Synthetic application and activity of cutinase in an aqueous, miniemulsion model system: Hexyl octanoate synthesis

Article abstract of DOI:10.1016/j.cattod.2011.05.039

Cutinase has been shown to be a promising biocatalyst for applications in processes targeted for industrial scale. This work aims to further contribute for highlighting such role, by bestowing detailed insight on the application of cutinase for the synthesis of value added compounds in miniemulsion environments (oil-in-water). The synthesis of hexyl octanoate was used as model system, due to the relevance of this compound for industrial applications as flavor and fragrance agent. The nature, specificity and selectivity of the catalyst enable operation under mild reaction conditions, without undesirable side reactions, leading to a very pure product. A high conversion yield, about 86%, for an enzyme concentration of 5 mg ml^-1, was achieved. Hexyl octanoate synthesis and cutinase activity for different acid: alcohol molar ratio (R), under different pH environments, were evaluated. A maximum cutinase activity of 2.20 μmol mg^-1 min^-1 was observed for R = 0.5, which was tentatively ascribed to the stabilizing effect of hexanol on the miniemulsion. It is thus considered that the accumulation of the hydrophobic hexyl octanoate inside the droplets stabilize the miniemulsion system.

Full text of DOI:10.1016/j.cattod.2011.05.039

Catalysis Today 173 (2011) 95–102
Contents lists available at ScienceDirect
Catalysis Today
journal homepage: www.elsevier.com/locate/cattod
Synthetic application and activity of cutinase in an aqueous, miniemulsion model
system: Hexyl octanoate synthesis
a,b,∗
, Pedro Fernandesa,b, Joaquim M.S. Cabrala,b, Luís P. Fonsecaa,b
Dragana P.C. de Barros
a
Department of Bioengineering, Instituto Superior Técnico (IST), Av. Rovisco Pais, 1049-001 Lisboa, Portugal
IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, IST, Lisboa, Portugal
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Cutinase has been shown to be a promising biocatalyst for applications in processes targeted for industrial
scale. This work aims to further contribute for highlighting such role, by bestowing detailed insight on
the application of cutinase for the synthesis of value added compounds in miniemulsion environments
Received 23 December 2010
Received in revised form 5 April 2011
Accepted 11 May 2011
(oil-in-water). The synthesis of hexyl octanoate was used as model system, due to the relevance of this
Available online 5 July 2011
compound for industrial applications as flavor and fragrance agent. The nature, specificity and selectivity
of the catalyst enable operation under mild reaction conditions, without undesirable side reactions, lead-
Keywords:
Cutinase
Miniemulsion
Hexyl octanoate
Activity
−
1
ing to a very pure product. A high conversion yield, about 86%, for an enzyme concentration of 5 mg ml
was achieved.
,
Hexyl octanoate synthesis and cutinase activity for different acid: alcohol molar ratio (R), under dif-
−
1
−1
ferent pH environments, were evaluated. A maximum cutinase activity of 2.20 ␮mol mg min was
observed for R = 0.5, which was tentatively ascribed to the stabilizing effect of hexanol on the miniemul-
sion. It is thus considered that the accumulation of the hydrophobic hexyl octanoate inside the droplets
stabilize the miniemulsion system.
©
2011 Elsevier B.V. All rights reserved.
microbial processes, the whole clearly favors the use of biocatalysts
5,6]. Furthermore, biological production processes have a better
1
. Introduction
[
public acceptance, particularly when targeted for products used
as ingredients or in formulations related to personal- and health-
care and food and feed sectors [7]. Major applications of enzymes
are in medicine, textile industry, and food and beverage industry.
Recently enzyme technology has also become prominent in the
development of analytic aids [8]. Esterases and lipases, particularly
the later, are among the enzymes that are widely used in industri-
ally relevant processes, namely when esterification reactions are
aimed at [9–12]. The use of cutinases for such applications presents
an interesting option, since they do not require interfacial activa-
tion, unlike classical lipases [13]. Moreover, cutinase may display
activity over a substrate irrespectively of its being in highly aggre-
gated or soluble states, which are otherwise typical behaviors of
lipases and esterases, respectively, cutinase being therefore able
to hydrolyze soluble esters as well as emulsified triacylglycerols
Low- and medium-chain molecular esters constitute a signifi-
cant part of aroma compounds. Many of such esters contribute to
the fruity nature of flavors or are used as emollients in the cosmetic
industry [1].
Biocatalysis may provide advantageous alternatives to purely
chemical routes for industrial processes, among them those involv-
ing synthesis of esters [2–4]. The use of enzymatic processes for
ester synthesis have cost-effective, industrially appealing features,
such as the mild reaction conditions, along with the high selectivity
and specificity, which enable the synthesis of flavor molecules with
high quality and purity. Moreover, and although from the chemist
point of view, there is no difference between a compound synthe-
sized in nature and the identical molecule produced by chemical
synthesis, there is a consistently growing trend towards the intro-
duction of biological and environmentally friendly processes for
the production of a wide plethora of molecules. Together with
strict US and Europeans regulation, that define that “natural” flavor
substances can only be prepared either by physical, enzymatic or
[
14,15]. Cutinases can thus be considered an intermediate between
those other two enzymes [16]. Besides, cutinases have been shown
to display stability and activity under adverse conditions, viz. pres-
ence of organic solvents, ionic liquids, detergents, proteases and
oxidizing agents, and their activity in hydrolysis, esterification and
transesterification has been highlighted in recent years. Recently,
the thermal stability of cutinases in aqueous solution has actu-
ally been considered higher than that of any lipase [17,18]. When
considering ester synthesis, cutinases present a sound alternative
to lipases, since the use of the later can be hampered by their
∗ Corresponding author at: Institute for Biotechnology and Bioengineering, Center
for Biological and Chemical Engineering, Depertment of Bioengineering, Instituto
Superior Técnico, Av. Rovisco Pais, 1049-001 Lisbon, Portugal.
E-mail addresses: dragana@ist.utl.pt, draganabarros@gmail.com
D.P.C. de Barros).
(
0
920-5861/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.cattod.2011.05.039

9
6
D.P.C. de Barros et al. / Catalysis Today 173 (2011) 95–102
relatively large size and relatively low stability under industrial
process conditions [16]. Given these features, it is therefore under-
standable that cutinases find application in several areas, among
them fine chemicals and pharmaceuticals, laundry, detergents and
personal care products, food industry, textiles, polymer chemistry
and in pesticide and insecticide degradation [16,18]. The catalytic
triad Ser-120, Asp-175, His-188, is accessible to the solvent and
presents catalytic activity with different substrates [16]. Particu-
lar attention has been recently given to the use of cutinases in
The catalytic activity of cutinase for ester synthesis in a
miniemulsion system was uncovered in a previous work [50]. This
prior study shows the high potential and advantages of using
water-based miniemulsions as a green reaction environment for
the biosynthesis of ethyl- and specially hexyl acid esters (acid chain
length from C6 to C18) by lipases and Fusarium solani pisi cutinase.
On the aftermath of these promising results, the present work
aims to evaluate the effect of the concentration of the substrates
on enzyme activity and stability of the reaction media. The focus
was on structural and catalytic aspects of cutinase in oil-in-water
miniemulsion. Given the conformational change of cutinase during
the time course of the reaction, it was possible to establish the effect
of the reaction media on enzyme activity.
(
trans)esterification reactions of fat or oils in low water activity
environments [17–19].
Several reports on the use of cutinases in different reaction
media, often dissolved in aqueous solution, but also suspended as
a powder or in immobilized form, have been published [17–20].
Immobilization has been commonly achieved by adsorption onto
solid supports [21,22]; and encapsulation in reverse micelles
of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) [23–26], phos-
phatidylcholine [27], or cetyltrimethylammonium bromide (CTAB)
The synthesis of hexyl octanoate was used as model system,
since this ester is a flavor compound incorporated in a wide range
of aromas such as apple, banana, cider, grape and melon [52].
2. Experimental
[
28]. Lyophilized cutinase was used in fundamental studies on
the hydrolysis of triglycerides [29]; transesterification reactions
30–32], esterification reactions [33–35]; and in the elucidation
2.1. Catalyst
[
2.1.1. Production of cutinase
of the reaction mechanisms of cutinase related to the stereo-
selectivity and specificity of this enzyme [36,37].
F. solani pisi cutinase wild-type was biosynthesized by recom-
binant Saccharomyces cerevisiae SU50 strain as described by Calado
et al. [53]. The cutinase producing S. cerevisiae SU50 strain (Mata,
leu2–3, ura3, gal1: URA3, MAL-8, MAL3, SUC3) contains the expres-
sion vector pUR7320 constructed and provided by the Unilever
Research Laboratory, Vlaardingen, The Netherlands. The strain was
Water-in-oil microemulsions, or reverse micelles, are often used
for bioconversion processes, involving either cutinases or lipases
[
38]. The application of microemulsions in several chemical and
industrial processes is due to key properties of these systems, such
as the very low interfacial tension, the large interfacial area and
the thermodynamic stability [39]. When organic synthesis is con-
sidered, organic solvents used as reaction media account for about
◦
stored at −80 C in frozen tubes containing 50% (v/v) selective
medium and 50% (v/v) of glycerol (Merck, Dannstadt, Germany).
The inoculum (0.4 l) was cultivated in cotton-stopped shake flasks
8
0% of mass utilization in typical pharmaceutical and fine chemical
◦
(
4 × 1l) at 30 C and 200 rpm in an orbital shaker (Agitorb 160E;
processes [40]. Many organic solvents used within such scope are
toxic and/or volatile. In the scale-up of industrial processes, the use
of organic solvents may therefore raise safety and environmental
concerns.
Aralab, Lisboa, Portugal) in a selective medium (medium lack-
ing leucine) composed of 20 gl d(+)-glucose anhydrous (Merck,
−
1
−
1
Germany), 6.7 gl
of yeast nitrogen base without amino acids
(
Difco, Sparks, MD, USA) and 20 mg l 1 l-histidine (Merck) until a
−
Water as reaction medium is, on the other hand, an environ-
mentally compatible solvent and since most biological processes
and enzymatic reactions take place in water, implementation of
synthetic biocatalytic processes in aqueous media would be clearly
advantageous [41–43]. Oil-in-water emulsions, with droplets in
the size range of about 200 nm, often referred to in the lit-
erature as miniemulsions, have been investigated, especially as
nanoreactors for polymerization [44]. Recently aqueous miniemul-
sion systems have been shown to provide promising media for
enzymatic reactions catalyzed by lipase such as enzymatic poly-
merization [45,46], preparation of optical active organic compound
−
1
biomass concentration between 1.1 and 1.8 gdcw
l
was attained.
This concentration was established based on a correlation curve
relating optical density at 600 nm and cell dry weight, determined
previously. This inoculum was transferred into the cultivation
media for enzyme biosynthesis, and corresponded to 10% (v/v) of
the working volume. These cultivations were performed in a 5 l
bioreactor (Biostat MD; B. Braun, Melsungen, Germany) containing
4
l working volume adjusted at pH 6.0 (by automatic addition of
NaOH or HCl solutions, both at 2 N). The fermentation for biocata-
◦
lyst production was performed at 30 C, with a minimum dissolved
−
1
oxygen tension of 30%. A constant air flow rate of 4.4 l min (equiv-
alent to 1.1 vvm) was established during the cultivation.
[
47,48], sterification [49], and by cutinase [50], for the synthesis of
alkyl esters.
Aqueous miniemulsions (oil-in-water) are two phase systems
where small droplets (organic phase) with high stability are dis-
persed in a continuous phase (water), by using high shear forces
originated by ultrasonication. The water phase contributes with
about 80% (w/w). The oil phase is composed mostly of the sub-
strates (16.5%, w/w), a hydrophobic agent for osmotic stabilization
of the droplets, and a surfactant for colloidal stability [44]. The
droplets are of uniform size and stabilized against coagulation by
the presence of ionic or nonionic surfactant, and against diffusion
degradation (Ostwald–Ripening) by the addition of a co-stabilizing,
extremely hydrophobic, agent (ex. hexadecane). The water pro-
duced in the dehydrative esterification [51] is expelled from the
continuous aqueous phase, favoring product formation. The other
very important factors that favor the formation of esters are the
enormous interfacial area, readily available for interfacial cataly-
sis, and the stability of the miniemulsion throughout the reaction,
which is increased with increasing hydrophobicity of the oil phase
2.1.2. Purification and lyophilization of cutinase
The isolation and purification of cutinase excreted by the recom-
binant S. cerevisiae SU50 strain was carried out by expanded bed
adsorption (EBA) [54]. Frontal adsorption experiments were carried
out in an EBA column Streamline 25, with a settled bed adsor-
bent height of 15 cm and the column top adjusted to 45 cm. The
cation adsorbent Streamline SP XL (Amersham Pharmacia Biotech,
Sweden) was used to isolate cutinase directly from the fermenta-
tion broth. The adsorbent was washed and equilibrated previously
with 20 mM citrate buffer pH 4.5. During the expanded bed oper-
ation, and in order to stabilize bed expansion, a volumetric flux of
−
1
270 cm h was maintained. Once the bed expansion was stabilized
and the adsorbent equilibrated with the buffer, the fermentation
broth (previously diluted with water 4:1), was loaded through
the EBA column. After frontal feedstock adsorption, the bed was
washed until the effluent was devoided of yeast cells. The elution
was carried out in packed bed mode in downward flow, using a
[
49,50].

D.P.C. de Barros et al. / Catalysis Today 173 (2011) 95–102
97
Table 1
The protein concentration was determined by the method of
Pierce (BCA, bicinchoninic acid, assay) from Thermo Scientific
(Rockford, USA) with reference to Bovine Serum Albumin (BSA)
Relative amounts of the compounds in miniemulsion systems.
Miniemulsion (ME) Water (%) Substrates (%) Hexadecane (%) Lutensol (%)
(Merck) as standard solution [56].
ME 80% (w/v)
81.2
16.5
0.7
1.6
Specific activities of lyophilized cutinase preparations were
calculated as the ratio between estereolytic activity and protein
−
1
constant volumetric flux of 122 cm h−1 with the starting buffer con-
taining 150 mM NaCl. The pH of all effluent fractions was corrected
with NaOH (1 M) to pH 6.0–7.0, the optimal values for enzyme sta-
bility. Cutinase activity and protein concentration of the collected
effluent fractions from the column were determined after each run,
as described elsewhere [55].
concentration (170 U mg ).
2.2. Enzymatic esterification reaction in miniemulsion system
Substrates, hexadecane, water and Lutensol, that make up the
esterification reaction media, were mixed in different relative
amounts, w/w (Table 1), and thoroughly mixed by magnetic stir-
ring for 1 h. The two phase system was ultrasonicated for 120 s
(total sonication time) in pulses of 10 s and pauses of 5 s, at 65%
amplitude (SONOPULS, Bandelin, Berlin, Germany) with ice cool-
ing. Unless otherwise stated, a typical esterification reaction (Fig. 1)
was carried out in 20 ml rubber-capped flasks with 10 ml of work-
ing volume, using a solution with an equimolar concentration of
the substrates (0.650 M), and taking into account the phase rela-
tionship as depicted in Table 1. 10 ml of the miniemulsion were
added into the reaction vessel containing the appropriate amount
of enzyme.
The pool of elution fractions exhibiting the highest cutinase
activity were firstly dialyzed against 20 mM phosphate buffer pH
◦
7
.0 and then frozen at −80 C and lyophilized overnight (Christ
Alpha 2-4 lyophilizer, B.BRAUN Biotech, Melsungen, Germany).
Lyophilized pure cutinase was stored at −20 C before use in the
◦
esterification reactions.
2.1.3. Characterization of the cutinase preparations
The esterolytic activity of cutinase and the protein content were
established, thus allowing the characterization of the lyophilized
cutinase preparations [55,56].
The esterolytic activity of cutinase was assayed using a spec-
trophotometric method based on monitoring the hydrolysis of
p-nitrophenylbutyrate (p-NPB) to nitrophenol (p-NP), a yellow
compound easily quantified by absorbance at 400 nm [55]. Briefly,
The esterification reactions were performed in a thermostated
incubator (Advanced ChemTec PLS 4 × 4, Louisville, Kentucky, USA,
◦
400 rpm magnetic stirring) at 40 C unless otherwise stated. This
setup enabled parallel experiments by the simultaneous use of 5
reactors against at least one blank without enzyme.
Samples were withdrawn periodically (100 ␮l) using a needle
(as to preserve the rubber cap) and then mixed with methanol
(quenching agent) prior to HPLC analysis.
2
0 ␮l samples of cutinase solution were added to 980 ␮l of a reac-
tion mixture containing 0.56 mM p-NPB, 11.3 mM sodium cholate
and 0.43 M tetrahydrofuran, in 50 mM potassium phosphate buffer
pH 7.0.
One unit of cutinase estereolytic activity was defined as the
amount of enzyme required to convert 1 ␮mol of p-NPB to p-NP
per minute, at 30 C and pH 7.
The miniemulsion systems and experiments were performed in
duplicate or triplicate and the results are the respective average
values.
◦
Fig. 1. Principle of an esterification reaction in miniemulsion.

9
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D.P.C. de Barros et al. / Catalysis Today 173 (2011) 95–102
2.3. Determination of the esterification yield in the miniemulsion
system
The reaction mixture was analyzed by reverse-phase liquid
chromatography (HLPC) using LiChroCART 250-4 (Purospher®
®
RP-18 column, VWR, Germany). The mobile phase was composed
−
1
of acetonitrile/water (90:10, v/v) at a flow rate of 0.8 ml min
.
Detection was performed with a UV detector at 220 nm. The con-
centrations of octanoic acid and hexyl octanoate were established
using as reference a calibration curve.
The concentration of hexanol, was determined by GC, in a
Hewlett–Packard model 5890 gas chromatograph, equipped with a
flame ionization detector (FID) and a WCOT Fused Silica coating CP
Chirasil-Dex CB column, 25 m × 0.25 mm, DF = 0.25 (Varian Inc.).
The reaction yield was calculated according to the molar ratio
between the hexyl ester and respective limiting substrate, alcohol
or acid.
Fig. 2. The effect of the lyophilized cutinase concentration on the synthesis of hexyl
octanoate. Reaction conditions: acid/alcohol molar ratio R = 1; T = 40 C.
◦
media so that enzyme concentrations ranging from 1 to 10 mg ml 1
were obtained, for equimolar substrates concentration of 0.65 M.
The amount of enzyme present in the bioconversion medium
clearly influenced the esterification yield and the reaction rate
−
The rate of ester synthesis, V, was calculated as the amount of
ester formed per mg of protein and per min from aliquots collected
−
1
−1
in the first hour of reaction (␮mol of ester mg prot min ).
(
Fig. 2). The esterification yield increased from 64%, for an enzyme
2
.4. Fluorescence measurements
−
1
concentration of 1 mg ml , to 86%, for an enzyme concentration of
5
influence on ester yield (85% for 10 mg ml ). On the other hand,
the reaction rate increased consistently within the range of enzyme
concentrations evaluated, up to 0.250 Mh
roughly linear manner.
Given that above 2.5 mg ml enzyme concentration there is no
significant increase in the esterification yield, and considering that
the amount of enzyme required is often a critical feature when over-
all production process costs are considered, further experiments
−
1
mg ml . A further increase of enzyme concentration showed no
Samples (1 ml) were collected periodically from the reaction
−
1
mixture using a needle, and centrifuged for 60 min at 14,000 rpm at
4
phase was used for fluorescence analysis. 100 ␮l of a sample was
added to 900 ␮l of distilled water and fluorescence measurement
was done using fluorescence cuvettes.
◦
C (Centrifuge 5417R, Eppendorf) for phase separation. The water
−
1
−1
for 10 mg ml , in a
−
1
Fluorescence measurements were performed by excitation at
80 nm (excitation of both tryptophan and tyrosine residues),
2
and emission was collected within 290–450 nm, with excitation
end emission slits set at 5 nm. Readings were performed on a
Cary Eclipse fluorescence spectrophotometer (Varian Inc.) with 90
geometry, equipped with a thermostated cuvette holder set up at
3
−
1
were performed with 2.5 mg ml enzyme concentration.
◦
3
.2. Effect of substrate molar ratio on ester synthesis
◦
0 C and an agitation device. The instrumental response at each
As it is well known, in order to shift the equilibrium of a reaction
wavelength was automatically corrected using in-built procedures,
according to the manufacturer.
towards the product side, one of the reactants can be applied in
excess.
However, increasing the amount of alcohol or acid above a
certain concentration could result in an inhibitory effect on the
enzyme. As an outcome, lower reaction rates/final product yields
are foreseen.
Without changing the relative amounts (Table 1), acid/alcohol
molar ratio (R) was increased from 0.1 to 10 (Fig. 3). The indi-
vidual effect of the acid and alcohol concentration on enzyme
activity was studied starting with a small concentration of the acid
Cutinase spectra were established by subtracting a baseline
obtained with a miniemulsion solution prepared in the same con-
ditions without the enzyme. The spectrum obtained of both Trp
and Tyr residues was normalized relatively to the fluorescence
intensity at the ꢀmax (wavelength where the maximum intensity
occurs).
2.5. Dynamic light scattering (DLS)
(
R = 0.1). By increasing R from 0.1 to 0.5, the initial rate of reaction
The micellar size was measured by DLS (Nano-Zetasizer,
−1
−1
increases and reaches a maximum value of 2.20 ␮mol min mg
◦
◦
Malvern Instruments) at 20 C under a scattering angle of 173 at a
wavelength of 633 nm. The samples were added to a glass cuvette
before the measurement. Particle sizes and PDIs are given as the
average of three measurements. PDI (dimensionless) is a measure
of the particle size distribution, displaying the heterogeneity of the
sample. As such, it can range from 0 (monodisperse) to 1 (polydis-
perse). The Z-average diameter is the mean diameter based on the
intensity of scattered light.
3
. Results and discussion
3.1. Effect of the lyophilized enzyme concentration on ester
synthesis
To establish the optimal amount of catalyst for carrying out
the targeted esterification, different quantities of lyophilized cuti-
nase were used. The lyophilized cutinase was added to the reaction
Fig. 3. The effect of the acid/alcohol molar ratio (R) on the synthesis of hexyl
octanoate by cutinase.

D.P.C. de Barros et al. / Catalysis Today 173 (2011) 95–102
99
at R = 0.5. For R = 1 (equimolar concentration of substrates) the ini-
−
1
−1
tial rate of reaction decreases to 1.31 ␮mol min mg . Further
increase of R showed further deleterious effect on the enzyme
activity.
When the final ester yield is addressed, a slight but steady
increase is observed from R = 0.1 to R = 1, where a 76.4% yield is
achieved.
With an excess of alcohol (R = 0.4), the initial rate of the reac-
tion was the highest, however a lower final ester yield was
obtained. Nevertheless the increasing acid concentration from
R = 1 up to R = 10 decreases the activity and ester yield. This inhi-
bition effect of acids was previously reported for CTAB/reverse
micelles as well as for organic solvents [28,57], and was
ascribed to the polar acid microenvironment surrounding the
enzyme surface. The inhibition effect of the alcohol is smaller,
however it could be observed, (Fig. 3), until R = 1. When address-
ing the esterification of nonanoic acid with 3-phenylpropanol,
Aschenbrenner et al. [49] also observed a similar profile, since
reactions with either an excess of acid or an equimolar sub-
strate ratio showed lower reaction rates than reactions with an
excess of alcohol. One reason for the enhanced activity in the
presence of an excess of hexanol may be a stabilizing effect
of hexanol on cutinase, reported before for reverse micellar
systems [28].
Fig. 4. Normalized spectra of cutinase in miniemulsion at R = 1 (acid/alcohol molar
ratio).
co-surfactant, increasing the interface flexibility and interdroplet
interaction [60].
On the other hand, with the increase of octanoic acid concen-
tration (R = 2.5, 5 and 10), the red shift was almost immediate,
suggesting a faster denaturation of the cutinase.
In both cases the fluorescence results are in good agreement
with the time course of the reactions, since the conversion of the
substrates roughly stops coincidently with the occurrence of the
red shift (data not shown). For R = 2.5, the red shift is noticeable at
t = 30 min, but for higher concentration of octanoic acid, the enzyme
no longer presents its active form after 10 min. These correlate
neatly with the very low esterification yield and activity of cuti-
nase observed for R values from 2.5 to 10 (Fig. 3), and could be due
to the low pH of the system, since the optimum pH for cutinase
was found to be 7–8 [25]. Hence further work was performed to
assess the effect of the variation of pH (ꢁpH), resulting from differ-
ent acid/alcohol molar ratio, on enzyme activity and on the stability
of the miniemulsion.
3.3. Fluorescence studies of enzyme activity
A steady state fluorescence study was performed to evaluate
and gain some insight in changes in cutinase conformation during
the time course of the esterification, in a miniemulsion system for
different acid/alcohol molar ratios.
Fluorescence emission of cutinase is originated from the aro-
matic amino acids, one tryptophan (Trp) and six tyrosines (Tyr)
[
58].
The intensity of the tryptophan fluorescence emission is weak
when exciting native cutinase and it could be due to the single
tryptophan residue of cutinase at position 69, which is strongly
quenched by an adjacent disulfide bridge between cysteines 31
and 109 [58–60]. Therefore the emission of native cutinase is dom-
inated by the six tyrosines [60] and its maximum intensity is at
By previous work in reverse micelles system [28,60] it was found
that cutinase deactivation and denaturation is a reversible process,
the next step would be to study reutilization of the cutinase in
miniemulsion system.
ꢀ
max = 303 nm [51,52].
The denaturation of cutinase can be followed by an increase in
3.4. Stability and Influence of pH of the system
tryptophyl emission, because this residue is released from the vicin-
ity of the disulfide bridge when cutinase unfolds. It was shown that
with the unfolding of cutinase, a shift in the emission maximum is
also observed towards a higher wavelength [59]. For unfolding pro-
tein, the spectra became dominated by tryptophyl emission with
the maximum occurring at 335 nm [60].
For an acid/alcohol molar ratio R = 1 (Fig. 4), a gradual red shift
was observed, characteristic of a spectra representative of a denat-
uration process involving Trp residues along time. Data suggest that
under the experimental conditions used, the unfolding of cutinase
sets off after 1 h, given the red shift from 303 nm, for t = 10 min up
to 1 h (considered native cutinase) to 319 nm after 2 h of reaction.
The trend towards a red shift, characteristic of denaturation,
under exposure to a miniemulsion environment was further eval-
uated, for the remaining values of R used (Fig. 5).
Enzyme activity depends on the pH of the system, which in
the case of a miniemulsion system is determined by the continu-
ous phase [49]. The higher esterification yields and activities were
obtained for R = 0.4 and R = 1, that correspond to initial pH of 4.22
and 4.06, respectively (Table 2). Maximum ꢁpH (change of pH from
t = 0 min to t = 24 h) observed in these cases can be related with the
higher amount of ester formed, which results in an increase in the
pH of the reaction medium. On the other hand, low enzyme activ-
ity with relatively low esterification yield (Fig. 3) was observed
Table 2
pH profile of the bioconversion system for different acid/alcohol ratios and a 24 h
incubation period.
Molar ratio
pH
ꢁpH
In the case of hexanol excess (Fig. 5a), the red shift is noticeable
after 70, 120 and 240 min for acid/alcohol molar ratios of R = 0.4,
R = 0.2 and R = 0.1, respectively. Data suggest that with the increase
of the alcohol concentration denaturation of cutinase evolves at a
slower pace along the reaction time even if the ester yields decrease
slightly (around 10%). The reason could be due to a stabilizing
effect of hexanol, previously described in AOT reversed micellar
system [24,59,60]. Hexanol, as a medium chain alcohol, acts as a
t = 0 min
t = 24 h
0
0
0
1
2.5
5
.1
.2
.4
5.10
4.63
4.22
4.06
4.23
3.72
3.78
5.74
5.30
5.27
5.01
4.39
4.18
4.26
0.64
0.67
1.05
0.95
0.16
0.46
0.48
10

1
00
D.P.C. de Barros et al. / Catalysis Today 173 (2011) 95–102
Fig. 5. Fluorescence spectra for different acid/alcohol molar ratio (a) excess of hexanol and (b) excess of octanoic acid.
for R = 0.1, corresponding to an initial pH of the system of 5.10
Table 2). The lower yield with initial pH higher than 5, was reported
by other authors [49], for the esterification of nonanoic acid with
-phenylpropanol in miniemulsion (pH = 6.8) using Amano lipase
PS. These authors showed that with a higher pH of the system,
the presence of non-ionic surfactant could tamper with enzyme
activity. This could take place by interaction with the active site
of the enzyme, adsorption on the enzyme or modification of the
conformation of the active site. At lower pH, the carboxylic acid
is in protonated form and cannot behave as a surfactant. With an
excess of the acids, the pH of the system decreases and an inhibitory
effect takes place, a pattern also observed in organic solvent [57]
and reverse micellar [28] systems, too.
confirmed in fluorescence studies (Fig. 5). In the stable miniemul-
sion the reaction surface is larger and ester yield is higher.
From the Z-average diameter (nm) of the miniemulsion, it was
possible to observe the evolution in the size of droplets during time
course of the reaction.
Before the addition of the enzyme the Z-average size of droplets
was 220 ± 20 nm for all molar ratios (Fig. 7). By increasing the time
(
3
A possibility to explain the difference in ester yields and cuti-
nase activity alongside the range of acid/alcohol molar ratio (Fig. 3)
could be a change in the reaction microenvironment, induced by
phase separation in the different miniemulsion system. Previous
works [49,50] suggested that the stability of miniemulsion sys-
tems is related to the hydrophobicity of substrates. Esterification
reactions in miniemulsion favor hydrophobic substrates due to the
formation of very stable hydrophobic droplets [50].
The stability of the miniemulsion is also influenced by changes
of the R (Fig. 6). The miniemulsion systems with R above 2.5 clearly
show phase separation after a 24 h incubation period. This could be
due to the low pH of the system and inhibition effect on the catalyst
already referred (Table 2).
Apparently miniemulsions with lower acid/alcohol molar ratio
(
due to excess of hexanol) are more stable and phase separation
was not observed after 24 h. It was confirmed that the esterifica-
tion yield in these cases is higher than with the system with higher
acid/alcohol molar ratio (excess of acid). The stabilizing effect of
hexanol already referred for the reverse micellar systems [28] was
Fig. 6. Stability of miniemulsion.

D.P.C. de Barros et al. / Catalysis Today 173 (2011) 95–102
101
P. Fernandes acknowledges Funda c¸ ão para a Ciência e Tecnolo-
gia (FCT) for contract under Programme Ciência 2007.
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