Optimized hybrid nanospheres immobilizing Rhizomucor miehei lipase for chiral biotransformation

Article abstract of DOI:10.1016/j.procbio.2015.11.020

In this study, the immobilization of Rhizomucor miehei lipase into hybrid nanospheres containing a liposomal core was reported. Organic internal liposomal enzyme phase was protected by inorganic silica matrix, obtained with and without surfactant, that st

Full text of DOI:10.1016/j.procbio.2015.11.020

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Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Optimized hybrid nanospheres immobilizing Rhizomucor miehei
lipase for chiral biotransformation
F. Verria,∗, U. Diazb, A. Macarioa, A. Cormab, G. Giordanoa
a Environmental and Chemical Engineering Department, Università della Calabria, via Pietro Bucci, Rende, Cosenza 87036, Italy
b Instituto de Tecnología Química (UPV-CSIC), Universidad Politécnica de Valencia, Avd. de los Naranjos s/n, Valencia 46022, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 June 2015
Received in revised form 23 October 2015
Accepted 16 November 2015
Available online xxx
In this study, the immobilization of Rhizomucor miehei lipase into hybrid nanospheres containing a lipo-
somal core was reported. Organic internal liposomal enzyme phase was protected by inorganic silica
matrix, obtained with and without surfactant, that stabilizes the internal organic phase and isolates
and protects the bioactive molecules. The optimized heterogeneous catalyst thus prepared was used for
enantioselective esterification of (R,S)-ibuprofen. The influence of several catalytic parameters on the
activity of hybrid nanospheres (type of solvent, nature of the alcohol, reaction temperature, etc.,) was
investigated. The heterogeneous biocatalysts best performed at 37 ◦C, using isooctane as solvent and
1-propanol as alcohol (with ester yield ranging between 78 and 93%). High activity and stability (up to
nine reaction cycles) of enzyme-immobilized hybrid nanospheres, with respect to the free form, were
observed. R. miehei lipase, both in its free and immobilized forms, reacts only with the S(+) enantiomer
of (R,S)-ibuprofen, in all the tested reaction conditions.
Keywords:
Rhizomucor miehei lipase
Liposome
Heterogeneous hybrid biocatalysts
(R,S)-ibuprofen
Biotransformation
© 2015 Elsevier Ltd. All rights reserved.
1. Introduction
their protection, allowing the enzymatic action and preserving
their associated catalytic activity and selectivity [11–23]. Liposome
The enzymes are very active and highly specific biocatalysts
that contribute to the continuing research that aim to obtain cat-
alytic systems with behaviors comparable with that of natural
catalysts [1,2]. Attempts to imitate the enzymatic action through
the synthesis of artificial homogeneous and heterogeneous cata-
lysts have largely been made [3–6]. However, their specificities
are far less than the well-known and reported catalytic activi-
ties of enzymatic systems, which exhibit high sterospecificity and
regioselectivity attributed to their characteristic shape, charge, and
hydrophobic–hydrophilic properties [7,8]. The direct use of natu-
ral enzymes or their derivatives could help enhance the specificity
and reactivity of several reactions. However, their poor physic-
ochemical stability limits their application as effective catalysts,
because the reaction media conditions denaturalize them, and
consequently, reduce their activity. Another associated drawback
wouldbe their impossiblerecoveryand reuse in successivecatalytic
cycles [9,10]. The aforementioned drawbacks would be overcome
by immobilizing the isolated and stabilized active enzymes into
inorganic matrices or organic systems, which presumably increase
systems have been widely used for the effective encapsulation of
enzymes [24–27]. However, liposomes also have drawbacks such
as poor hydrothermal and chemical stability that would favor rapid
denaturation of the encapsulated enzymes [28–31]. The alterna-
tive approach could be the protection of the liposomal phase with
external inorganic shells, such as porous silica [32–35], whose
hydrothermal stability would protect the internal enzymatic lipo-
somal system, and its external porosity would allow the interaction
with the reaction media. Furthermore, the associated structural
role of organized phosphatidylcholine micelles, which form the
liposomes, as surfactants, would facilitate the generation of exter-
nal porous silica layers around of spherical liposomes. Particularly,
these nanospheres would be composed of a purely organic internal
liposomal phase in which the bioactive enzymes are encapsulated.
An external self-assembled porous silica shell would be covering
this part, stabilizing the internal liposomal phase and, conse-
quently, isolating and protecting the enzymes present in it [36]. This
methodology was successfully used to obtain organic–inorganic
nanospheres with responsive external molecular gates, localized
in the outer silica shell, for effective storage and controlled release
of drugs [37].
Recently, a method for the preparation of an effective biocata-
lyst by the encapsulation of liposome-lipase systems onto porous
∗
Corresponding author at: Environmental and Chemical Engineering Department,
Università della Calabria, via Pietro Bucci, Rende, Cosenza 87036, Italy.
E-mail address: francescaverri85@libero.it (F. Verri).
http://dx.doi.org/10.1016/j.procbio.2015.11.020
1359-5113/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: F. Verri, et al., Optimized hybrid nanospheres immobilizing Rhizomucor miehei lipase for chiral
biotransformation, Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2015.11.020

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silica nanoparticles for biodiesel production was proposed [38]. In
spite of the previous work, in this study, we optimized the proce-
dure for the synthesis of heterogeneous biocatalyst to improve the
stability of immobilized Rhizomucor miehei lipase. The influence of
several experimental factors, such as silica/liposome weight ratio
and mixing time between liposome and lipase during the prepa-
ration process, was studied. The addition of a templating agent
(hexadecylamine) to the silica covering the core liposome resulted
in the creation of a mesoporous silica shell with properties facili-
tating the diffusion of reactants and products.
In the last two decade, there was an increasing trend toward
the use of lipase for the production of enantiomerically pure
compounds [39–48], particularly in a chiral resolution of (R,S)-
ibuprofen [49–59]. In particular, immobilized R. miehei lipase
exhibited sufficient stability and biosynthetic ability for the chi-
ral resolution of (R,S)-ibuprofen [60–62]. Therefore, the catalytic
performance and stability of the optimized heterogeneous catalysts
included in this study were evaluated in the enantioselective ester-
ification of racemic ibuprofen (Fig. 1) to facilitate the production of
active enantiomers from the racemic product.
2.2. Synthesis of hybrid nanospheres: enzyme immobilization
procedure
The hybrid nanospheres can be synthesized by two consecutive
steps. The first step consists of the preparation of liposomal phase
containing R. miehei lipase and the evaluation of the influence of
two important synthesis parameters: silica/liposome weight ratio
and mixing time of liposome/lipase solution. In the second step,
a porous silica shell was formed around the liposomal phase by
adding a certain amount of TEOS to the liposome/lipase solution
at room temperature for 24 h. After this time, 7.1 mg of sodium
fluoride was added to the mixture and stirred for 48 h at room tem-
perature to initialize the condensation of silane groups. In order
to synthesize different samples, a templating agent was added to
the silica covering the core liposome. A certain amount of hex-
adecylamine (TEOS/hexadecylamine molar ratio equal to 4) was
dissolved in 40 mL of ethanol. The hexadecylamine solution was
added drop by drop, during the 24 h after the addition of TEOS,
with vigorous stirring at room temperature. After this time, 7.1 mg
of NaF was added to the mixture and stirred for 48 h at room tem-
perature. Then, the sample was centrifuged and the recovered solid
was washed with distilled water and dried at 30 ◦C overnight. All
the prepared catalysts were activated by washing with 100 mL of
solvent (isooctane) and 900 mL of distilled water, and dried at 30 ◦C
overnight.
2. Materials and methods
2.1. Materials
The total protein concentration of the initial and final solu-
tions was calculated using ultraviolet (UV) absorption method at
235/280 nm [63], and the quantity of protein adsorbed on the sup-
port was determined by achieving a mass balance between the
initial and final solutions.
2.1.1. Organic nanospheres preparation
For the preparation of the organic parts of nanospheres, L-␣-
phosphatidylcholine, purchased from Sigma–Aldrich, was used as
the lecithin liposome precursor, whereas commercial lipase solu-
tion, PALATASE 20000L, purchased from Novo Nordisk, Denmark,
was used as the enzyme. This enzyme is a purified 1,3-specific lipase
(EC 3.1.1.3) from R. miehei.
2.3. Catalyst characterization
2.3.1. Thermogravimetric and differential thermal analysis
TGA–DTA curves were recorded in nitrogen stream using a
Metler-Toledo TGA/SDTA 851E instrument. Measurements were
made in a temperature range of 20–800 ◦C, with a heating rate of
10 ◦C/min and a synthetic air stream flow rate of 50 mL/min.
2.1.2. Silica porous shell preparation
For the preparation of silica porous shell, 99% tetraethyl
orthosilicate (TEOS, silica source), 98% hexadecylamine (template),
and 99% sodium fluoride (mineralizing agent), purchased from
Sigma–Aldrich, were used.
2.3.2. Fluorescence confocal microscopy
Leica TCS SL is the imaging core’s point-scanning laser confocal
system, which was used to accurately determine the exact position
of the enzyme inside the nanospheres. In order to perform the anal-
ysis, during the synthesis of nanospheres, lipase was mixed with a
fluorescent compound (fluorescein isothiocyanate (99%)) for 2 h.
2.1.3. Reactants for catalytic test
Alcohols such as methanol (99.9%), 1-propanol (99.9%), and
1-butanol (99.9%), racemic ibuprofen (98%) were tested as reac-
tants, whereas isooctane (99.9%) and dimethylformamide (DMF)
(99%) were tested as reaction solvents. All products used, included
enantiomers (R and S) of ibuprofen, were purchased from
Sigma–Aldrich.
2.3.3. Transmission electron microscopy
Micrographs of transmission electron microscopy (TEM) were
obtained with a Philips CM10 electron microscope operating at
100 KeV.
Fig. 1. Enantioselective esterification of (R,S)-ibuprofen catalyzed by Rhizomucor miehei lipase.
Please cite this article in press as: F. Verri, et al., Optimized hybrid nanospheres immobilizing Rhizomucor miehei lipase for chiral
biotransformation, Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2015.11.020

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2.4. Catalytic test
3
Table 1
Characteristics of prepared samples.
2.4.1. Enantioselective esterification procedure
Mixing
time [h]
Sample code Lecithin amount Silica amount
SiO2/liposome
weight ratio
[gr]
(TEOS) [gr]
The standard reaction mixture was composed of an organic sol-
vent (10 mL), racemic ibuprofen (66 mM), and alcohol (66 mM),
without water. The reaction was initiated by the addition of a pre-
pared heterogeneous biocatalyst (7 %wt of lipase with respect to
ibuprofen) to the solution in a 50-mL conical flask, under orbital
magnetic stirring at 135 rpm and at different temperatures. Sam-
ples of 50 ␮L of the solution were withdrawn at different times
and diluted with 50 ␮L of isooctane. The amount of ester (ester
yield) formed during the reaction and the enantiomeric excess were
determined by gas chromatography and chiral gas chromatogra-
phy, respectively.
1
NS1
NS2
NS3
0.44
0.44
0.88
0.88
0.44
0.44
2
1
0.5
2
NS1
NS2
NS3
0.44
0.44
0.88
0.88
0.44
0.44
2
1
0.5
4
NS1
NS2
NS3
0.44
0.44
0.88
0.88
0.44
0.44
2
1
0.5
6
NS1
NS2
NS3
0.44
0.44
0.88
0.88
0.44
0.44
2
1
0.5
2.4.2. Analytical procedures of reaction products
12
NS1
NS2
NS3
0.44
0.44
0.88
0.88
0.44
0.44
2
1
0.5
2.4.2.1. Gas chromatography analysis. This technique was per-
formed in an Agilent 7890A, equipped with a flame ionization
detector (FID) and a mass spectrometry detector. The column
used was a BP5MS with low-polarity phase (5%-phenyl-95%-
polysilphenylene-siloxane; 30 m × 250 ␮m × 0.25 ␮m). The injec-
tor and detector temperatures were 280 and 300 ◦C, respectively.
Nitrogen was used as the carrier gas, with a flow rate of 25 mL/min.
The temperature program of the column was as follows: 2 min at
50 ◦C and 30 ◦C/min until 280 ◦C. Ester yield was calculated using
the following equation:
NS1: hybrid nanospheres characterized by 0.44 g of liposome; 40 mL of 0.2 M phos-
phate buffer pH 7 containing 7.1 mL of Rhizomucor miehei lipase (RML); 0.88 g of
SiO2; 7.1 mg of NaF.
NS2: hybrid nanospheres characterized by 0.44 g of liposome; 40 mL of 0.2 M phos-
phate buffer pH 7 containing 7.1 mL of Rhizomucor miehei lipase (RML); 0.44 g of
SiO2; 7.1 mg of NaF.
NS3: hybrid nanospheres characterized by 0.88 g of liposome; 40 mL of 0.2 M phos-
phate buffer pH 7 containing 7.1 mL of Rhizomucor miehei lipase (RML); 0.44 g of
SiO2, 7.1 mg of NaF.
ꢀ
ꢁ
The values shown in the table are the average of the values obtained after 3 tests.
(AE/PME)
(AE/PME) + (AE/PMib
Ester yield % =
( )
× 100
(1)
)
2.3.4. 13C NMR and 29Si NMR
where AE and PME are the peak area and molecular weight of the
(S)-ester of ibuprofen (desired product), respectively, and Aib and
PMib are the peak area and molecular weight of ibuprofen, respec-
tively. The amounts of ester produced and the remaining acid were
quantitatively measured using internal standardization.
Turnover number (TON) and turnover number of frequency
(TOF) were calculated with respect to a reaction time of 1 h as
follows:
Nuclear magnetic resonance (NMR) spectra were recorded at
room temperature under magic-angle spinning (MAS) using a
Bruker AV-400 spectrometer. The single-pulse 29Si spectra were
obtained at 79.5 MHz using a 7-mm Bruker BL-7 probe with pulses
of 3.5 ␮s corresponding to a flip angle of 3/4 ꢀ radians, and a recycle
delay of 240 s. Pulses of 0.5 ␮s to flip the magnetization ␲/20 rad,
and a recycle delay of 2 s were used. The 13C spectra were recorded
using a 7-mm Bruker BL-7 probe at a sample spinning rate of 5 kHz.
13C and 29Si spectra were referred to adamantine and tetramethyl-
silane, respectively.
%ester yield
100
moliburpofen
molenzyme
TON = (
) × (
)[/]
(2)
(3)
TON
Time
TOF =
[h−1
]
2.3.5. N2 adsorption/desorption
2.4.2.2. Chiral analysis. This analysis was performed using a chi-
ral gas chromatograph (Agilent 8000S), equipped with an FID and
a Beta-DEXTM 120 column (35%-phenyl-65%-dimethylsiloxane;
30 m × 0.25 mm × 0.25 ␮m). The injector and detector tempera-
tures were 280 and 300 ◦C, respectively. Nitrogen was used as the
carrier gas, with a flow rate of 25 mL/min. The temperature pro-
gram of the column was as follows: 20 min at 50 ◦C and 5 ◦C/min
until 140 ◦C; 20 min at 140 ◦C and 5 ◦C/min until 210 ◦C; and 20 min
at 210 ◦C. The enantiomeric excess (ee) observed by retention times
was calculated as follows:
Nitrogen adsorption isotherms were measured at −196 ◦C using
a Micromeritics ASAP 2010 volumetric adsorption analyzer. Before
analysis, all samples were calcinated at 600 ◦C under vacuum for
8 h, and outgassed for 12 h at 100 ◦C.
2.3.6. Powered X-ray diffraction (XRD)
The samples were analyzed by powered X-ray diffraction (XRD)
technique using Philips X’Pert diffractometer. Data were collected
stepwise over the angular region of 2◦ ≤ 2ϑ ≤ 20◦, with steps of
0.01◦ 2ϑ, accumulation time of 20 s/step, and radiation of Cu K␣
(ꢁ = 1.54 Å).
R − S
ee% =
× 100ForR > S
(4)
S + R
Table 2
Enzyme immobilization results at optimized conditions of hybrid nanospheres prepared with surfactant (sample NSH1).
Sample
Lecithin amount
[gr]
Silica amount
(TEOS) [gr]
SiO2/liposome
weight ratio
SiO2/hexadec
molar ratio
Liposome/lipase
mixing time [h]
mg of
immobilized
enzyme
mg immobilized
enzyme/gr SiO2
NSH1
0.44
0.88
2
4
2
2.2
3
NSH1: hybrid nanospheres characterized by 0.44 g of liposome; 40 mL of 0.2 M phosphate buffer pH 7 containing 7.1 mL of Rhizomucor miehei lipase (RML); 0.88 g of SiO2; an
amount of hexadecylamine (SiO2/hexadecylamine equal to 4); 7.1 mg of NaF.
The values shown in the table are the average of the values obtained after 3 tests.
Please cite this article in press as: F. Verri, et al., Optimized hybrid nanospheres immobilizing Rhizomucor miehei lipase for chiral
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Fig. 2. (a) Milligram of immobilized enzyme and (b) mg of immobilized enzyme/grams of SiO2 vs lipase/liposome mixing time.
Fig. 3. TEM images of: NS2 sample at 2 h (a) and 12 h (b) of mixing time between lipase and liposome and (c) TEM image of NS3 at 12 h of mixing time between lipase and
liposome.
Fig. 4. TEM images of NS1 sample at 2 h (a) and 12 h (b) of mixing time between liposome and lipase.
Fig. 5. Fluorescence confocal microscopy images of sample NS1 (different resolution).
Please cite this article in press as: F. Verri, et al., Optimized hybrid nanospheres immobilizing Rhizomucor miehei lipase for chiral
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Fig. 7. TEM images of NSH1 sample (nanoparticles with dimension 20÷70 nm).
Fig. 6. Fluorescence confocal microscopy images of sample NSH1.
where R is the peak area for the R(−) enantiomer of ibuprofen
(retention times equal to 76 min) and S is the peak area for the
S(+) enantiomer of ibuprofen (retention times equal to 76.4 min).
Table 3
Esterification of (R,S)-ibuprofen results obtained using different alcohols and NS1
as catalyst, after 72 h of reaction. 66 mM of ibuprofen; 66 mM of alcohol, 10 mL of
isooctane as a solvent and 7 %wt of immobilized lipase with respect to ibuprofen,
was used. Each data point represents the average of 3 experiments.
3. Results and discussion
Alcohol
Yield in estera [%]
eeb
TOFc
Stereopreferenced
3.1. Characterization catalysts results
Methanol
1-Propanol
1-Butanol
13
78
18
28
60
39
54
660
215
S
S
S
The influence of the two important synthesis parameters, sil-
ica/liposome weight ratio and mixing time of liposome/lipase
solution, on the quantity of immobilized enzyme and the mor-
phology of nanospheres was evaluated. Table 1 summarizes these
specifications for each prepared catalyst. The effect of the quantity
of immobilized enzyme on the mixing time of liposome/lipase solu-
tion, for each liposome/silica ratio tested, is reported in Fig. 2. It is
evident from the figure that the mixing time between liposome
and lipase strongly affects the amount of immobilized enzyme
(Fig. 2a). Particularly, for each silica/liposome ratio tested, the max-
imum amount of lipase-immobilized enzyme was obtained at a
mixing time of 2 h. With further increase of the mixing time, the
amount of retained enzyme decreases, which might be due to
the damage/breaking of liposomal shell. Moreover, for the same
liposome/lipase mixing time, the specific amount of immobilized
enzyme firmly increased when the amount of SiO2 used was lower,
as it was expected (Fig. 2b).
The result of TEM analysis (Fig. 3) shows that the morphology
of the samples was also found to be strongly affected by the two
parameters. The use of equal or lower amount of SiO2 than that
of the liposome (NS2 and NS3 samples) does not permit the full
coverage of single liposome sphere by the silica shell (Fig. 3a). In
fact, more spheres are included in a unique silica shell. With an
increase of the mixing time between lipase and liposome, the dam-
age/breaking and opening of the liposomal shells occur (Fig. 3b and
c for NS2 and NS3 samples, respectively).
a
Yield in ester is given as a percentage of (S)-ibuprofen esterified after the reaction
time.
b
Enantiomeric excess calculated at 72 h of reaction.
Calculated at 1 h of reaction.
Configuration of the enantiomer found in the ester.
c
d
ting the mixing time between liposome and lipase to 2 h (Fig. 4a),
as represented by the sample NS1 that was selected as the refer-
ence catalyst and analyzed with more details. Primarily, in order
to accurately determine the exact position of the enzyme inside
the optimized nanospheres of sample the NS1, fluorescence con-
focal microscopy analysis was performed, which confirmed that
the effective enzyme was distributed inside the internal liposomal
phase, and the organic liposome/lipase phase was protected by the
inorganic matrix (Fig. 5).
The optimized procedure (SiO2/liposome weight ratio of 2 and
the liposome/lipase solution mixing time of 2 h) was also used
to produce different hybrid nanospheres (NSH1), using a surfac-
tant to create mesoporous silica shell. The synthesis conditions are
reported in Table 2. The use of surfactant did not affect the determi-
nation of the position of the enzyme inside the nanospheres, which
resulted in a perfect confinement inside the liposomal shell (Fig. 6).
In order to get more information about the external silica structure
of this sample, XRD and TEM analyses were performed (for XRD
results, see Supporting information: section A). With respect to the
nanospheres synthesized without surfactant (in which the exter-
nal silica shell was amorphous, as it was corroborated by the XRD
pattern—data not provided), the silica shell of the sample NSH1
exhibited a typical worm-hole structure characterized by parallel
channels to the support surface (Fig. 7).
On the contrary, perfect coverage of liposomal spheres by the
silica shell can be observed when the amount of silica is double
that of the liposome (sample NS1), both for lower (2 h) and higher
(12 h) mixing times (Fig. 4a and b, respectively). However, in this
latter case, the quantity of immobilized enzyme is very low, which
might be due to the few remaining unaltered liposome cells. The
nanospheres are rather homogeneous with an average diameter
between 20 and 250 nm.
Other characterization tests were conducted to provide more
information about optimized biocatalysts structure (Supporting
information: section A).
To conclude, the optimal hybrid nanospheres were obtained by
doubling the amount of silica than that of the liposome and set-
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Fig. 8. Influence of the solvent nature in the reaction catalyzed by NS1 catalyst. Reaction conditions: 66 mM of ibuprofen, 66 mM of 1-propanol as a alcohol, 10 mL of solvent,
7 %wt of immobilized lipase with respect to ibuprofen, T = 37 ◦C. Each data point has been calculated by gas chromatography analysis and represents the average of three
experiments.
Fig. 9. Catalytic performance of NS1 and NSH1 catalyst after 9 reaction cycles. Reaction conditions: 66 mM of ibuprofen with 66 mM of 1-propanol, in 10 mL of isooctane,
7 %wt of immobilized lipase with respect to ibuprofen, for 72 h for each cycle at T = 37 ◦C, was carried out.
3.2. Catalytic test results
whose results are summarized in Table 3. The stereobias (S-(+)-
preference) was the same for all the nucleophiles tested (see the
Supporting information, section 1B, where all figured clearly show
that only the (S)-enantiomer of ibuprofen was converted to the
desired product, while the (R)-enantiomer is not chemically mod-
ified.)
However, the catalyst performance was strongly influenced by
the chain length of the alcohol used. With 1-butanol and methanol,
immobilized R. miehei lipase showed lower activity, which might
be due to the different substrate specificity of the lipase and/or
the different substrate solvation of the alcohol, as suggested by
previous studies [65]. Enantiomeric excess (ee), ester yield, TON,
and TOF were found to be maximum when 1-propanol was used
as alcohol. In the Supporting Information: Section B, the progress
and the whole time profile of the reaction carried out in the pres-
ence of 1-propanol, monitored by chiral gas chromatography, were
reported.
3.2.1. Influence of catalyst composition
Catalytic test was conducted to evaluate the role of liposome and
hexadecylamine in the reaction (see Supporting information: sec-
tion B for more details), whose results confirmed that no reaction
between ibuprofen and liposome or ibuprofen and hexadecylamine
took place.
3.2.2. Influence of alcohol
It is recognized that lipase from R. miehei is more advan-
tageous in the esterification of primary alcohols, whereas its
activity decreases with secondary alcohols and is inactive with
tertiary alcohols [64]. With the aim of studying the effect of the
nature of alcohol on the esterification reaction, our attention was
directly focused on the influence of primary alcohols with differ-
ent chain lengths, such as methanol, 1-propanol, and 1-butanol,
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Table 4
Effect of temperature on the conversion and enantiomeric excess in the reaction cat-
alyzed by NS1 after 72 h of reaction. Reaction conditions: 66 mM ibuprofen, 66 mM
1-propanol in 10 mL of isooctane, 7 %wt of immobilized lipase respect to ibuprofen.
Each data point represents the average of 3 experiments.
Temperature[◦C]
Yield in estera[%]
eeb
TOFc
Stereopreferenced
27
37
50
80
26
78
71
43
16
393
660
297
159
S
S
S
S
60
58
9
a
Yield in ester is given as a percentage of (S)-ibuprofen esterified after the reaction
time.
b
Enantiomeric excess calculated at 72 h of reaction.
Calculated at 1 h of reaction.
Configuration of the enantiomer found in the ester.
c
d
3.2.3. Influence of temperature
The effect of temperature, in the range of 27–80 ◦C, on the
enzyme activity in the enantioselective esterification of (R,S)-
ibuprofen was examined. In order to obtain optimum results,
all catalytic tests were conducted using 1-propanol as alcohol.
At all temperatures tested, R. miehei lipase showed S-(+)-
enantiorecognition, and the results are reported in Table 4. It can
be observed that the highest ester yield and TONs were obtained
at 37 ◦C, decreasing at higher temperature due to the modification
of the active center geometry of the enzyme. In order to avoid the
evaporation of the organic solvent and to obtain a higher ester yield
of the desired product, the optimum temperature was fixed at 37 ◦C
in the esterification of ibuprofen.
Fig. 10. TEM image of NSH1 sample after 9 cycles of reaction.
mation: section C). When the NS1 catalyst was removed, the ester
yield did not increase significantly after the first hour of reaction
(<5%). Therefore, no significant enzyme leaching was detected.
At this point, the residual esterification activity under optimal
reaction conditions of NS1 and NSH1 catalysts, after successive cat-
alytic uses, was analyzed. The results of stability of both catalysts
after nine reaction cycles are reported in Fig. 9. The immobilized
lipase in the NS1 catalyst retained its activity with moderate loss:
<3% after 4 reaction cycles and <11% after 5 reaction cycles. After
nine cycles, the lipase immobilized into the NS1 catalyst retained
almost 45% of its initial esterification activity. For the sample NSH1,
the activity loss was <3% after 4 reaction cycles and <18% after
8 reaction cycles. After nine cycles, the lipase immobilized into
the NSH1 catalyst retained almost 60% of its initial esterification
activity, and the external mesoporous structure of the catalyst was
also preserved (Fig. 10). The reason behind the exhibition of the
most favorable catalytic performance by the NSH1 catalyst might
be the facilitation of mass transfer of the substrate and products
by the mesoporous structure of its external silica shell. However,
for both samples, the immobilized enzyme was stable after succes-
sive catalytic cycles and it remained attached to the phospholipids
layer inside the liposome, being still present and stabilized onto the
nanospheres (Fig. 11).
3.2.4. Influence of the solvent nature
The influence of the nature of the solvent on the catalytic effi-
ciency of enzymes was also studied. Rather than using apolar
isooctane solvent, the activity of immobilized R. miehei lipase in the
presence of polar solvent, DMF, was tested. The polar DMF totally
deactivated the enzyme contained in the NS1 catalyst (Fig. 8),
because of the removal of water necessary for maintaining the
native and active conformation of the enzyme. On the contrary,
hydrophobic solvents, because of their lower tendencies to strip
essential water in the microenvironment of enzymes, allowed pre-
serving their activity.
3.2.5. Stability of catalysts
After determining the best reaction conditions—temperature
37 ◦C, 1-propanol as alcohol, isooctane as solvent—a “leaching test”
was conducted to verify whether enzymatic leaching has taken
place after the first catalytic use of the optimized catalysts. There-
fore, after 1 h, the reaction was stopped, the catalyst was separated
from the liquid reaction media and the reaction was again per-
formed without catalyst. The time reaction profile of this reaction
was compared with that of standard reaction (see Supporting infor-
3.2.6. Comparison with free lipase
The performances of the free and immobilized enzymes were
compared, whose results are reported in Fig. 12a and b. Both the
catalysts, NS1 and NSH1, showed better performance with respect
to the free lipase. R. miehei lipase showed better performance only
Fig. 11. Fluorescence confocal microscopy images: (a) of NS1 and (b) of NSH1 after 9 reaction cycles.
Please cite this article in press as: F. Verri, et al., Optimized hybrid nanospheres immobilizing Rhizomucor miehei lipase for chiral
biotransformation, Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2015.11.020

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ARTICLE IN PRESS
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F. Verri et al. / Process Biochemistry xxx (2015) xxx–xxx
Fig. 12. (a) Comparison among reaction time profiles of NS1, NSH1 and free lipase. Reaction conditions: 66 mM of ibuprofen, 66 mM of 1-propanol as a alcohol, 10 mL of
isooctane as a solvent, 7 %wt of immobilized lipase with respect to ibuprofen, T = 37 ◦C. Each data point has been calculated by gas chromatography analysis and represents
the average of 3 experiments. (b) Comparison between TOF numbers of NS1, NSH1 and free lipase after 9 reaction cycles. Each cycle is of 72 h at 37 ◦C.
with the S-(+) enantiomer of (R,S)-ibuprofen, both in its immobi-
lized and free forms.
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