Optimized synthesis of novel prenyl ferulate performed by feruloyl esterases from Myceliophthora thermophila in microemulsions

Article abstract of DOI:10.1007/s00253-017-8089-8

Five feruloyl esterases (FAEs; EC 3.1.1.73), FaeA1, FaeA2, FaeB1, and FaeB2 from Myceliophthora thermophila C1 and MtFae1a from M. thermophila ATCC 42464, were tested for their ability to catalyze the transesterification of vinyl ferulate (VFA) with prenol in detergentless microemulsions. Reaction conditions were optimized investigating parameters such as the medium composition, the substrate concentration, the enzyme load, the pH, the temperature, and agitation. FaeB2 offered the highest transesterification yield (71.5?±?0.2%) after 24?h of incubation at 30?°C using 60?mM VFA, 1?M prenol, and 0.02?mg FAE/mL in a mixture comprising of 53.4:43.4:3.2?v/v/v n-hexane:t-butanol:100?mM MOPS-NaOH, pH 6.0. At these conditions, the competitive side hydrolysis of VFA was 4.7-fold minimized. The ability of prenyl ferulate (PFA) and its corresponding ferulic acid (FA) to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was significant and similar (IC₅₀ 423.39?μM for PFA, 329.9?μM for FA). PFA was not cytotoxic at 0.8–100?μM (IC₅₀ 220.23?μM) and reduced intracellular reactive oxygen species (ROS) in human skin fibroblasts at concentrations ranging between 4 and 20?μM as determined with the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay.

Full text of DOI:10.1007/s00253-017-8089-8

Appl Microbiol Biotechnol
DOI 10.1007/s00253-017-8089-8
BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Optimized synthesis of novel prenyl ferulate performed
by feruloyl esterases from Myceliophthora
thermophila in microemulsions
Io Antonopoulou1 & Laura Leonov2 & Peter Jütten3 & Gabriella Cerullo4
&
Vincenza Faraco4 & Adamantia Papadopoulou5 & Dimitris Kletsas5 & Marianna Ralli6
&
Ulrika Rova1 & Paul Christakopoulos1
Received: 2 November 2016 /Revised: 13 December 2016 /Accepted: 21 December 2016
#
The Author(s) 2017. This article is published with open access at Springerlink.com
Abstract Five feruloyl esterases (FAEs; EC 3.1.1.73),
FaeA1, FaeA2, FaeB1, and FaeB2 from Myceliophthora
thermophila C1 and MtFae1a from M. thermophila ATCC
42464, were tested for their ability to catalyze the
transesterification of vinyl ferulate (VFA) with prenol in
detergentless microemulsions. Reaction conditions were opti-
mized investigating parameters such as the medium composi-
tion, the substrate concentration, the enzyme load, the pH, the
temperature, and agitation. FaeB2 offered the highest
transesterification yield (71.5 0.2%) after 24 h of incubation
at 30 °C using 60 mM VFA, 1 M prenol, and 0.02 mg FAE/
mL in a mixture comprising of 53.4:43.4:3.2 v/v/v n-hexane:t-
butanol:100 mM MOPS-NaOH, pH 6.0. At these conditions,
the competitive side hydrolysis of VFA was 4.7-fold mini-
mized. The ability of prenyl ferulate (PFA) and its correspond-
ing ferulic acid (FA) to scavenge 1,1-diphenyl-2-
picrylhydrazyl (DPPH) radicals was significant and similar
(IC50 423.39 μM for PFA, 329.9 μM for FA). PFA was not
cytotoxic at 0.8–100 μM (IC50 220.23 μM) and reduced in-
tracellular reactive oxygen species (ROS) in human skin fi-
broblasts at concentrations ranging between 4 and 20 μM as
determined with the dichloro-dihydro-fluorescein diacetate
(DCFH-DA) assay.
.
.
Keywords Feruloyl esterase Transesterification
.
.
.
Myceliophthora thermophila Prenyl ferulate Antioxidant
DPPH DCFH-DA
.
Introduction
Ferulic acid (FA), along with other hydroxycinnamic acids (p-
coumaric, caffeic, sinapic), has a widespread industrial poten-
tial due to its strong antioxidant activity. It is ubiquitous in
nature as a component of plant cell walls offering linkage with
lignin; in arabinoxylans, FA is esterified to the C-5 of α-L-
arabinofuranose; in pectins, it is esterified to the C-2 of α1➔5-
linked arabinofuranose or to the C-6 of β1➔4-linked
galactopyranose, while in xyloglucans, it is found attached
to the C-4 of α-D-xylopyranose (Kikugawa et al. 2012). FA
is present in grains, fruits, and vegetables (Zhao and
Moghadasian 2008). It has a broad spectrum of attractive bi-
ological properties including UVabsorptive, antibacterial, an-
tiviral, antiinflammatory, antithrombosis, and antitumor ef-
fects, while during the recent years, it is revealed that it may
have beneficial effects against Alzheimer’s disease (Huang
et al. 1988; Graf 1992; Kanski et al. 2002; Suzuki et al.
2002; Ou and Kwok 2004; Sultana et al. 2005; Vafiadi et al.
2008a; Barone et al. 2009). It is also suggested to suppress
melanin generation by antagonizing tyrosine, which makes it
a potential skin-whitening agent (Briganti et al. 2003; Chandel
* Paul Christakopoulos
paul.christakopoulos@ltu.se
1
Division of Chemical Engineering, Department of Civil,
Environmental and Natural Resources Engineering, Luleå University
of Technology, 97187 Luleå, Sweden
2
DuPont Industrial Biosciences, Nieuwe Kanaal 7-S, 6709
PA Wageningen, The Netherlands
3
Taros Chemicals GmbH & Co. KG, Emil Figge Str 76a,
44227 Dortmund, Germany
4
Department of Chemical Sciences, University of Naples BFederico
II^, Via Cintia 4, 80126 Naples, Italy
5
Institute of Biosciences and Applications NCSR BDemokritos,^
Laboratory of Cell Proliferation and Aging, T. Patriarchou Grigoriou
& Neapoleos, 15310 Athens, Greece
6
Korres Natural Products, 57 Km National Road,
32011 Lamia, Athens, Greece

Appl Microbiol Biotechnol
et al. 2011). Nevertheless, a major disadvantage of FA and
other natural antioxidants is their poor solubility in both oil
and aqueous media limiting their application in formulations
intended for food, cosmetic, cosmeceutical, or pharmaceutical
products.
StFae-C from Sporotrichum thermophile ATCC 34628,
FoFae-I from Fusarium oxysporum, AnFaeA from A. niger,
multienzymatic preparations such as Ultraflo L or Depol
740 L from Humicola insolens, and Depol 670 L from
Trichoderma reesei resulting in varying yields (3–97%)
(Topakas et al. 2003, 2004, 2005; Vafiadi et al. 2008a, b).
The same reaction reached a conversion up to 90% when
performed in a single solvent system (1-butanol:buffer) by
immobilized Depol 740 L (Thörn et al. 2011). FAEs are gen-
erally less stable in non-conventional media and low water
content than lipases, whereas they are more selective having
higher substrate specificity (Zeuner et al. 2011).
(Trans)esterification can be carried out by lipases only if the
aromatic ring is not para-hydroxylated and the lateral chain is
saturated. Thus, enzymatic (trans)esterification of
hydroxycinnamoyl substrates can be obtained efficiently only
by FAEs (Vafiadi et al. 2008a).
Myceliophthora thermophila (previously known as
S. thermophile) is a thermophilic filamentous fungus that ex-
presses a powerful consortium of enzymes able to break down
lignocellulosic biomass (Karnaouri et al. 2014; Kolbusz et al.
2014). Its genome, which was entirely sequenced and anno-
tated in 2011, encodes over 200 secreted carbohydrate-active
enzymes (CAZy) and other enzymes of industrial interest
(Berka et al. 2011). M. thermophila was developed into a
mature protein production platform named C1. The main fea-
tures of C1 include a low-viscosity morphology and high pro-
duction levels (up to 100 g/L protein) in fed-batch fermenta-
tions providing thus an alternative to traditional fungal protein
production hosts for cost-effective industrial applications
(Visser et al. 2011). The genome of M. thermophila possesses
six genes encoding enzymes belonging to the CE1 family of
the CAZy database; four of which are FAEs (Hinz et al. 2009;
Karnaouri et al. 2014). Three FAEs (FaeA1, FaeA2, and
FaeB2) have been over-expressed in M. thermophila C1 and
characterized (Kühnel et al. 2012). Sharing the same primary
sequence with FaeB2, the type B FAE from M. thermophila
ATCC 42464 (MtFae1a) has been heterologously expressed in
Pichia pastoris and characterized (Topakas et al. 2012).
In the present study, we synthesized a novel feruloylated
derivative, namely, prenyl ferulate (PFA), by investigating
different reaction parameters (medium composition, substrate
concentration, enzyme concentration, pH, temperature, time,
and agitation) in relation with the rate, the yield, and the prod-
uct selectivity. Transesterification was performed using vinyl
ferulate (VFA) as activated donor and prenol as acceptor,
while a competitive side reaction of hydrolysis was observed
with presence of water (Fig. 1). Prenol (3-methyl-2-buten-1-
ol) is a natural occurring alcohol found in citrus fruits, berries,
hops, tomato, grapes, passion fruit, and coffee, where it serves
as a building block for terpenoids. In industry, it is used as a
fragrance ingredient due to its fruity odor (Kabera et al. 2014).
Polyprenols are abundant in wood and needles, such as pine
A common way to alter solubility is by esterification or
transesterification, with the latter requiring a prior activation
of FA into an esterified derivative. Modification with sugars or
glycerol results to more hydrophilic derivatives, whereas
modification with fatty compounds results to more lipophilic
products. Additionally to solubility, lipophilization has been
shown to enhance the antioxidant activity of alkyl ferulate
derivatives (Vafiadi et al. 2008b). Classic methods of esterifi-
cation involve use of strong acids (concentrated sulfuric acid,
hydrogen chloride) or expensive and toxic reagents as cata-
lysts (boron trifluoride, aluminum chloride, trifluoroacetic an-
hydride, polyphosphate ester, neodymium oxide,
dicyclohexylcarbodiimide, graphite bisulfate, etc.), high tem-
peratures (150–250 °C), long reaction times, low yields, and
tedious operations (Li et al. 2009). Process limitations include
the heat sensitivity and oxidation susceptibility of FA; safety
concerns for human health and the environment; and the high-
energy consumption for purification, deodorization, and
bleaching due to low selectivity (Kiran and Divakar 2001).
The requirement for greener processes and the consumers’
preference for natural products demand the development of
biotechnological sustainable and competitive processes for the
production of interesting compounds with biological activities
such as antioxidants. Enzymatic (trans)esterification is an at-
tractive alternative as it offers mild conditions, use of greener
solvents, and high selectivity.
During the past 15 years, the potential of feruloyl esterases
(FAEs; EC 3.1.1.73) as biosynthetic tools has been
underlined. FAEs represent a subclass of carboxylic acid es-
terases that are generally known to catalyze the hydrolysis of
the ester bond between hydroxycinnamic acids and sugars as
accessory plant cell wall-degrading enzymes. Based on their
specificity towards monoferulates and diferulates, for substi-
tutions on the phenolic ring and on their amino acid sequence
identity, they have been classified into four types (A–D)
(Crepin et al. 2004). The reported FAE-based modifications
of hydroxycinnamic acids and their esters include their
(trans)esterification with primary alcohols, e.g., 1-butanol,
glycerol, or sugars in non-conventional media such as
microemulsions of organic solvents characterized by low wa-
ter content, solvent-free systems where the substrates function
as reaction medium or single organic solvents. Specifically,
lipophilization of FA has been performed by esterification
with 1-pentanol using a type A FAE from Aspergillus niger
in water-in-oil microemulsions (Giuliani et al. 2001).
Transesterification of methyl ferulate (MFA) with 1-butanol
has been reported in detergentless microemulsions of n-hex-
ane:1-butanol:buffer using various FAEs such as StFae-A and

Appl Microbiol Biotechnol
(Kühnel et al. 2012; Visser et al. 2011). The C1-production
strains were cultured aerobically in 2-L fermentors in a medi-
um containing glucose, ammonium sulfate, and trace min-
erals. Protein production was done in a fed-batch system ac-
cording to Verdoes et al. (2010). After fermentation, the
enzyme-containing broth was separated from the biomass by
centrifugation (15,000×g for 1 h at 4 °C) and filtration. The
crude enzyme supernatant was concentrated eightfold, dia-
lyzed against 10 mM potassium phosphate (pH 6.5), and
was further lyophilized. MtFae1a from M. thermophila
ATCC 42464 was recombinantly expressed in P. pastoris
strain X33 as reported previously (Topakas et al. 2012).
Samples of the culture broth were withdrawn after 3 days of
incubation in 250-mL flasks at 20 °C; the biomass was re-
moved by centrifugation (7000×g for 30 min at 4 °C); and
the supernatant was 100-fold concentrated and dialyzed
against 100 mM MOPS-NaOH, pH 6.0 using a tangential flow
filtration system (10-kDa cutoff; PALL, NY, USA).
Biochemical properties of the used FAEs are presented in
Table 1.
Fig. 1 Scheme of a transesterification of VFA (donor) with prenol
(acceptor). b Hydrolysis of VFA (natural reaction when water is
present, observed as side reaction during transesterification)
and birch. They have attractive pharmacological effects that
are based on their substitutive effect in the case of dolichol
deficits, which are observed with chronic inflammatory, de-
generative, and oncological diseases (Khidyrova and
Shakhidoyatov 2002). The use of prenol as acceptor offers
synthesis of a highly lipophilic derivative utilizing substrates
of natural origin. In the same time, the use of novel FAEs
derived from the thermophilic fungus M. thermophila offers
an insight into their potential for efficient transesterification.
These FAEs have been proved to be excellent hydrolytic en-
zymes; however, this is the first time that they are evaluated
for their transesterification efficiency.
Enzyme and protein assays
Protein concentration was determined by the Pierce™ BCA
Protein Assay (ThermoFisher Scientific, Waltham, USA).
The FAE content (w/w) of the enzymatic preparations was
determined by SDS-PAGE using a Novex Sharp pre-stained
protein standard (ThermoFisher Scientific, Waltham, USA),
followed by quantification using the JustTLC software
(Sweday, Lund, Sweden). For the assessment of hydrolytic
activity, a stock solution of substrate was prepared in dimethyl
sulfoxide (DMSO). The activity was assayed using 1 mM
MFA or PFA in 100 mM MOPS-NaOH, pH 6.0 and
0.005 mg FAE/mL enzyme load. Samples were incubated
for 10 min at 45 °C without agitation. Reaction was ended
by incubating the reaction mixtures at 100 °C for 5–10 min.
All reactions were performed in duplicate and concomitant
with appropriate blanks. One unit (1 U) is defined as the
amount of enzyme (mg) releasing 1 μmol of FA per minute
under the defined conditions. No substrate consumption was
observed in the absence of esterase.
Materials and methods
Materials
VFA was prepared in 52% overall yield after three steps
starting from FA. The procedure was modified from
Mastihubova and Mastihuba (2013), offering >95% purity
1
after column chromatography on silica gel based on H
NMR and LC-MS. Methyl ferulate (MFA) was purchased
from Alfa Aesar (Karlsruhe, Germany), while FA, prenol
(99%), n-hexane (<0.02% water), t-butanol (anhydrous,
≥99.5%), MOPS solution 1 M, and other materials were pur-
chased from Sigma-Aldrich (Saint Louis, USA).
Transesterification reactions
Transesterification reactions were performed in a ternary
system of n-hexane:t-butanol:buffer forming detergentless
microemulsions. Reactions were prepared by diluting ad-
equate amount of donor in the mixture of n-hexane and t-
butanol, followed by the addition of prenol and by vigor-
ous shaking. Reaction was initiated by introducing the
enzyme in the form of concentrated stock solution in buff-
er, followed by vigorous shaking until a stable one-phase
solution was obtained. Transesterification was carried out
Enzymes
Feruloyl esterases FaeA1, FaeA2, FaeB1, and FaeB2 from
M. thermophila C1 were over-expressed individually in low-
background C1-expression hosts as reported previously

Appl Microbiol Biotechnol
in sealed glass vials at microscale (500 μL) in a
temperature-controlled water bath. Medium composition,
VFA concentration, prenol concentration, enzyme concen-
tration, pH, and temperature were optimized. The effect of
medium composition was studied at systems chosen ac-
cording to previous reports (Topakas et al. 2005; Vafiadi
et al. 2006b, 2008b) based on the phase diagram by
Khmelnitsky et al. (1988). The effect of pH was studied
using the following buffers at 100 mM concentration: so-
dium acetate (pH 4–6), MOPS-NaOH (pH 6–8), and Tris-
HCl (pH 8–10). Optimal conditions obtained from each
study were applied in subsequent experiments. Unless
otherwise stated, reactions were performed at fixed con-
ditions (50 mM VFA, 200 mM prenol, 0.02 mg FAE/mL
FaeA1, FaeA2, 0.002 mg FAE/mL FaeB1, FaeB2,
0.04 mg FAE/mL MtFae1a, 40 °C, 100 mM MOPS-
NaOH, pH 6.0, 8 h of incubation). Experiments that in-
cluded agitation were performed in an Eppendorf
thermomixer (Eppendorf, Hamburg, Germany). All reac-
tions were performed in duplicate and concomitant with
appropriate blanks. No donor consumption (<1%) was
observed in the absence of esterase.
Quantitative analysis
Analysis was performed by HPLC on a 100–5 C18 Nucleosil
column (250 × 4.6 mm) (Macherey Nagel, Düren, Germany).
Reaction mixtures were diluted with acetonitrile before anal-
ysis. Elution was done with 7:3 v/v acetonitrile:water for
10 min at a flow rate of 0.6 mL/min and room temperature.
Absorbance was measured at 300 nm with a PerkinElmer
Flexar UV/Vis detector (Waltham, USA). Retention times
for FA, MFA, VFA, and PFA were 4.5, 6.1, 7.4, and
8.7 min, respectively. Calibration curves were prepared using
standard solutions of feruloyl compounds in acetonitrile (0.1–
2 mM). The sum of molar amounts of the donor and products
at the end of reaction was always within a 5% error margin
compared to the starting molar amount of the donor. The
transesterification yield (or PFA yield) was calculated as the
molar amounts of generated PFA compared to the initial
amount of donor, expressed as a percentage. The overall yield
was calculated as the molar amounts of PFA and FA compared
to the initial amount of donor, expressed as percentage.
Product selectivity was defined by the PFA/FA ratio (the mo-
lar concentration of produced PFA divided by the molar con-
centration of produced FA).
Isolation of products and enzyme recovery
At the optimum conditions and after the reaction had been
completed, the reaction mixture was diluted fivefold with
buffer (100 mM MOPS-NaOH, pH 6.0), followed by an
equal volume of n-hexane. After vigorous handshaking,

Appl Microbiol Biotechnol
two liquid layers were produced. The upper layer (organ-
ic, containing the lipophilic compounds) was separated
and evaporated under vacuum, whereas the lower phase
(aqueous, containing the hydrophilic compounds and the
enzyme) was collected, buffer exchanged, and subse-
quently analyzed for activity. All obtained fractions were
analyzed by HPLC.
Results
Effect of medium composition
Transesterification was tested in four different compositions
of the ternary system n-hexane:t-butanol:100 mM MOPS-
NaOH, pH 6.0 monitoring the competitive hydrolysis of
VFA as side reaction. Systems offering highest PFA concen-
tration, rate, and yield were considered as optimal, system IV
(19.8:74.7:5.5 v/v/v) for FaeA1 and FaeA2, system III
(47.2:50.8:2.0 v/v/v) for FaeB1, and system II
(53.4:43.4:3.2 v/v/v) for FaeB2 and MtFae1a (Fig. 2a).
Generally, the highest PFA/FA molar ratio was achieved by
FaeB2, while it was <1 for all tested FAEs and systems (0.095
for FaeA1, 0.034 for FaeA2, 0.301 for FaeB1, 0.513 for
FaeB2, and 0.297 for MtFae1a at optimum conditions).
Product selectivity was highest at lowest water content (sys-
tem III, 2% water) for all tested FAEs; however, it was shown
that such low water content was detrimental to the yield (ex-
cept in the case of FaeB1). FaeB2 offered highest rate
(0.321 mol PFA/g FAE L h), followed by FaeB1
(0.187 mol PFA/g FAE L h).
Structural characterization of prenyl ferulate
NMR spectroscopy was performed in DMSO-d6 with a
Bruker Ascend Eon WB 400 spectrometer (Bruker BioSpin
AG, Fällanden, Switzerland), 1H NMR (DMSO-d6,
400 MHz):δ 9.59 (s, 1H, ArOH), 7.53 (d, 1H, J = 16 Hz,
−CHCHCOOR), 7.32 (d, 1H, J = 4 Hz, ArH), 7.11 (dd, 1H,
J1 = 2 Hz, J2 = 8.4 Hz, ArH), 6.79 (d, 1H, J = 8.4 Hz, ArH),
6.46 (d, 1H, J = 16 Hz, −CHCHCOOR), 5.39–5.34 (m, 1H,
−OCH2CHC<), 4.63 (d, 2H, J = 7.2 Hz, −OCH2CHC<), 3.81
(s, 3H, −OCH3), and 1.72 (dd, 6H, J1 = 1.2 Hz, J2 = 14 Hz,
−CHC(CH3)2).
Antioxidant activity and cytotoxicity
The antioxidant activity of synthesized PFA as well as of
its corresponding FA was monitored by the reduction in
the optical density of 2,2-diphenyl-1-picrylhydrazyl
(DPPH) radical. Serial dilutions of the test compounds
were mixed with an equal volume of 1 mM DPPH in
ethanol in U-bottomed 96-well plates and kept in the
dark and at ambient temperature until measurement of
absorbance at 520 nm at various time points. Human
skin fibroblasts (HSFs; strain AG01523) were purchased
from Coriell Institute for Medical Research (Camden, NJ,
USA). Cells were cultured in monolayers and the possi-
ble cytotoxicity of PFA and FA was estimated by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay as previously described
(Guldbrandsen et al. 2015). The antioxidant activity,
expressed as the capacity to reduce the intracellular
levels of reactive oxygen species (ROS), was assessed
using the 2′,7′-dichlorofluorescein diacetate (DCFH-DA)
assay. When HSFs were confluent, the medium was
changed to serum-free Dulbecco’s modified Eagle medi-
um (SF DMEM), and 18 h later, it was aspirated and
renewed with phenol red and SF DMEM along with
10 μM of DCFH-DA. Following incubation with
DCFH-DA for 1 h, serial dilutions of the test compounds
were added and the fluorescence was measured in differ-
ent time intervals for 480-nm excitation and 530-nm
emission. The antioxidant activity of the compounds
was visualized as reduction of DCF fluorescence and
expressed as percent of control. Each experiment was
conducted in triplicates.
Effect of VFA concentration
Under the optimal medium composition for each enzyme, the
effect of VFA concentration on the transesterification was ex-
amined. Product selectivity was not affected by the variation
in donor concentration (coefficient of variance ~2%), while
higher concentrations resulted in higher rate but lower yield
(Fig. 2b). The concentration of VFA that resulted in highest
PFA concentration and rate was used as optimal donor con-
centration in subsequent experiments for each enzyme
(80 mM for FaeA1, 50 mM for FaeA2 and FaeB1, 60 mM
for FaeB2, and 100 mM for MtFae1a). Highest rate was
0.346 mol PFA/g FAE L h for FaeB2, followed by
0.183 mol PFA/g FAE L h for FaeB1 at optimal conditions.
Effect of prenol concentration
The rate and yield increased remarkably by increasing the
acceptor concentration. FaeB2 transesterified at highest rate
(0.412 mol/g FAE L h) when 1 M prenol was used, followed
by FaeB1 at 0.8 M prenol (0.246 mol/g FAE L h) (Fig. 3a).
Optimal prenol concentration was implemented in subsequent
experiments for each enzyme (1 M for FaeA1, FaeA2, and
FaeB2 and 0.8 M for FaeB1 and 0.6 M for MtFae1a).
Increase in prenol concentration remarkably increased
transesterification against the side hydrolytic reaction, as more
prenol molecules were available near the interface between
the organic and water phase of the microemulsion, allowing
more frequent transesterification instead of hydrolysis
(Fig. 3b). Highest PFA/FA ratio (>1) was observed when

Appl Microbiol Biotechnol
Fig. 2 a Effect of medium composition on the rate. Reactions were
performed in n-hexane:t-butanol:100 mM MOPS-NaOH using 50 mM
VFA, 200 mM prenol at 40 °C, pH 6.0 for 8 h. Black system I
(37.8:57.2:5.0 v/v/v), gray system II (53.4:43.4:3.2 v/v/v), striped
system III (47.2:50.8:2.0 v/v/v), and white system IV (19.8:74.7:5.5 v/v/
v). b Effect of VFA concentration on the rate. Reactions were performed
at optimal medium composition for each enzyme, 200 mM prenol, 40 °C,
pH 6.0 for 8 h. FaeA1 (black circle), FaeA2 (white circle), FaeB1 (black
square), FaeB2 (white square), and MtFae1a (black rectangle) (rate is
expressed as ×5 for FaeA1, FaeA2, and MtFae1a)
FaeB2 was used, followed by MtFae1a. Nevertheless, higher
product selectivity is generally observed at acceptor concen-
trations higher than 1.5 M, where rates are generally reduced.
For instance, in the case of FaeB2, highest rate and yield were
observed at 1 M prenol, but highest PFA/FA ratio was found at
1.5 M, where the overall yield decreased by 23.8%. On the
contrary, MtFae1a seemed to have higher tolerance for prenol
as the rate was practically constant between 0.5 and 2 M,
although optimal performance was observed at only 0.6 M
prenol.
and was most efficient catalyst for both substrates (highest Km/
kcat). MtFae1a had lower affinity for VFA and was approxi-
mately 300 times less efficient catalyst comparing to FaeB2.
Type A FAEs had the lowest affinity towards prenol, showing
low efficiency in the synthesis of PFA (Fig. 3).
Effect of enzyme concentration
In most industrial applications, cost of enzyme production and
operational costs are usually the limiting factors.
Improvement on the yield and reduction of reaction time occur
when the enzyme concentration increases, leading to a reduc-
tion of operation costs; however, the process cost in terms of
enzyme production increases significantly, too. With the addi-
tion of more enzyme, the rate (mol/g FAE L h) decreased
exponentially (data not shown), but the PFA yield reached a
peak at different enzyme concentration for each tested FAE
(Fig. 4a). FaeB2 was able to synthesize PFA at highest yield
(32.5%) and rate (0.122 mol/g FAE L h) at minimal enzyme
load of 0.02 mg FAE/mL. FaeA1, FaeB1, and MtFae1a
achieved similar PFA yields (~21%) at optimal FAE concen-
tration of 0.1 mg/mL for FaeA1 and FaeB1 and 0.2 mg/mL for
Enzyme kinetics
The apparent kinetic constants for each substrate were deter-
mined by fitting data regarding the effect of VFA and prenol
concentration under standard reaction conditions (40 °C, pH
6.0) on the Michaelis-Menten equation using nonlinear re-
gression (p < 0.0001) (Table 2). FaeB1 had highest affinity
towards VFA and prenol (lowest Km), while FaeB2 had sim-
ilar affinity with FaeB1 towards VFA and lower affinity to-
wards prenol comparing to FaeB1 and MtFae1a. However,
FaeB2 catalyzed fastest the transesterification (highest vmax
)
Fig. 3 Effect of prenol
concentration on the a rate and b
product selectivity. Reactions
were performed at optimal
medium composition and VFA
concentration for each enzyme,
40 °C, pH 6.0 for 8 h. FaeA1
(black circle), FaeA2 (white
circle), FaeB1 (black square),
FaeB2 (white square), and
MtFae1a (black rectangle) (rate is
expressed as ×5 for FaeA1,
FaeA2, and MtFae1a in a)

Appl Microbiol Biotechnol
MtFae1a. Regarding product selectivity, only FaeB2 offered a
ratio >1, while MtFae1a and FaeB1 achieved a value of
0.938 at optimal conditions (Fig. 4b). In low enzyme concen-
trations, an increase substantially affected the PFA/FA ratio, as
more enzyme molecules were available near the interface of
the organic and aqueous phase being able to catalyze the
transesterification. However, for most FAEs, the yield and
product selectivity stabilized or decreased over 0.1–
0.2 mg FAE/mL. Optimal conditions were implemented in
subsequent experiments.
Effect of pH
Although the water content in the reaction is low, the pH of the
aqueous solution may influence the ionization state of the
residues of the enzymes’ active site. Rate, yield, and product
selectivity were observed in transesterification reactions per-
formed at pH range of 4–10. Environment that offered highest
PFA concentration, yield, and rate was considered as optimal
and proved to be 100 mM MOPS-NaOH buffer at pH 6.0 for
FaeB2 and FaeA2, pH 7.0 for FaeB1, and pH 8.0 for FaeA1
and MtFae1a. In these conditions, the PFA/FA ratio was equal
to 0.692 for FaeA1, 0.295 for FaeA2, 1.119 for FaeB1, 2.298
for FaeB2, and 1.670 for MtFae1a. Product selectivity was
affected by pH, as highest PFA/FA ratio was observed at pH
4.0 for FaeA2, pH 5.0 for FaeB1, pH 6.0 for FaeB2, and pH
8.0 for FaeA1 and MtFae1a. However, in the cases of FaeA2
and FaeB1, low pH was detrimental to the yield.
Effect of temperature
Transesterification was monitored at different temperatures
(25–60 °C) with respect to time (Fig. 5). Among all, FaeB2
had the highest PFA yield (71.5%), rate (0.211 mol PFA/
g FAE L h), and product selectivity (2.373) at 30 °C after
24 h of incubation, achieving a 100% conversion of VFA to
products. FaeA1 and FaeA2 appeared to be more thermophilic
with optimal performance at 55 and 45 °C, respectively.
Interestingly, although both FaeB2 and MtFae1a performed
optimally at 30 °C, FaeB2 appeared to inactivate at 35 °C,
while MtFae1a had the same profile as at 30 °C during
transesterification. At optimal temperature, FaeA1, FaeB1,
and MtFae1a reached a PFA yield of 40–48% out of 93.9,
83.1, and 63.2% overall yield after 24 h, respectively.
Lowest yield was demonstrated by FaeA2 15.2% after 48 h
out of 82.2% overall yield. A summary on the obtained pa-
rameters for each enzyme is presented in Table 3.
Effect of other donors and agitation
The effect of different donors and agitation was studied for
96 h at optimal conditions using FaeB2 (Fig. 6). As expected,
VFA was a more reactive donor, as the by-product vinyl

Appl Microbiol Biotechnol
Fig. 4 Effect of enzyme
concentration on the a yield and b
product selectivity. Reactions
were performed at the optimum
medium composition and
substrate concentration for each
enzyme, 40 °C, pH 6.0 for 8 h.
FaeA1 (black circle), FaeA2
(white circle), FaeB1 (black
square), FaeB2 (white square),
and MtFae1a (black rectangle)
alcohol tautomerizes to acetaldehyde, shifting the equilibrium
towards transesterification. Agitation offered higher selectivi-
ty towards synthesis during the first 2 h of incubation probably
due to the better contact of the biocatalyst with substrates and
better mass and heat transfer. At these conditions, the PFA/FA
ratio decreased with respect to time, while it was observed that
it is stabilized at ~2.36 after 48 h independently of agitation.
The same value is reached when MFA was used as donor,
Fig. 5 Effect of temperature on
the yield. Reactions were
performed at optimal medium
composition, substrate
concentration, enzyme
concentration, and pH for each
enzyme. a FaeA1, b FaeA2, c
FaeB1, d FaeB2, and e MtFae1a

Appl Microbiol Biotechnol
Table 3 Summary of optimal conditions and obtained parameters
Enzyme
FaeA1
FaeA2
FaeB1
FaeB2
MtFae1a
Optimized conditionsa
Water content (%)
5.5
80
1
5.5
50
1
2.0
50
0.8
0.1
7
3.2
60
1
3.2
100
0.6
0.2
8
VFA concentration (mM)
Prenol concentration (M)
Enzyme concentration (g FAE/L)
pH
0.1
8
0.1
6
0.02
6
Temperature (°C)
55
24
45
48
40
24
30
24
30
24
Time (h)
Obtained parameters
PFA concentration (mM)
PFA yield (%)
32.9 (2.4)
7.6 (0.3)
24.1 (0.2)
42.9 (0.1)
42.7 (2.0)
41.1 (3.0)
15.2 (0.5)
48.1 (0.4)
71.5 (0.2)
42.7 (2.0)
Overall yield (%)
93.9 (5.4)
82.2 (0.5)
83.1 (3.0)
102.0 (4.7)
0.089 (0.000)
0.182 (0.008)
2.373 (0.362)
63.2 (1.6)
Rate (mol PFA/g FAE L h)
Initial rate (mol PFA/g FAE L h)
PFA/FA ratio
0.014 (0.001)
0.053 (0.003)
0.778 (0.021)
0.0016 (0.0001)
0.0057 (0.001)
0.227 (0.011)
0.010 (0.000)
0.030 (0.002)
1.378 (0.093)
0.0089 (0.0004)
0.022 (0.004)
1.700 (0.041)
Numbers in the parentheses are the estimates of standard errors
a Concentrations are expressed as in total volume of reaction (500 μL)
revealing that the synthetic specificity of FaeB2 is not affected
significantly by the substitution on the feruloyl donor.
Regarding the yield, VFAwas fully converted to products fast
(71.5% PFA after 24 h), while the reaction appeared much
slower (25.8% PFA after 72 h) when MFA was used. Direct
esterification of FA was negligible (1.4 after 96 h).
and methods). FA was collected after buffer exchange, while
FaeB2 maintained its initial specific activity by 83%. In that
context, detergentless microemulsions are proved to be an
attractive system with environmentally friendly prospects as
the high lipophilicity of PFA combined with the total conver-
sion of VFA allows product separation with low energy costs.
Moreover, the recovery and potential reusability of FaeB2
could reduce the cost of enzyme production.
Isolation of products and recovery of enzyme at optimal
conditions
Antioxidant activity and cytotoxicity
At the end of 24 h, the reaction mixture (500 μL) was sepa-
rated into two phases. The upper organic phase contained only
PFA, as VFA was converted completely (100%) by FaeB2,
while the lower aqueous phase contained FA and the enzyme.
The organic phase was evaporated, and PFA was recovered
successfully (98.5%, 5.542 mg) and was subjected to identifi-
cation by 1H NMR (chemicals shifts are reported in materials
The free radical scavenging activity of synthesized PFA as
well as of its corresponding FA was investigated with the
DPPH cell-free assay. Both compounds have significant scav-
enging activity, while FAwas found slightly more potent. The
half maximal inhibitory concentration (IC50), estimated after
3 h of incubation, was 329.9 μM for FA and 423.39 μM for
Fig. 6 Effects of different donors
and agitation on the a yield and b
product selectivity. Reactions
were performed by FaeB2 at
optimal conditions. VFA (black
circle), VFA and agitation (white
circle) (1000 rpm), MFA (black
square), and FA (white square)

Appl Microbiol Biotechnol
PFA. Our observations are in accordance with previous stud-
ies reporting the scavenging ability of FA and its related esters
(Kikuzaki et al. 2002). Following, the study of the antioxidant
activity was aimed in a cell system based on HSFs. To this
end, cytotoxic activity was determined by the MTT assay.
Data indicate that both PFA and FA at concentrations ranging
from 0.8 to 100 μM do not affect HSFs’ viability. At higher
concentrations, PFA displayed a cytotoxic action (IC50
220.23 μM), while FA was non-cytotoxic even at 500 μM,
the highest concentration tested. These results are in agree-
ment with previous reports on the effect of FA on the viability
of human skin fibroblasts (Cos et al. 2002) and human em-
bryonic kidney 293 (HEK293) cells (Bian et al. 2015). The
DCFH-DA assay was employed at non-cytotoxic concentra-
tions in HSFs. As shown in Fig. 7, FA reduced significantly
the intracellular levels of ROS in a concentration-dependent
manner, in accordance with previous data from a murine mac-
rophage cell line (Nadal et al. 2016). PFA also provoked a
reduction of intracellular ROS at concentrations ranging from
4 to 20 μM, while at 100 μM, no significant antioxidant effect
was observed.
these enzymes. Subsequently, they showed low affinity to-
wards lipophilic prenol comparing to type B FAEs and proved
to be less efficient biocatalysts for the synthesis of PFA.
Interestingly, each type B FAE demonstrated similar turnover
rates (kcat) for both substrates, while each type A FAE had
higher turnover rate for prenol comparing to VFA (Table 2).
After optimization, the PFA/FA ratio was <1 only for FaeA1
and FaeA2 (0.782 and 0.239, respectively), while they appear
to be more thermophilic enzymes with optimal temperature of
55 and 45 °C. On the contrary, type B FAEs are more
mesophilic.
The synthetic activity and potential of tested FAEs are not
directly correlated with their hydrolytic activity. All FAEs
showed specific hydrolytic activities of the same magnitude
(Table 1), while the transesterification potential of type B
FAEs performed in detergentless microemulsions was gener-
ally twofold to threefold higher than the one of type A. This
substantial difference could be attributed to structural differ-
ences. In most lipases, a mobile hydrophobic lid covers the
substrate-binding site, and in this closed structure, the lipase is
assumed to be inactive. Upon activation of the lipase by con-
tact with a hydrophobic solvent or at a hydrophobic interface,
the lid opens offering accessibility to substrate binding (Rehm
et al. 2010). Although very few structures are available for
FAEs, it is proved that the catalytic triad of the type A
AnFaeA and the active site cavity is confined by a lid and a
loop that confers plasticity to the substrate-binding site in
analogy with lipases (Hermoso et al. 2004). What is surprising
is the fact that FAE’s lid exhibited a high ratio of hydrophilic
residues, keeping it in an open conformation that gives the
typical preference of catalyzing the hydrolysis of hydrophilic
substrates compared to lipases. According to this, the low
product selectivity (PFA/FA ratio) of type A FAEs and the
preference for environments with higher water content could
be attributed to the presence of a higher percentage of hydro-
philic amino acids in their lid comparing to the type B FAEs.
Glycosylation can affect the transesterification along with
the enzyme properties. Among tested enzymes, FaeB2 and
MtFae1a share the same primary sequence, while the latter
Discussion
In this work, we demonstrated the potential of five FAEs de-
rived from M. thermophila to synthesize the novel compound
prenyl ferulate. Among them, FaeB2 proved to be the most
efficient biocatalyst with attractive properties including high
product selectivity (2.373), high yield (71.5% PFA), high
overall yield (100%), low enzyme load (0.02 mg FAE/mL),
reduced reaction time (24 h), and low operation temperature
(30 °C). Results on the effect of solvent composition revealed
that type A FAEs (FaeA1 and FaeA2) prefer an environment
with higher water content (5.5%; system IV) than type B
FAEs. Moreover, it was observed that in every optimization
step, the product selectivity (PFA/FA ratio) was quite low for
type A FAEs, without meaning that they are not active overall
since the side hydrolysis was observed to be very robust for
Fig. 7 Antioxidant activity of a
PFA and b FA determined with
the DCFH-DA assay. Black 0.5 h,
light gray 1 h, striped 3 h, white
6 h, and dark gray 24 h

Appl Microbiol Biotechnol
has undergone glycosylation resulting to an observed MW of
39 kDa, instead of 33 kDa. The difference of the post-
translational modification in their respective production hosts
results in two biocatalysts with common origin but different
performance. Specifically, both FAEs showed preference in
the same media composition and water content (3.2%), while
MtFae1a had 2.6-fold lower affinity towards VFA, is required
at a 10-fold higher concentration in the reaction mixture, and
operates optimally at alkaline pH. Interestingly, its affinity
towards prenol is slightly higher (1.22-fold), and it appears
more stable at very high acceptor concentrations (1.5–3 M).
Finally, although both enzymes perform optimally at 30 °C,
FaeB2 appears to inactivate at 35 °C, while MtFae1a has the
same profile as at 30 °C. Overall, glycosylation offered in-
creased stability regarding prenol, pH, and temperature but
influenced negatively the process in terms of efficiency and
cost, as the transesterification was characterized by lower sub-
strate affinity for VFA, lower biocatalytic efficiency (260–
560-fold decrease), limited yields (1.7-fold), and product se-
lectivity (1.4-fold decrease).
In transesterification reactions catalyzed by FAEs, only the
use of methyl activated donors is reported (Antonopoulou
et al. 2016). In this work, for the first time, a vinyl donor
was used in a FAE-catalyzed reaction. The use of vinyl acti-
vated donors is a common practice as they are more reactive
offering high rates and reduced reaction times. Moreover, un-
der normal conditions, the by-product vinyl alcohol
tautomerizes to acetaldehyde and is easier to be removed com-
paring to methanol when MFA is used as donor. FaeB2 was
the only enzyme that fully converted VFA to products after
24 h of incubation, while other enzymes such as FaeA1
reached very high overall yield (93.9%) at given time. As
VFA and the transesterification product PFA have high and
similar lipophilicity, a total conversion of donor relieves the
process from laborious purification steps. On the other hand, it
is evident that when MFA is used as donor, only 25.8% are
converted to PFA after 72 h, indicating that separation, recov-
ery, and reuse of substrate are essential additionally to in-
creased operational costs (Fig. 6). There are numerous reports
on the use of vinyl donors in lipase-catalyzed reactions
(Chigorimbo-Murefu et al. 2009; Yang et al. 2010; Yu et al.
2010; Schär and Nyström 2016). Recently, a cutinase from
F. oxysporum catalyzed the acylation of tyrosol using various
vinyl esters with good yields (up to 60.7%) (Nikolaivits et al.
2016). However, the cost of the synthesis of an activated do-
nor such as VFA is an issue to be addressed.
esterification of FA with glycerol yielded 81%, while esterifica-
tion of p-coumaric acid yielded approximately 60% after 72 h
using the same enzyme (Tsuchiyama et al. 2006, 2007). The
transesterification of methyl sinapate with 1-butanol using
AnFaeA resulted to 78% conversion after 120 h (Vafiadi et al.
2008b). All aforementioned modifications though were
employed in systems where the acceptor is used in very high
concentrations as solvent component. Although high concentra-
tions of prenol lead to increased product selectivity, a limiting
factor in the reaction is the inactivation of FaeB2 and other
enzymes at concentrations higher than 1 M. Given this, a
71.5% yield of PFA after optimization is quite promising.
Detergentless microemulsions are a reaction system used in
the vast majority of synthetic reactions based on FAEs as they
do not affect enzyme activity (Topakas et al. 2003, 2004,
2005; Vafiadi et al. 2005, 2006a, b, 2008a, b; Couto et al.
2010, 2011). They form in ternary systems consisting of a
hydrocarbon, a polar alcohol, and water representing thermo-
dynamically stable and optically transparent dispersions of
aqueous microdroplets in the hydrocarbon solvent. The drop-
lets are stabilized by alcohol molecules adsorbed at their sur-
face and possess spherical symmetry (Khmelnitsky et al.
1988). In the present reaction, we propose that the enzyme is
enclosed and protected in the microdroplet, while the lipophil-
ic donor VFA is present in the organic phase (comprising of n-
hexane, t-butanol, and prenol). Microdroplets are stabilized by
t-butanol, while the acceptor prenol (logP equal to 0.91) is
mostly present in the organic phase as it is less polar than t-
butanol (log P equal to 0.584). Given that, the target reaction
takes places in the interface of the microdroplet when the
enzyme gets in contact with VFA and prenol leading to
transesterification. The side reaction of hydrolysis occurs
when the enzyme gets in contact with VFA and water mole-
cules. We propose that the transesterification product (PFA) is
transferred in the organic phase immediately after its produc-
tion due it its increased lipophilicity, which subsequently pro-
tects it from further hydrolysis. This is the main reason why
this reaction keeps moving towards synthesis until equilibri-
um is reached.
An ideal solvent should offer attractive characteristics to
the process such as low toxicity, easy product recovery,
aiding substrate solubility, aiding the targeted reaction, and
not affecting enzyme activity (Wei et al. 2003). The
transesterification of VFA with prenol in detergentless
microemulsions offers easy product separation and recovery,
recycling of solvents, enzyme recovery, and reuse. Shifting
the physicochemical equilibrium of the microemulsion and
causing the formation of two separate phases, the lipophilic
product PFA is encountered in the upper organic phase, while
the hydrophilic FA and enzyme are found in the lower aque-
ous phase. Enzyme can be recovered and reused by ultrafil-
tration, while PFA and FA can be recovered by vacuum evap-
oration from their respective phase. A drawback of the
Using VFA as donor and at optimal conditions, the obtained
yield is comparable with other reports on the synthesis of lipo-
philic feruloyl or other hydroxycinnamic acid derivatives cata-
lyzed by FAEs. Very high yield (95% after 12 h) has been
reported regarding the synthesis of diglycerol ferulates catalyzed
by a FAE from A. niger (purified from the commercial prepara-
tion Pectinase PL BAmano^) (Kikugawa et al. 2012). The

Appl Microbiol Biotechnol
esterase biocatalysts for cost-effective industrial production
(OPTIBIOCAT)^ grant agreement no. 613868, co-funded within the
FP7 Knowledge Based Bio-Economy (KBBE). Dr. Shubhankar
Bhattacharyya (Luleå University of Technology, Luleå, Sweden) is ac-
knowledged for his substantial help with the NMR measurements and
product characterization.
composition of microemulsions is the lower solubility of VFA
at room temperature, while they appear to enable a robust
hydrolytic side reaction additionally to the target reaction.
Nevertheless, the presence of water is essential for FAEs as
they tend to be inactivate in pure hydrophobic media contrary
to lipases. In enzymatic (trans)esterification, an amount of
donor inevitably will get hydrolyzed due to the presence of
water constituting product separation and purification
essential.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
A methodological dilemma in transesterification is whether
as optimum parameter should be considered the one that offers
higher concentration of desired product (or yield or rate) or the
one that offers highest product selectivity. In the present opti-
mization study, conditions that offered highest concentration
of PFA were chosen as optimal, as parameter values that of-
fered highest product selectivity (PFA/FA ratio) in most cases
were detrimental to the yield. For instance, it was observed
that lower water content (2%) offered higher PFA/FA ratio but
decreased the rate and yield up to 50% for all FAEs except for
FaeB1. Donor concentration did not affect product selectivity,
probably because the presence of more VFA molecules close
to the interface of the microemulsions has the same chance for
transesterification and hydrolysis. Prenol concentration and
enzyme load were factors that affected product selectivity
the most. When prenol concentration is increased, more mol-
ecules are available near the interface of the organic and water
phase of the microemulsion, allowing more frequent
transesterification instead of hydrolysis. However, very high
prenol concentration (>1 M) seemed to cause enzyme inacti-
vation. In the case of enzyme concentration, optimal values
for yield and product selectivity coincided. Finally, pH values
offering high PFA/FA ratio resulted in lower yields for FaeA2
and FaeB1, while temperature, time, and agitation did not
affect significantly the selectivity. Although FA production
could be further minimized in an optimization study focused
on highest selectivity, still some hydrolysis would be observed
due to the inevitable presence of water sustaining the need of a
purification step. In the same time, higher enzyme loads
would be needed increasing the cost. Using FaeB2 at optimal
conditions, hydrolysis was reduced 4.7-fold compared to the
initial conditions. Moreover, detergentless microemulsions of-
fer easy separation between PFA and FA, allowing the utiliza-
tion of the latter for its beneficial effects as food additive,
dietary supplement, etc.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appro-
priate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
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