-diglyceride surfactant compounds (Benavides
et al. 2004). The results we obtained are in concordance
with those found in the literature. These findings suggest
that Bz-Arg-NHCn would be less skin irritant than cetrim-
ide, turning them into very interesting compounds to be
tested in skin formulations.
Conclusions
Syntheses of two arginine-based cationic surfactants (Bz-
Arg-NHC10 and Bz-Arg-NHC12) were achieved in mild
conditions by means of enzymatic catalysis. Purification
steps involved low cost materials and minimal amount of
1 3

M. E. Fait et al.
Castillo JA, Infante MR, Manresa A et al (2006) Chemoenzymatic
synthesis and antimicrobial and haemolytic activities of amphi-
philic bis (phenylacetylarginine) derivatives. ChemMedChem
1:1091–1098. doi:10.1002/cmdc.200600148
Castillo-Expósito JA (2006) Studies on antimicrobial activity of argi-
nine-based surfactants and chemo- enzymatic synthesis of novel
amphiphiles based on l-arginine and d-fagomine. Ph D Thesis,
Universidad Autónoma de Barcelona
Castro M, Griffiths D, Patel A et al (2004) Effect of chain length on
transfection properties of spermine-based gemini surfactants.
Org Biomol Chem 2:2814–2820. doi:10.1039/B410240A
Chamani J, Heshmati M (2008) Mechanism for stabilization of the
molten globule state of papain by sodium n-alkyl sulfates: spec-
troscopic and calorimetric approaches. J Colloid Interface Sci
322:119–127. doi:10.1016/j.jcis.2008.03.001
Chimutengwende-Gordon M, Pendegrass C, Bayston R, Blunn G
(2014) Preventing infection of osseointegrated transcutaneous
implants: incorporation of silver into preconditioned fibronectin-
functionalized hydroxyapatite coatings suppresses Staphylococ-
cus aureus colonization while promoting viable fibroblast growth
in vitro. Biointerphases 9:031010. doi:10.1116/1.4889977
Clapés P, Morán C, Infante MR (1999) Enzymatic synthesis of argi-
nine-based cationic surfactants. Biotechnol Bioeng 63:333–343.
doi:10.1002/(SICI)1097-0290(19990505)63:3<333:AID-BIT10>
3.0.CO;2-G
solvents with low toxicity. Antimicrobial activity of Bz-
Arg-NHC10 and Bz-Arg-NHC12 was proved against both
Gram-positive and Gram-negative bacteria: in this context,
both compounds demonstrated to have broad biocidal abil-
ity, revealing their potential use as disinfectants. In addi-
tion, cytotoxic effect was compared to that of a commercial
cationic surfactant (cetrimide) using both human erythro-
cytes and cell cultures. The results obtained in both assays
showed the following order in the magnitude of cytotoxic-
ity: cetrimide > Bz-Arg-NHC12 > Bz-Arg-NHC10. In gen-
eral, it can be concluded that both synthesized compounds
were less eye-irritating and less toxic than cetrimide. More-
over, Bz-Arg-NHC10 can be considered as non haemolytic
(HC50 > 1000 μg/mL), whereas both Bz-Arg-NHCn are
non-eye-irritants (L/D > 100). Overall, the evidence from
this preliminary study would indicate that these arginine-
based surfactants represent a promising alternative to com-
mercial cationic surfactants, with biological properties that
make them interesting candidates as additives in topical
formulations.
Cornellas A, Perez L, Comelles F et al (2011) Self-aggregation and
antimicrobial activity of imidazolium and pyridinium based ionic
liquids in aqueous solution. J Colloid Interface Sci 355:164–171.
doi:10.1016/j.jcis.2010.11.063
Fité M, Clapés P, López-Santín J et al (2002) Integrated process for
the enzymatic synthesis of the octapeptide PhAcCCK-8. Bio-
technol Prog 18:1214–1220. doi:10.1021/bp0256465
Florence AT, Attwood D (2006) Physicochemical principles of phar-
macy, 4th edn. Pharmaceutical Press, London
Hanefeld U, Gardossi L, Magner E (2009) Understanding enzyme
immobilisation. Chem Soc Rev 38:453–468. doi:10.1039/
b711564b
Acknowledgments This research was supported by projects PIP 0150
(CONICET), X-613 (UNLP), CTQ2012-31605 and BIO2013-44973-R
(MINECO, Spain). Mass spectra were performed in the Unidad de
Microanálisis y Métodos Físicos Aplicados a la Química Orgánica
(UMYMFOR), CONICET-FCEN-UBA, Buenos Aires, Argentina. The
valuable contribution of Dr. Alicia S. Cánepa (LADECOR, Depto. de
Química, Fac. Cs. Exactas, UNLP) in the analysis of NMR spectra is
also acknowledged. MEF was awarded CONICET fellowship. GLG
and SRM are members of CONICET Researcher Career.
Conflict of interest The authors declare that they have no conflict
of interest.
Kelly JH, Darlington GJ (1989) Modulation of the liver specific phe-
notype in the human hepatoblastoma line Hep G2. Vitr Cell Dev
Biol 25:217–222. doi:10.1007/BF02626182
Klein JU, Cerovský V (1996) Protease-catalyzed synthesis of Leu-
enkephalin in a solvent-free system. Int J Pept Protein Res
47:348–352
References
Lang A, Hatscher C, Kuhl P (2007) Papain-catalysed synthesis of
Z-l-aminoacyl-antipyrine amides from Z-protected amino acid
esters and 4-aminoantipyrine. Tetrahedron Lett 48:3371–3374.
doi:10.1016/j.tetlet.2007.03.067
Lartigue DJ (1975) Characteristics of free vs. immobilized enzymes.
In: Messing RA (ed) Immobilized enzymes for industrial reac-
tors. Academic Press, New York, pp 125–127
Martinez V, Corsini E, Mitjans M et al (2006) Evaluation of eye and
skin irritation of arginine-derivative surfactants using different in
vitro endpoints as alternatives to the in vivo assays. Toxicol Lett
164:259–267. doi:10.1016/j.toxlet.2006.01.005
Messing RA (1975) Introduction and general history of immobilized
enzymes. In: Messing RA (ed) Immobilized enzymes for indus-
trial reactors. Academic Press, New York, pp 2–3
Miletić N, Nastasović A, Loos K (2012) Immobilization of biocata-
lysts for enzymatic polymerizations: possibilities, advantages,
applications. Bioresour Technol 115:126–135. doi:10.1016/j.
biortech.2011.11.054
Mitin Y, Braun K, Kuhl P (1997) Papain catalyzed synthesis of glyceryl
esters of N-protected amino acids and peptides for the use in trypsin
catalyzed peptide synthesis. Biotechnol Bioeng 54:287–290.
doi:10.1002/(SICI)1097-0290(19970505)54:3<287:AID-BIT9>
3.0.CO;2-B
Adlercreutz P (1991) On the importance of the support mate-
rial for enzymatic synthesis in organic media. Support effects
at controlled water activity. Eur J Biochem 199:609–614.
doi:10.1111/j.1432-1033.1991.tb16161.x
Arnon R (1970) Papain. In: Perlmann GE, Lorand L (eds) Methods in
Enzymology, vol 19., Proteolytic EnzimesAcademic Press, New
York, pp 226–244
Benavides T, Mitjans M, Martínez V et al (2004) Assessment of
primary eye and skin irritants by in vitro cytotoxicity and
phototoxicity models: an in vitro approach of new arginine-
based surfactant-induced irritation. Toxicology 197:229–237.
doi:10.1016/j.tox.2004.01.011
Bradford MM (1976) A rapid and sensitive method for the quan-
titation of microgram quantities of protein utilizing the prin-
ciple of protein-dye binding. Anal Biochem 72:248–254.
doi:10.1016/0003-2697(76)90527-3
Carmona-Ribeiro AM, de Melo Carrasco LD (2013) Cationic antimi-
crobial polymers and their assemblies. Int J Mol Sci 14:9906–
9946. doi:10.3390/ijms14059906
Castillo JA, Pinazo A, Carilla J et al (2004) Interaction of antimicro-
bial arginine-based cationic surfactants with liposomes and lipid
monolayers. Langmuir 20:3379–3387. doi:10.1021/la036452h
1 3

Biocatalytic synthesis, antimicrobial properties and toxicity…
Morán C, Infante MR, Clapés P (2001a) Synthesis of glycero amino
acid-based surfactants. Part 1. Enzymatic preparation of rac-1-O-
(Nα-acetyl-l-aminoacyl) glycerol derivatives. J Chem Soc Per-
kin Trans l 1:2063–2070. doi:10.1039/b103132p
Morán C, Clapés P, Comelles F et al (2001b) Chemical structure/
property relationship in single-chain arginine surfactants. Lang-
muir 17:5071–5075. doi:10.1021/la010375d
Piera E, Comelles F, Erra P, Infante MR (1998) New alquil amide
type cationic surfactants from arginine. J Chem Soc Perkin Trans
2:335–342. doi:10.1039/a705565j
Piera E, Infante MR, Clapés P (2000) Chemo-enzymatic synthe-
sis of arginine-based gemini surfactants. Biotechnol Bioeng
70:323–331. doi:10.1002/1097-0290(20001105)70:3<323:AID-
BIT9>3.0.CO;2-N
Morcelle SR, Liggieri CS, Bruno MA et al (2009) Screening of plant
peptidases for the synthesis of arginine-based surfactants. J Mol
Catal B Enzym 57:177–182. doi:10.1016/j.molcatb.2008.08.013
Mosmann T (1983) Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays. J
Immunol Methods 65:55–63. doi:10.1016/0022-1759(83)90303-4
Nakaoka H, Miyajima Y, Morihara K (1998) papain-catalyzed synthe-
sis of aspartame precursor: a comparison with thermolysin. J Fer-
ment Bioeng 85:43–47. doi:10.1016/S0922-338X(97)80351-0
Narai-Kanayama A, Koshino H, Aso K (2008) Mass spectromet-
ric and kinetic studies on slow progression of papain-catalyzed
polymerization of l-glutamic acid diethyl ester. Biochim Bio-
phys Acta 1780:881–891. doi:10.1016/j.bbagen.2008.03.009
Nogueira DL, Mitjans M, Infante MR, Vinardell MP (2011) Compar-
ative sensitivity of tumor and non-tumor cell lines as a reliable
approach for in vitro citotoxicity screening of lysine-based sur-
factants with potential pharmaceutical applications. Int J Pharm
420:51–58. doi:10.1016/j.ijpharm.2011.08.020
Ota S, Moore S, Stein WH (1964) Preparation and Chemical Proper-
ties of Purified Stem and Fruit Bromelains. Biochemistry 3:180–
185. doi:10.1021/bi00890a007
Pape WJW, Pfannenbecker U, Hoppe U (1987) Validation of the red
blood cell test system as in vitro assay for the rapid screening of
irritation potential of surfactants. Mol Toxicol 1:525–536
Patiny L, Borel A (2013) ChemCalc: a building block for tomor-
row’s chemical infrastructure. J Chem Inf Model 53:1223–1228.
doi:10.1021/ci300563h
Sanchez L, Mitjans M, Infante MR, Vinardell MP (2004) Assessment
of the potential skin irritation of lysine-derivative anionic sur-
factants using mouse fibroblasts and human keratinocytes as an
alternative to animal testing. Pharm Res 21:1637–1641
Sanchez L, Mitjans M, Infante MR, Vinardell MP (2006) Poten-
tial irritation of lysine derivative surfactants by hemolysis and
HaCaT cell viability. Toxicol Lett 161:53–60. doi:10.1016/j.
toxlet.2005.07.015
Schechter I, Berger A (1967) On the size of the active site in pro-
teases. I Papain Biochem Biophys Res Commun 27:157–162.
doi:10.1016/S0006-291X(67)80055-X
Scudiero DA, Shoemaker RH, Paul KD et al (1988) Evaluation of a
soluble tetrazolium/formazan assay for cell growth and drug sen-
sitivity in culture using human and other tumor cell lines. Cancer
Res 48:4827–4833
Singh A, Van Hamme JD, Ward OP (2007) Surfactants in microbiol-
ogy and biotechnology: part 2. Application aspects. Biotechnol
Adv 25:99–121. doi:10.1016/j.biotechadv.2006.10.004
Tischer W, Wedekind F (1999) Biocatalysis—from discovery to appli-
cation 200:95–126. doi:10.1007/3-540-68116-7
Torres JL, Piera E, Infante MR, Clapés P (2001) Purification of non-
toxic, biodegradable arginine-based gemini surfactants, bis
(Args), by ion exchange chromatography. Prep Biochem Bio-
technol 31:259–274. doi:10.1081/PB-100104908
Valivety R, Jauregi P, Gill I, Vulfson E (1997) Chemo-enzymatic
synthesis of amino acid-based surfactants. J Am Oil Chem Soc
74:879–886. doi:10.1007/s11746-997-0232-8
Vieira OV, Hartmann DO, Cardoso CMP et al (2008) Surfactants
as microbicides and contraceptive agents: a systematic in vitro
study. PLoS One 3:e2913. doi:10.1371/journal.pone.0002913
Walsh KA, Wilcox PE (1970) Proteolytic enzymes. Methods Enzy-
mol 19:31–41. doi:10.1016/0076-6879(70)19005-7
Pérez L, Torres JL, Manresa A et al (1996) Synthesis, aggregation,
and biological properties of a new class of gemini cationic
amphiphilic compounds from arginine, bis (Args). Langmuir
12:5296–5301. doi:10.1021/la960301f
Pérez L, García MT, Ribosa I et al (2002) Biological properties of
arginine-based gemini cationic surfactants. Environ Toxicol
Chem 21:1279–1285
Pérez L, Pinazo A, Pons R, Infante M (2014) Gemini surfactants from
natural amino acids. Adv Colloid Interface Sci 205:134–155.
doi:10.1016/j.cis.2013.10.020
1 3