Enzymatic synthesis of N-10-undecenoyl-phenylalanine catalysed by aminoacylases from Streptomyces ambofaciens

Article abstract of DOI:10.1016/j.procbio.2020.09.009

Due to its physico-chemical and biological activities, N-10-undecenoyl-phenylalanine (C11'F) is one of the most interesting lipoaminoacids used in cosmetic and pharmaceutical industries. Its production is currently based on the Schotten-Baumann chemical reaction, which shows some environmental issues in terms of effluents. As a possible biocatalytic alternative, this study presents the evaluation of the reactional and process conditions allowing the production of C11'F using aminoacylases from Streptomyces ambofaciens culture. These aminoacylases showed the best activity at 45 °C and pH between 7 and 8 with a moderate thermal stability. The influence of substrates concentrations on the kinetic parameters of C11'F synthesis showed a more important impact of the phenylalanine concentration as compared to the 10-undecenoic acid concentration. As a reactional product, C11'F appeared to have an inhibitory effect on the enzymatic N-acylation. Cobalt addition allowed an eleven-fold increase of the reaction rate. Batch reactors were used with free aminoacylases without impact on the final C11'F concentration. For the first time, enzymatically produced C11'F was finally purified at the gram scale as a 99% purity white powder. The evaluation of the biological activity on melanocytes cultures showed the presence of skin lightening activity similar to the one obtained with the chemically produced C11'F.

Full text of DOI:10.1016/j.procbio.2020.09.009

Process Biochemistry 99 (2020) 307–315
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Enzymatic synthesis of N-10-undecenoyl-phenylalanine catalysed by
aminoacylases from Streptomyces ambofaciens
Mohamed Chafik Bourkaiba, Stephane Delaunaya, Xavier Framboisiera, Catherine Humeaua,
b
b
b
a
a,
´ ˆ
Jerome Guilbot , Cecile Bize , Estelle Illous , Isabelle Chevalot , Yann Guiavarc’h *
a
´
ˆ
LRGP, UMR 7274 CNRS-Universite de Lorraine, 2 avenue de la Foret de Haye, TSA 40602, F-54518, VANDŒUVRE Cedex, France
b R&D laboratory of Seppic, Air Liquide Healthcare Specialty Ingredients, 50 boulevard National - CS 90020, 92257, La Garenne Colombes Cedex, France
A B S T R A C T
Due to its physico-chemical and biological activities, N-10-undecenoyl-phenylalanine (C11’F) is one of the most interesting lipoaminoacids used in cosmetic and
pharmaceutical industries. Its production is currently based on the Schotten-Baumann chemical reaction, which shows some environmental issues in terms of ef-
fluents. As a possible biocatalytic alternative, this study presents the evaluation of the reactional and process conditions allowing the production of C11’F using
aminoacylases from Streptomyces ambofaciens culture. These aminoacylases showed the best activity at 45 ◦C and pH between 7 and 8 with a moderate thermal
stability. The influence of substrates concentrations on the kinetic parameters of C11’F synthesis showed a more important impact of the phenylalanine concentration
as compared to the 10-undecenoic acid concentration. As a reactional product, C11’F appeared to have an inhibitory effect on the enzymatic N-acylation. Cobalt
addition allowed an eleven-fold increase of the reaction rate. Batch reactors were used with free aminoacylases without impact on the final C11’F concentration. For
the first time, enzymatically produced C11’F was finally purified at the gram scale as a 99% purity white powder. The evaluation of the biological activity on
melanocytes cultures showed the presence of skin lightening activity similar to the one obtained with the chemically produced C11’F.
1. Introduction
some fatty acid traces is obtained. Despite a high yield of synthesis, the
corresponding down-stream processing of the reaction medium leads to
large volumes of saline effluents requiring an expensive reprocessing
step for the industrials [6]. Nowadays, the environmental issues caused
by such process encouraged the authorities to promote the industrial
development of sustainable technologies by switching from waste
remediation and pollution control by end-of-pipe solution to waste
prevention thus reducing the reprocessing cost [7]. Enzymes represent
an alternative for such reactions to avoid by-products and salts gener-
ation. Enzymes were widely used for acylation reaction especially for
O-acylation but much less for N-acylation of amino acids. The ability of
some enzymes to perform the N-acylation of amino acids were studied
by only few authors using free fatty acids [8–15]. Lipases and proteases
are hydrolases that can perform the acylation reaction in non-aqueous
media like organic solvents or in solvent-free conditions in order to
shift the thermodynamic equilibrium of the reaction. However, the low
solubility of amino acids in organic solvents limits the mass transfer of
the substrates to the active site, which decreases the performance of the
reaction [8,16,17]. In this context, the use of acylases like aminoacylases
that are able to catalyse the N-acylation reaction in aqueous media with
better solubility of the substrates, especially of the acyl acceptor, has
been yet considered [18,19]. Several enzymes were evaluated for
Amino acids surfactants (AAS) are eco-compatible surface-active
agent with very interesting techno-functional properties and, for some of
them, with attractive bioactivities. There are molecules of choice for
food, pharmaceutical and cosmetic industries [1]. Non-ionic AAS, like
phenylalanine and leucine derivatives, are very attractive AAS as in-
gredients at industrial scale because of their good detergency and
foaming properties [2,3]. In addition to these properties,
phenylalanine-based surfactants were described as presenting cytotox-
icity against several human cancer cells thus widening their application
scope [4]. Bisset et al., 2009 described the ability of phenylalanine-fatty
acid derivatives like N-10-undecenoyl-L-phenylalanine to reduce
melanin production in melanocytes. In their study, an emulsion pre-
pared by the combination of 1 % N-10-undecenoyl-phenylalanine with 5
% niacinamide increased significantly the efficiency in reducing the
appearance of facial hyperpigmentation in vivo [5]. However, such AAS
are produced at industrial scale using the Schotten-Baumann chemical
reaction where the amino acid reacts with a fatty acid chloride under
highly alkaline conditions by using NaOH in water and sulfuric acid to
stop the reaction [4]. In some cases, some organic solvents are used to
facilitate the reaction. In this last case a high purity powder with only
* Corresponding author.
E-mail address: yann.guiavarch@univ-lorraine.fr (Y. Guiavarc’h).
https://doi.org/10.1016/j.procbio.2020.09.009
Received 27 April 2020; Received in revised form 4 September 2020; Accepted 7 September 2020
Available online 10 September 2020
1359-5113/© 2020 Elsevier Ltd. All rights reserved.

M.C. Bourkaib et al.
Process Biochemistry 99 (2020) 307–315
amidation reaction but much less for the N-acylation of amino acids. The
most used enzymes performing this reaction were described by Koreishi
et al. who identified four acylases from Streptomycess mobaraensis [10,
11,20,21]. Three of them were aminoacylases characterized as mono-
meric metallo-enzymes with different regioselectivities. Indeed, the
aminoacylase of 100 kDa and the second one of 55 kDa (called Sm-AA)
had regioselectivity towards the alpha amino group of the amino acid
while the third one of 60 kDa (called Sm-ELA) was specific for the lysine
epsilon amino group [10,12,20,21]. The fourth acylase was a penicillin
V acylase (Sm-PVA), which is a dimeric enzyme able to perform the
N-acylation of several amino acids with lauric acid showing a better
2.2. Enzymatic N-acylation of L-Phenylalanine assays
The evaluation of the operational conditions influencing the activity
of the crude extract of aminoacylases were conducted in test tubes of
2 mL final reaction medium containing 100 mM of each substrate
(except for substrate concentration influence) and 1 g/L enzymes con-
centration (except for enzyme concentration influence) prepared in
25 mM Tris-HCl, 50 mM NaCl buffer, at 45 ◦C (except for temperature
influence), pH 8 (except for pH influence) and 500 rpm magnetic
agitation.
The influence of substrate concentration was investigated by using
concentrations from 25 to 200 mM of L-phenylalanine and from 25 to
200 mM of 10-undecenoic acid setting the other substrate’s concentra-
tion at 100 mM. The influence of the pH of the reaction was studied in
the pH range [6-9.5] using NaOH 7.5 M and HCl 1 M to control the pH.
Temperature effect on the reaction was evaluated in two steps. The first
one was the evaluation of the optimal temperature of the enzymes by
performing the synthesis reaction in a temperature range of [30-55 ◦C].
The second step was to evaluate the stability of the enzymes by per-
forming the standard reaction after incubation at different temperature
(37, 45 and 55 ◦C) and during different incubation time (16 and 25 h).
The enzymes concentration influence was evaluated by using (0.25, 0.5,
1 and 2 g/L) protein concentration at the standard conditions. The in-
hibition by the product of the reaction was investigated by adding
different concentrations of a chemically produced commercial N-10-
undecenoyl-phenylalanine. The relative activity was then calculated
using the initials rates of the reactions with (Vi C11’F) and without (Vi0)
activity with arginine, cysteine and lysine with a in ε position [11,22]. In
another study, the ability of a crude extract from Streptomycess ambo-
faciens ATCC23877 to perform the N-acylation of lysine and peptides
containing lysine on their
α position was described [13]. By genome
comparison, a very close sequence identity between the genes encoding
Sm-AA, Sm-ELA and Sm-PVA from S. mobaraensis and the ones respon-
sible for the acylation activity from S. ambofaciens was shown, with 86,
80 and 69 % sequence identity respectively. Another gene was also
identified with 55% identity with Sam-AA (Sam-AA like) but this had a
very low
the enzyme similar to Sm-AA, named SamAA, was responsible of 85 % of
the N- -acetyl-L-lysine hydrolysis activity [13]. However, no informa-
α-acylation activity. In addition, the authors demonstrated that
α
tion is available until now about the condition influencing the activity of
the acylases from S. ambofaciens ATCC23877. In a recent study, Bour-
kaib et al. (2020) described the substrate specificity of the crude extract
of Streptomyces ambofaciens was investigated toward the 20 natural
proteogenic amino acids and different fatty acids for the enzymatic
synthesis of many AAS in water. In the same study authors also inves-
tigated the impact of some operational conditions on the production of
alpha-lauroyl lysine in batch reactor [23]. They also compared the
performance of the crude extract from the wild strain of S. ambofaciens
and the one from a mutant strain which express only Sam-AA. The re-
sults showed that the substrates specificity was similar between both
strains confirming the position that Sam-AA is responsible of the
the addition of the product (relative activity (%)
=
Vi C11’F).
Vi 0
We investigated the effect of metal ions by using 0.1 mM of CuCl2
(from Fluka Chemika), CoCl2, ZnSO4 (from Sigma), and Ethylene-
Diamine-Tetra-acetic Acid (EDTA) in the standard conditions. The in-
fluence of CoCl2 concentration was evaluated by using CoCl2 concen-
trations in the range [0-0.5 mM]. In all case, the reaction was started
after one-night agitation, in order to check if there was a reaction
without enzymes, by adding 1 g/L of the crude extract of aminocaylases
from S. ambofaciens.
N-α-acylation activity of the crude extract from the wild strain Strepto-
myces ambofaciens.
At appropriate times, 50 or 100 μL aliquot of the reaction mixture
The aim of the present work was to focus on the enzymatic synthesis
of one AAS, the N-10-undecenoyl-phenylalanine, using aminoacylase
from a crude extract from Streptomyces ambofaciens culture. As above
mentioned, the N-10-undecenoyl-phenylalanine (C11’F) is an AAS of
direct industrial interest, used in cosmetics and pharmaceutics as a skin
lightening agent. To date, no study was reported on the enzymatic
synthesis of this AAS in water and using enzymes. In this study, the in-
fluence of the operational conditions on the synthesis reaction of C11’F
was investigated. An up-scaling of production using different reactor
configurations as well as a convenient way to purify C11’F as a white
powder was also presented. The inhibition of melanin production of the
purified product was evaluated using melanocyte cell line and compared
to the activity of the chemically produced C11’F.
was withdrawn and diluted with 80% Methanol for HPLC-ELSD analysis.
Finally, all reactions were stopped by freezing directly the mixture at
ꢀ 18 ◦C. Each reaction run was performed at least twice.
2.3. Reactor configuration
Different batch reactor with different reaction medium volumes and
agitation systems were also evaluated and compared to the classic batch
reaction performed in a standard vessel filled with 2 mL of the reaction
medium. The first one was a 100 mL glass spinner flask with double
sidearms (Wheaton® 356875 Celstir®) used as reactor equipped with
suspended magnetic agitation filled with 20 mL of reaction medium final
volume. The second one was a reactor of 500 mL equipped with auto-
matic pH, temperature and agitation monitoring (OptiMax, METTLER
TOLEDO©). The agitation module consisted of a four blades propeller,
upwards (M8) of 45 mm diameter and 45◦ tilt. The reactor was filled
with 300 mL of final reaction medium. In both cases the reaction me-
dium contained 100 mM of each substrate and 1 g/L enzymes concen-
tration in 25 mM Tris-HCl, 50 mM NaCl buffer, at 45 ◦C, pH 8 and
2. Materials and methods
2.1. Aminoacylases of S. ambofaciens production
The production of aminoacylases from S. ambofaciens was performed
as described in detail in [23]. Aminoacylases from S. ambofaciens were
finally obtained as a protein crude extract whose specific activity was
evaluated using a lauroyl-lysine synthesis reaction. For this reaction,
lauric acid and L-lysine were used as substrates at equimolar concen-
tration (100 mM) in 25 mM Tris-HCl buffer, NaCl 50 mM, at pH 8 and
45 ◦C at 500 rpm magnetic stirring (2 mL reaction volume) in a simple
glass tube.
500 rpm agitation. At appropriate times, 100 μL aliquot of the reaction
mixture was withdrawn and diluted with 80% Methanol for HPLC-ELSD
analysis. Finally, all reactions were stopped by freezing directly the
mixture at ꢀ 18 ◦C. Each reaction was performed at least twice. The
product of the reaction performed in the OptiMax was purified following
the procedure described below.
308

M.C. Bourkaib et al.
Process Biochemistry 99 (2020) 307–315
2.4. Purification of the final product
intial rates of the reaction by using a third order polynomial model
applied to the derivate of the first experimental data points exhibited in
the figures. The maximum velocity (Vmax) and the Michaelis-Menten
constant (Km) for each substrate were determined using a standard
In order to get, for the first time, a pure enzymatically produced N-
10-undecenoyl-pheylalanine, a purification process was developed. The
300 mL final reaction medium containing 1.53 g of the produced C11’F,
residual 10-undecenoic acid (4.64 g) and L-phenylalanine (4.16 g) and
the enzymes was used for the development of the purification protocol.
The first stage was an ultrafiltration with a 10 kDa membrane (Amicon
ultra-15; Merck) made of regenerated cellulose in order to eliminate
small molecules coming from the aminoacylases crude extract. Two
diafiltration steps and a 10-fold concentration step of the initial mixture
were performed by centrifugation during 30 min and 5000 rpm, using a
fixed angle rotor (Centrifuge 5804 R, Eppendorf). The second stage of
the protocol consisted in removing the proteins by precipitation with
methanol. To do this the 30 mL concentrated solution was diluted 10
times with a 80%/20% methanol/water solution. After 3 hours magnetic
agitation the precipitated proteins were removed by centrifugation
(20 min at 8400 rpm and 15 ◦C). The methanol present in the superna-
tant part was evaporated using a rotating evaporator (Laborota 4002
control, Heidolph, Germany). The third stage consisted in removing
residual impurities and salts by size exclusion chromatography (Bio-
Michaelis-Menten model based on the equation of the graphic repre-
( )
1
1
1
Km
1
senting
= f
=
∗
+
Vmax where V is the initial rate and S is
V
S
S
Vmax
the substrate concentration.
2.5.2. Mass spectrometry analysis
We performed the qualitative and semi-quantitative analysis of N-10-
undecenoyl-phenylalanine by using an HPLC-MS-MS system (Thermo-
Fisher Scientific, San Jose, CA, USA) consisting in a binary pump con-
nected to a photodiode array detector (PDA) and a Linear Trap
Quadrupole mass spectrometer (LTQ). An atmospheric pressure ioniza-
tion interface operating in positive electrospray mode (ESI+) was used.
The chromatographic separation and mass spectrometric conditions that
were used as described in details in [23]. Raw data were processed using
Xcalibur software program (version 2.1, http://www.thermoscientific.
com).
SepTM SEC-S2000 300 × 21.2 mm, 5
μm particle diameter, Phenomesex,
2.6. Biological activity evaluation
USA). The elution of the 5 mL injected volume was performed using an
isocratic system (45% acetonitrile) with a 5 mL/min flow rate, during
37 min. The mixture C11’/C11’F was collected after a 23 min elution
time. The recovered mixture was then concentrated by evaporation for
the next stage of purification by liquid chromatographic method.
Different high pressure reverse phase liquid chromatography methods
were tested using a “classical” C18 column (Grace/Alltech, Darmstadt,
The evaluation of the melanin production by melanocytes was per-
formed using B16 murine melanoma cells (B16F1 cells) following the
method described by Chung et al., 2019 with some adaptations [24].
B16F1 cells were maintained in a CO2 incubator in Dulbecco’s modified
Eagle medium (DMEM), without phenol red, supplemented with 10%
fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 μg/ml).
Germany, 150 mm × 2.1 mm, 5
μ
m particle size) or less classical C30
m particle size,
Cells were incubated in CO2 for 24 h after being seeded in 96-well plate
(6000 cells/well). Then cells were treated with SepiwhiteTM MSH
(SEPPIC) or enzymatic N-10-undecylenoyl-phenylalanine initially dis-
solved in ethanol or the culture medium. Two different concentrations of
phases (LC Acclaim™ C30, 250 mm × 2.1 mm, 5
μ
ThermoFisher scientific, USA) and also a more specific phase containing
phenyl functional groups on its surface (Force Biphenyl
5
5
μ
m,
m,
150 × 2.1 mm for analytic column and Ultra Biphenyl
μ
each was tested (10 and 50
μ
g/mL) and kojic acid at 50
μ
g/mL in the
250 × 21.2 mm for preparative column, Restek, USA). However, the
very close physicochemical properties of the product and the residual
fatty acid, like hydrophobicity (logP C11’ = 3.99, logP C11’F = 5.05),
did not allow their proper separation. Also, although the biphenyl
analytical scale column showed its capability to separate the two mol-
ecules with about 2 min difference in retention times, the same phase
used in a preparative scale column did not allow any separation
anymore even after several different trials. Actually, normal phase
chromatography using Kieselgel G 60 powder under atmospheric pres-
sure appeared to be the most suitable method leading to the pure
product. The mixture 50% cyclohexane/50% ethyl acetate was selected
to elute the residual fatty acid, selectively. The product was then eluted
by adding around 1% (v/v) acetic acid to the previous eluting system.
The elimination of the residual fatty acid and the recovery of the product
were followed using Kieselgel G 60 Plates and iodine chamber. The
collected fractions containing the product were concentrated using a
rotary evaporator and dissolved again in water for the lyophilisation
step, leading to a white powder. The product purity was assessed by
HPLC-ELSD, LC-MS and proton NMR analyses.
culture medium was used as a positive reference. 200
μL of the cell
culture medium was transferred to a 96-well plate after 72 h incubation.
The absorbance of the samples and standard curve of synthetic melanin
(0 to 500
μ
g/mL) was measured at 450 nm. The inhibition activity (%)
concentration of melanin with the tested molecule
was expressed as
× 100. In order to
concentration of melanin without tested molecule
evaluate the cells viability, the latters were isolated and lysed in PBS
buffer prior concentration determination using a protein assay kit (BCA
Protein Quantitation Kit). The cells viability was expressed as
concentration of proteins with the tested molecule
× 100. The measured melanin con-
concentration of proteins without tested molecule
tents were then normalised by the protein amount and expressed as
μ
g/mg of cell’s proteins. The evaluation was done at least 5 times and
the statistical analysis was done using Student test to compare the results
with the control (XLSTAT software, Addinsoft Inc., Paris, France).
3. Results and Discussion
3.1. Influence of substrate concentration on enzymatic activity
In a recent study [23], the ability of the aminoacylases from
S. ambofaciens to catalyse the N-acylation of the amino acids on alpha
amino group position was demonstrated. 10-undecenoic acid is a
mono-unsaturated fatty acid described as having antifungal, antiviral
and insect-repelling activity which makes it useful to treat superficial
infections [25,26]. It was also described as having neuroprotective and
radical-scavenging activity [27]. The N-undecyl-10-enoyl-L-phenylala-
nine (C11’F) generated by the condensation of the phenylalanine with
the 10-undecenoic acid was described as having very interesting prop-
erties for cosmetic applications. However, the enzymatic synthesis of
C11’F has never been reported. In order to study the operational con-
ditions influencing the activity of the aminoacylases from S. ambofaciens
the synthesis of C11’F was used as reference reaction. In this study all
2.5. Analytical methods
2.5.1. Quantitative analyses by HPLC
We monitored the reaction conversion by using the same analytical
method as the one previously described in [23], based on the use of an
HPLC (LC 10 AD-VP, Shimadzu, France) equipped with a UV detector at
214 nm followed by a light-scattering low temperature evaporative de-
tector (Shimadzu, France). Calibrations were performed using a chem-
ically synthetized N-10-unecenoyl-phenylalanine named SepiwhiteTM
MSH kindly provided by SEPPIC, France. The substrate conversion yield
was determined using the following equation: conversion (%) =
(([C11’F] mol/L produced / [C11’] mol/L initial) × 100. We calculated the
309

M.C. Bourkaib et al.
Process Biochemistry 99 (2020) 307–315
experiments were carried out using the same crude extract from
S. ambofaciens containing 21 ± 0.7 g/L of proteins [28]. The apparent
specific activity of the proteins crude extract was evaluated using
lauroyl-lysine synthesis reaction performed with an equimolar concen-
tration of the lauric acid and lysine (100 mM). The reaction was per-
formed using 1 g/L of enzymatic crude extract. The apparent specific
even the ionisation state of the substrates and enzymes and, subse-
quently, the enzymes activity. The results showed that the activity
remained nearly stable at pH range from 6.5 to 9 while no reaction was
obtained at pH 6 and very low activity was measured at pH 9.5 (Fig. 3
A). However, by following and comparing the kinetic of the reaction at
pH 8-9 (Fig. 3 B), the enzymes seems to be much less stable at pH 8.5 and
9 than at pH 8, which is very important in the perspective of an
extrapolation. For this reason, the pH of all the reaction performed using
the aminoacylases from S. ambofaciens at 45 ◦C were set at a range of 7-8.
Several studies evaluated the influence of the pH on the enzymatic ac-
tivity and showed nearly the same pH range for optimum activity, which
is widely used for industrial application. Koreishi et al., showed that the
optimum pH of the aminoacylase (SmAA) from Streptomyces mobaraensis
was between 7 and 8 while the second aminoacylase of 100 kDa had an
optimum pH of 6.5 [20,21]. Wada et al., showed that the acylase I from
Pig Kidney had an optimal pH for the reaction of around 7 and 7.5 [29].
activity of the acylases from S. ambofaciens was 33.4 ± 5 μM of α-laur-
oyl-lysine / min / mg of proteins.
All N-acylation (amidation) reactions were carried out in 25 mM
Tris-HCl buffer, NaCl 50 mM pH 8, containing 1 g/L acylases at 45 ◦C ±
3 ◦C. In order to evaluate the substrate concentration influence on
enzyme activity different reactions were performed. First of all, the best
substrate concentration, at equimolar conditions, was evaluated for the
further parameters evaluation by using different concentration (25 mM,
50 mM, 100 mM and 200 mM). The concentration produced after 48 h
(Fig. 1) showed a maximum reaction by using 100 mM of both substrates
while very low activity was observed in the other conditions. The in-
fluence of the substrate concentration on the performance of the ami-
noacylases from S. ambofaciens was then studied by fixing the
concentration of one of the substrates at 100 mM and by varying the
concentration of the other substrate from 25 mM to 200 mM. The in-
fluence of the concentration in 10-undecenoic acid was very low
compared to the influence of phenylalanine concentration (Fig. 2). The
initial rate of the reaction was increased by the increase of the con-
centration of the fatty acid from 25 mM to 125 mM followed by a sta-
bilization of the reaction rate. The evolution of the initial rate followed
standard Michaelis-Menten model allowing the determination of Km
3.3. Influence of the temperature
The influence of the temperature of the reaction on the enzymatic
activity is shown in Fig. 4.A. the best activity was obtained at 45 ◦C
while very low activity was observed at 55 ◦C. The loss of the activity at
55 ◦C can be related to a low stability of the enzymes due to a fast protein
denaturation. In order to evaluate the thermal stability of the enzymes,
the aminoacylases were incubated at different temperature (37, 45 and
55 ◦C) during 16 and 25 h before starting the reaction at 45 ◦C. As shown
in Fig. 4.B, the enzymes lost around 80 % of the activity after 16 h in-
cubation at 37 ◦C and around 90 % after 25 h and the higher is the in-
cubation temperature, the higher is the loss of activity. The results at
55 ◦C confirmed that the low activity observed when performing the
reaction at this temperature was due to protein denaturation. Similar
results were obtained by Koreishi, who studied the stability of the
aminoacylase (SmAA) from Streptomyces mobaraensis by performing the
hydrolysis of N-acetyl-L-methionine at 37 ◦C in Tris-HCl buffer at pH
7.5. They also showed that the enzymes lost almost all the activity after
only one-hour incubation at 55 ◦C [21].
and Vmax for the undecenoic acid. According to the equation of the
( )
1
1
graphic representing
= f
Vmax and Km of around 0.50 ±
V
S
0.05 mM / h and 19.2 mM were found. Above a C11’ concentration of
175 mM a slight decrease of the activity was observed which can be due
to a substrate inhibition. The influence of the amino acid was more
pronounced than the fatty acid where a continuous increase of the initial
rate was observed. Indeed, according to the equation of the graphic
( )
1
1
representing = f
Vmax and Km of around 0.50 ± 0.05 mM / h and
V
S
3.4. Influence of protein concentration
42 mM were found. Koreishi et al., 2005 observed nearly the same
relationship when performing the reaction of Nα-Feruloyl-L-lysine syn-
Reactions using different protein concentration in 25 mM Tris-HCl
buffer, NaCl 50 mM containing 100 mM of both substrates at 45 ◦C,
pH 8 and 450 rpm magnetic agitation were conducted. By following the
kinetics of the production of C11’F at the different protein concentra-
tions, the increase of the quantity produced after 3 days was propor-
tional to the protein concentration when a concentration between 0.25
and 1 g/L was used. However, the increase of the produced concentra-
tion was much less important from 1 to 2 g/L while the initial rate was
proportional to the quantity of protein used (Fig. 5 A and B). In all cases,
a decrease of the activity after a certain time was observed. This can be
attributed to a relatively low thermal stability of the enzymes or to an
inhibition by the product of the reaction knowing that the C11’F is
soluble in the operational condition. In order to face the problem of
thermal denaturation and to increase the amount of C11’F produced, a
reaction was performed using 1 g/L of enzymes with an additional 1 g/L
introduced to the reaction medium after 24 h. The kinetic of the reaction
was compared to the ones with 1 and 2 g/L (Fig. 5 C). The addition of
enzymes during the reaction caused an increase of the production to
reach the same concentration as with 2 g/L protein concentration. This
means that the protein denaturation is not the only factor controlling the
reaction. The second reason could then be the enzymes inhibition by the
C11’F itself or that the thermodynamic equilibrium of the reaction was
reached. The product inhibition of the reaction was evaluated by per-
forming the reaction in 25 mM Tris-HCl buffer, NaCl 50 mM containing
100 mM of both substrates and [0-10 mM] of C11’F at 45 ◦C, pH 8 and
450 rpm magnetic agitation. The results (Fig. 5 D) showed a clear
product inhibition where a progressive decay of the initial activity of the
thesis using the aminoacylase from Streptomyces mobaraensis. They
showed, like in this study, that the activity was almost unaffected by the
concentration of the acyl donor. However, the effect of L-lysine con-
centration on the activity was positively much more important [20].
3.2. Influence of the pH on aminoacylases activity
The pH of the reaction medium is one of the most important oper-
ational parameters affecting the performance of a reaction by changing
Fig. 1. Influence of substrates concentration at equimolar condition on the
synthesis of N-10-undecenoyl-phenylalanineafter after 72 h in 25 mM Tris-HCl
buffer, NaCl 50 mM containing 1 g/L acylases at 45 ◦C, pH 8 and 450 rpm
magnetic agitation (n = 2 or 3).
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Process Biochemistry 99 (2020) 307–315
Fig. 2. Influence of substrates concen-
tration on the initial rate for the syn-
thesis
of
N-10-undecenoyl-
phenylalanine in 25 mM Tris-HCl
buffer, NaCl 50 mM containing 1 g/L
acylases at 45 ◦C, pH 8 and 450 rpm
magnetic agitation, A. Effect of 10-
undecenoic acid concentration on the
initial rate of N-10-undecenoyl-phenyl-
alanine synthesis reaction. A reaction
mixture containing 0.025-0.2 M of 10-
undecenoic acid and 100 mM L-phenyl-
alanine. B. Effect of L-phenylalanine
concentration on the initial rate of N-
10-undecenoyl-phenylalanine synthesis
reaction. A reaction mixture containing
100 mM 10-undecenoic acid and
0.025–0.2 M of L-phenylalanine (n = 2).
Fig. 3. Influence of the pH on the enzymatic activity for the synthesis of N-10-undecenoyl-phenylalanine in 25 mM Tris-HCl buffer, NaCl 50 mM containing 100 mM
of both substrates, 1 g/L acylases at 45 ◦C, pH [6 - 9.5] and 450 rpm magnetic agitation, A. effect of the pH on the initial rate. B. Kinetic evolution of the synthesized
N-10-undecenoyl-phenylalaninesynthesis reaction at pH 8, 8.5 and 9 (n = 2).
Fig. 4. Effect of the temperature on the
enzymatic activity A. Influence of the
reaction temperature on the enzymatic
activity for the synthesis of N-10-unde-
cenoyl-phenylalanine in 25 mM Tris-
HCl buffer, NaCl 50 mM containing
100 mM of both substrates, 1 g/L acy-
lases at T◦C [30-55 ◦C], pH
8 and
500 rpm magnetic agitation B. Thermal
stability of the enzymes after 16 and
25 h incubation time at 37, 45 and 55 ◦C
compared to fresh enzymes reaction
conducted in 25 mM Tris-HCl buffer,
NaCl 50 mM containing 100 mM of both
substrates, 1 g/L acylases at 45 ◦C, pH 8
and 500 rpm magnetic agitation (n = 2).
enzymes was observed by increasing the concentration of the product.
coding the aminoacylase from S. mobaraensis (Sm-AA) described by
Koreishi et al. in 2009 with 86 % sequence identity. This enzyme was
described as belonging to a metal-dependant family which can be
influenced by the addition of metal ions playing a key role in the cata-
lytic process. In order to evaluate the effect of metal ions on the acylases
from S. ambofaciens activity, Co2+, Zn2+ and Cu2+ which were described
3.5. Influence of metal activator on the reaction
As described by Dettori et al., 2018, the sequence coding the ami-
noacylase from S. ambofaciens, named SamAA, is very close to the one
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Process Biochemistry 99 (2020) 307–315
Fig. 5. Influence of the protein con-
centration and the product concentra-
tion on the enzymatic activity for the
synthesis of N-10-undecenoyl-phenylal-
anine in 25 mM Tris-HCl buffer, NaCl
50 mM containing 100 mM of both
substrates, 1 g/L acylases (except for the
protein concentration influence) at
45 ◦C, pH
8 and 450 rpm magnetic
agitation, A. C11’F production kinetic at
different enzymes concentration [0.25-
2 g/L] B. Initial rate of the production at
different protein concentration C. C11’F
production kinetic with enzymes addi-
tion during the reaction compared to
the reaction performed using 1 and 2 g/
L initial enzymes concentration D. In-
fluence of the C11’F added concentra-
tion on the initial rate of the reaction
(n = 2 for Fig. 5A, 5B, 5C, 5D).
as the most influencing ones, were tested [10,11,20,21].
Ethylene-diamine-tetra-acetic acid (EDTA) was used as chelating agent
to check the metal-dependency of the enzyme. As shown in Fig. 6, for the
same concentration of the three metal ions (0.1 mM), Cobalt ion led to a
three time increase of the initial rate unlike Zn2+ and Cu2+ ions allowing
only a slight increase. No reaction was registered when EDTA was
added, which confirmed that the aminoacylases involved in the
N-acylation are metalloenzymes. However, by comparing the reaction
course, the thermodynamic equilibrium was reached with CoCl2 after
48 h while the thermodynamic equilibrium of the control reaction and
the reaction with Zn2+ as metal ion was not yet reached after 72 h. For
the reaction with Cu2+ the thermodynamic equilibrium was also reached
after 48 h but with around 25% less activity than with Co2+ as metal ion.
The influence of Cobalt concentration was therefore also investigated.
The increase of the Cobalt concentration allowed an important increase
of the initial rate of the reaction to reach around eleven-fold increase of
the initial rate by using 0.5 mM of CoCl2 (Table 1). When concentration
from 0.3 to 0.5 mM of CoCl2 was used, the thermodynamic equilibrium
was reached after only 24 h reaction time but the final product con-
centration was affected. Indeed, the increase of the CoCl2 concentration
led to an increase in the conversion rate until 0.3 mM CoCl2 with more
than 20 mM C11’F concentration. A 16 mM C11’F concentration was
reached when using a 0.5 mM CoCl2 concentration. Koreishi et al. 2005
studied the influence of metal ions on the hydrolytic activity of
S. mobaraensis aminoacylase. Their results were similar to the ones
observed in this study where three-fold increase of the activity by adding
0.1 mM CoCl2 and only a slight increase by adding Zn2+ ion was
observed. However, they showed a decrease of 80 % of the activity by
adding Cu2+ ion (Koreishi et al., 2005a), which was not the case with the
acylases from S. ambofaciens.
3.6. Influence of reactor configuration
Fig. 6. influence of different metal ions at 0.1 mM concentration on the
enzymatic activity for the synthesis of N-10-undecenoyl-phenylalaninein
25 mM Tris-HCl buffer, NaCl 50 mM containing 100 mM of both substrates, 1 g/
L acylases at 45 ◦C, pH 8 and 500 rpm magnetic agitation.
In a perspective of process intensification and to evaluate the best
way to produce the C11’F at gram scale, different reactor configurations
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Process Biochemistry 99 (2020) 307–315
Table 1
effect of metal ion and the concentration of CoCl2 on the initial rate of the reaction synthesis of N-10-undecenoyl-phenylalaninein and the C11’F produced con-
centration after 48 h in 25 mM Tris-HCl buffer, NaCl 50 mM containing 100 mM of both substrates, 1 g/L acylases at 45 ◦C, pH 8 and 500 rpm magnetic agitation.
Metal ion
No metal ions
ZnSO4
CuCl2
CoCl2
Concentration (mM)
0.1
0.1
0.1
0.05
0.2
0.3
0.4
0.5
Initial rate (mM / h)
C11’F concentration (mM)
0.4 ± 0.08
11.8 ± 1.5
0.73 ± 0.17
12.1 ± 3
0.61 ± 0.05
11.5 ± 2.2
1.01 ± 0.26
17 ± 1.2
0.69 ± 0.18
15.7 ± 1.5
1.64 ± 0.1
17.7 ± 2.5
2.48 ± 0.7
22 ± 0.35
3.22 ± 0.6
20 ± 2.9
4.34 ± 0.4
17.2 ± 2.7
were evaluated. CoCl2 was not added in this part in order to avoid re-
sidual Co2+ in the final product. Intensified designed reactors were used
by different authors for enzymatic intensification allowing an
enhancement of the activity [30,31]. In this kind of reactors free or
immobilized enzymes can be used. The immobilization of the acylases
from S. ambofaciens was evaluated on several supports with different
characteristics (hydrophilic, hydrophobic and surface modified sup-
ports) and using different immobilisation technics (adsorption, covalent
and entrapment) however the aminoacylases lost almost all of their
activity (results not shown).
retentate forming a polarization layer, while the apolar characteristic
and the high quantity of the residual C11’ increased its elimination in
the permeate. However, after the elimination of around 64 % of the fatty
acid, the membrane pores became more accessible for the C11’F, leading
to a loss of the product. A compromise that allowed the partial elimi-
nation of the residual C11’ while limiting the loss of the C11’F was found
by applying two diafiltration steps and a concentration step. This led to
the recovery of a solution containing around 1.1 g of the product and
equivalent quantity of the fatty acid in addition to the aminoacylases
used. The proteins were eliminated by precipitation with methanol, and
the product was separated from the residual fatty acid by size exclusion
chromatography. After lyophilisation 0.8 g of a 99 % pure white powder
of enzymatically produced N-10-undecenoyl-phenylalanine was ob-
tained, as determined by mass spectroscopy and proton NMR analysis
(Fig. 8 B).
Different batch reactor configurations with different volume and
agitation module were evaluated in order to reach a gram scale of pure
enzymatic C11’F. As shown in Fig. 7 A, there is no influence of the
reactor configuration on the final product concentration when per-
forming the reaction in the same conditions. Indeed, the same final
concentration was obtained when passing from 3 mL batch reactor with
magnetic stirrer to the 300 mL batch reactor (Optimax) equipped with
Rushton mechanical stirrer. The reaction using Optimax reactor was
carried out seven days allowing to reach around 1.58 g of produced
C11’F with around 4.6 g residual fatty acid (Fig. 7 B). The final reaction
mixture was used for the purification procedure in order to obtain the
pure product.
3.8. Evaluation of the biological activity of the purified product
N-10-undecenoyl-phenylalanine was described in the literature as
having a lightening effect on the skin. This activity is due to an inhibition
of the production of melanin by melanocytes as antagonist to alpha-
melanocyte stimulating hormone (MSH) receptor [5]. The enzymatic
and the chemical N-10-undecenoyl-phenylalanine solutions were pre-
pared in ethanol and culture medium at different concentrations (10 and
3.7. Purification of the reaction product
50
μ
g/mL) for the bioactivity evaluation. As represented in Table 2,
g/mL was higher with enzymatic C11’F than
As detailed in the Materials and Methods section, the C11’F was
purified using filtration and chromatographic methods. The first step
consisting in an ultrafiltration on a 10 kDa allowed the selective passage
of the product of interest and the residual fatty acid (331 Da and 184 Da
respectively) while eliminating few impurities. As shown in Fig. 8 A, the
three first steps of diafiltration allowed the elimination of 64 % of the
fatty acid with only 15 % loss of the product from a sample of the initial
reaction medium composed of 300 mg of C11’ and 127 mg of C11’F.
However, when more diafiltration steps were performed a higher loss of
the C11’F was observed (Fig. 8.A). This partial separation of the C11’
and the C11’F can be due to the amphiphilic property of the acylated
amino-acid which can interact with the proteins standing in the
inhibitory activity at 50
μ
with the chemical one when it was solubilized in ethanol, while it was
less active when it was solubilized in the medium. This can be explained
by the solubility of the enzymatic C11’F (data not shown), which was
slightly lower than the chemical one in the medium. The lower solubility
of the enzymatic product in the medium might be due to the presence of
silica particles traces from the last chromatographic step or acetic acid
used to recover the product from the column. The acetic acid can in-
fluence the ionization of the product resulting in a precipitation of a part
of the product in unbuffered medium. By comparing cells viability in
those conditions, the enzymatic product prepared in medium showed
less toxicity than the chemical one unlike the enzymatic product
Fig. 7. scaling-up of the C11’F synthesis reaction in 25 mM Tris-HCl buffer, NaCl 50 mM containing 100 mM of both substrates, 1 g/L acylases at 45 ◦C, pH 8 and
500 rpm magnetic agitation A. influence of batch reactor configuration B. kinetic of the C11’F production and the C11’ consumption for the reaction performed in
OptiMax reactor (n = 2).
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Process Biochemistry 99 (2020) 307–315
produced C11’F showed lower cell toxicity than the chemical one, which
might be related to the lower solubility reducing the accessibility of the
product to the cells and to the MSH receptor resulting in less toxicity and
activity. At 10 μg/mL, according to statistical analysis, non-significant
difference was observed between enzymatic and chemical C11’F pre-
pared in ethanol and culture medium for the depigmentation activity,
due to low concentration. However, cell toxicity was slightly more
pronounced with enzymatic C11’F for no explained reason. Further
study is needed in order to explain the reason of the observed
differences.
4. Conclusion
The aim of this study was to evaluate the operational conditions
influencing the performances of the aminoacylases from S. ambofaciens
for the N-acylation of the L-phenylalanine with 10-undecenoic acid as
acyl donor. The results showed a positive effect of the phenylalanine
concentration on the aminoacylases activity, unlike what was observed
with the 10-undecenoic acid. Investigations on operational conditions
showed a good N-acylation activity at 45 ◦C, pH [7–8] but low tem-
perature stability. The inhibitory effect of the product on the conversion
rate was also demonstrated showing the interest of immobilization
aiming to use these aminoacylases in continuous flow reactors. The in-
fluence of the metal ions was also studied confirming that the amino-
acylases used are metallo-dependant and showing a very interesting
acceleration of the reaction by adding CoCl2. Different reactor config-
urations were evaluated but the batch reactor was the most suitable for
the synthesis of N-10-undecenoyl-phenylalanine without any effect of
the agitation mode. A purification procedure was developed leading to
0.8 g of N-10-undecenoyl-phenylalanine with more than 99% purity,
under the form of a white powder. The biological activity of the product
was tested on melanocyte cell cultures and showed a clear inhibition of
the melanin production. When significant, the difference in the light-
ening activity and/or the cell toxicity observed between the C11’F of
chemical origin or that issued from the enzymatic process was likely to
be due to different impurities remaining in the two products. These re-
sults represent a step forward towards the development of a green bio-
process using S. ambofaciens aminoacylases in aqueous media, which
could constitute a promising alternative to existing chemical processes
for the production and the purification of lipoaminoacids (i.e. AAS) as
biosurfactants.
Fig. 8. Details on C11’F purification process A. quantity of fatty acid and
product removed at different diafiltration steps using a 10 kDa cut-off mem-
brane and an initial solution of 25 mL of the final reaction mixture (300 mg of
C11’ and 127 mg of C11’F) B. proton NMR of the final purified product.
CRediT authorship contribution statement
Mohamed Chafik Bourkaib: Conceptualization, Investigation,
Writing - original draft. Stephane Delaunay: Conceptualization,
Methodology. Xavier Framboisier: Resources, Investigation. Cath-
erine Humeau: Investigation, Methodology, Writing - review & editing.
prepared in ethanol, which showed higher toxicity as compared to the
chemical one. Knowing that ethanol and the medium do not show any
effect on the cell growth, this effect is related to the presence of the
product. This can also be explained by the slight solubility difference of
the enzymatic and chemical C11’F. In ethanol, enzymatic C11’F showed
higher cell toxicity than the chemical one but much better lightening
activity due to its good solubility. In the medium, the enzymatically
´ ˆ
Jerome Guilbot: Funding acquisition, Validation. Cecile Bize:
Ressources, Investigation, Formal analysis, Writing - review & editing.
Estelle Illous: Project administration, Writing - review & editing. Isa-
belle Chevalot: Project Administration, Supervision, Writing - review &
editing, Validation. Yann Guiavarc’h: Funding acquisition, Project
Table 2
melanin production inhibition and cells viability evaluation.
Dilution solution
Concentration (
μg/mL)
Depigmentation* (%)
p-value T test
Cells viability** (%)
p-value T test
Chemical C11’F
Enzymatic C11’F
Chemical C11’F
Enzymatic C11’F
50
10
50
10
9
26
9
0.0002 (***)
0.2101 (NS)
0.0463 (*)
84
75
95
82
97
0.0001 (***)
0.0100 (*)
0.0018 (**)
0.0205 (*)
Ethanol
6
98
67
17
48
13
76
Culture medium
0.2514 (NS)
100
LS: limit of significance; 0.05 < p < 0.1; *0.01 < p < 0.05; **0.001 < p < 0.01 and ***p < 0.001, n = 6.
*
**
97% inhibition of melanin production by melanocytes using Kojic acid as positive control without any cell toxicity.
100% cell viability correspond to the result with the control culture of melanocyte (without any added molecules).
314

M.C. Bourkaib et al.
Process Biochemistry 99 (2020) 307–315
aminoacylase activity from Streptomyces ambofaciens catalyzes the acylation of
administration, Supervision, Formal analysis, Writing - review & edit-
lysine on α-position and peptides on N-terminal position, Eng. Life Sci. 18 (2017)
ing, Validation.
589–599, https://doi.org/10.1002/elsc.201700173.
[14] C. Bernal, F. Guzman, A. Illanes, L. Wilson, Selective and eco-friendly synthesis of
lipoaminoacid-based surfactants for food, using immobilized lipase and protease
biocatalysts, Food Chem. 239 (2018) 189–195, https://doi.org/10.1016/j.
foodchem.2017.06.105.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgement
[15] Y. Takakura, Y. Asano, Purification, characterization, and gene cloning of a novel
aminoacylase from Burkholderia sp. strain LP5_18B that efficiently catalyzes the
synthesis of N-lauroyl-l-amino acids, Biosci. Biotechnol. Biochem. 83 (2019)
1964–1973, https://doi.org/10.1080/09168451.2019.1630255.
[16] P. Villeneuve, Lipases in lipophilization reactions, Biotechnol. Adv. 25 (2007)
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This work was mainly supported by the French National Research
Agency (ANR) through the “ISEAPIM3” project (ANR-15-CE07-0023-
01), and partly by the “Impact biomolecules” project of the « Lorraine
[17] R. Valivety, P. Jauregi, I. Gill, E. Vulfson, Chemo-enzymatic synthesis of amino
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10.1007/s11746-997-0232-8.
´
Universite d’Excellence (LUE) » (PIA-ANR) program. We also thank
[18] A. Ferjancic-Biagini, T. Giardina, M. Reynier, A. Puigserver, Hog Kidney and
Intestine Aminoacylase-Catalyzed Acylation of L-Methionine in Aqueous Media,
Biocatal. Biotransformation. 15 (1997) 313–323, https://doi.org/10.3109/
10242429709003197.
Emilien GIROT from LRGP for giving us access to the Optimax reactor
and for his valuable advices.
´
[19] S.R. Morcelle, C.S. Liggieri, M.A. Bruno, N. Priolo, P. Clapes, Screening of plant
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Streptomyces mobaraensis, Biosci. Biotechnol. Biochem. 69 (2005) 1914–1922,
https://doi.org/10.1271/bbb.69.1914.
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