Optimized hybrid nanospheres immobilizing Rhizomucor miehei lipase for chiral biotransformation

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Optimized hybrid nanospheres immobilizing Rhizomucor miehei lipase for chiral biotransformation F. Verri a,∗ , U. Diaz b , A. Macario a , A. Corma b , G. Giordano a a b

Environmental and Chemical Engineering Department, Università della Calabria, via Pietro Bucci, Rende, Cosenza 87036, Italy 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

Article history: Received 24 June 2015 Received in revised form 23 October 2015 Accepted 16 November 2015 Available online xxx Keywords: Rhizomucor miehei lipase Liposome Heterogeneous hybrid biocatalysts (R,S)-ibuprofen Biotransformation

a b s t r a c t 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 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. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction The enzymes are very active and highly specific biocatalysts that contribute to the continuing research that aim to obtain catalytic 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 catalysts have largely been made [3–6]. However, their specificities are far less than the well-known and reported catalytic activities 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 natural enzymes or their derivatives could help enhance the specificity and reactivity of several reactions. However, their poor physicochemical stability limits their application as effective catalysts, because the reaction media conditions denaturalize them, and consequently, reduce their activity. Another associated drawback would be their impossible recovery and reuse in successive catalytic 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

∗ Corresponding author at: Environmental and Chemical Engineering Department, Università della Calabria, via Pietro Bucci, Rende, Cosenza 87036, Italy. E-mail address: [email protected] (F. Verri).

their protection, allowing the enzymatic action and preserving their associated catalytic activity and selectivity [11–23]. Liposome 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 alternative 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 liposomal 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 external 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, consequently, 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 biocatalyst by the encapsulation of liposome-lipase systems onto porous

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 procedure 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 preparation 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 facilitating 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 chiral 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 esterification of racemic ibuprofen (Fig. 1) to facilitate the production of active enantiomers from the racemic product.

2. Materials and methods 2.1. Materials 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 solution, 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.

2.1.2. 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.

2.1.3. 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 reactants, 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.

2.2. 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 temperature 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 hexadecylamine (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 temperature. 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. The total protein concentration of the initial and final solutions was calculated using ultraviolet (UV) absorption method at 235/280 nm [63], and the quantity of protein adsorbed on the support was determined by achieving a mass balance between the initial and final solutions. 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. 2.3.2. 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 analysis, during the synthesis of nanospheres, lipase was mixed with a fluorescent compound (fluorescein isothiocyanate (99%)) for 2 h. 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

Mixing time [h]

Sample code Lecithin amount [gr]

Silica amount (TEOS) [gr]

SiO2 /liposome weight ratio

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

12

NS1 NS2 NS3

0.44 0.44 0.88

0.88 0.44 0.44

2 1 0.5

2.4.1. Enantioselective esterification procedure The standard reaction mixture was composed of an organic solvent (10 mL), racemic ibuprofen (66 mM), and alcohol (66 mM), without water. The reaction was initiated by the addition of a prepared 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. Samples 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 chromatography, respectively. 2.4.2. Analytical procedures of reaction products 2.4.2.1. Gas chromatography analysis. This technique was performed 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 injector 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 phosphate 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 phosphate 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 phosphate 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.



Ester yield (%) = 2.3.4. 13 C NMR and 29 Si NMR Nuclear magnetic resonance (NMR) spectra were recorded at room temperature under magic-angle spinning (MAS) using a Bruker AV-400 spectrometer. The single-pulse 29 Si 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 13 C spectra were recorded using a 7-mm Bruker BL-7 probe at a sample spinning rate of 5 kHz. 13 C and 29 Si spectra were referred to adamantine and tetramethylsilane, respectively.

(AE /PME ) (AE /PME ) + (AE /PMib )



× 100

(1)

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, respectively. 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: TON = ( TOF =

2.3.5. N2 adsorption/desorption 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.

%ester yield moliburpofen )[/] )×( 100 molenzyme

(2)

TON −1 [h ] Time

(3)

2.4.2.2. Chiral analysis. This analysis was performed using a chiral 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 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: 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:

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).

ee% =

R−S × 100ForR > S S+R

(4)

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.

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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).

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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). 3. Results and discussion 3.1. Characterization catalysts results The influence of the two important synthesis parameters, silica/liposome weight ratio and mixing time of liposome/lipase solution, on the quantity of immobilized enzyme and the morphology 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 solution, 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 maximum 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 damage/breaking and opening of the liposomal shells occur (Fig. 3b and c for NS2 and NS3 samples, respectively). 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. 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. 7. TEM images of NSH1 sample (nanoparticles with dimension 20÷70 nm).

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. Alcohol

Yield in estera [%]

eeb

TOFc

Stereopreferenced

Methanol 1-Propanol 1-Butanol

13 78 18

28 60 39

54 660 215

S S S

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. c Calculated at 1 h of reaction. d Configuration of the enantiomer found in the ester.

ting the mixing time between liposome and lipase to 2 h (Fig. 4a), as represented by the sample NS1 that was selected as the reference 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 confocal 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 surfactant to create mesoporous silica shell. The synthesis conditions are reported in Table 2. The use of surfactant did not affect the determination 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 external 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). Other characterization tests were conducted to provide more information about optimized biocatalysts structure (Supporting information: section A).

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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 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: section 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 advantageous 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 different chain lengths, such as methanol, 1-propanol, and 1-butanol,

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 modified.) 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 presence of 1-propanol, monitored by chiral gas chromatography, were reported.

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Table 4 Effect of temperature on the conversion and enantiomeric excess in the reaction catalyzed 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 [%]

27 37 50 80

26 78 71 43

eeb 16 60 58 9

TOFc

Stereopreferenced

393 660 297 159

S S S S

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. c Calculated at 1 h of reaction. d Configuration of the enantiomer found in the ester.

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. 3.2.4. Influence of the solvent nature The influence of the nature of the solvent on the catalytic efficiency 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 preserving 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. Therefore, after 1 h, the reaction was stopped, the catalyst was separated from the liquid reaction media and the reaction was again performed without catalyst. The time reaction profile of this reaction was compared with that of standard reaction (see Supporting infor-

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 catalytic 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 successive catalytic cycles and it remained attached to the phospholipids layer inside the liposome, being still present and stabilized onto the nanospheres (Fig. 11). 3.2.6. 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.

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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 immobilized and free forms. 4. Conclusion Optimized organic–inorganic nanospheres formed by a hybrid silica shell (with an internal liposomal core) containing bioactive molecules (lipase enzyme) were successfully synthesized. The maximum amount of immobilized enzyme and the best nanospheres morphology were obtained with a SiO2 /liposome ratio of 2 and a lipase/liposome mixing time of 2 h. Hybrid nanospheres with mesoporous external silica shell were also synthesized using hexadecylamine as surfactant in the polymerization of silica shell. Heterogeneous biocatalysts were used in the enantioselective esterification of ibuprofen. Many factors affected the enzyme activity in the esterification reaction. The optimal reaction conditions for the esterification of ibuprofen using hybrid nanospheres containing R. miehei lipase as biocatalyst were as follows: temperature 37 ◦ C, 1-propanol as alcohol, isooctane as solvent, and ibuprofen/alcohol molar ratio equal to 1. Catalysts with mesoporous external silica shell perform better than those synthesized without surfactant, which might be attributed to the facilitation of the mass transfer of the substrate and product. Finally, the stability of the immobilized lipase was also found to be very high, because of the maintenance of its activity up to at least 9 reaction cycles. This high stability of the heterogeneous biocatalyst resulted in a 15–16 times higher immobilized enzyme productivity than that of its free form. The optimized catalysts and the best reaction conditions result in a simple and efficient enantioselective synthesis of (S)enantiomer of ibuprofen, which has a potential application in the pharmaceutical industry. The high-efficiency catalytic system proposed in this article could be used in the near future for different enzymatic/chemical processes, such as multienzymatic cascade reaction, drug and gene delivery, and biosensors. Acknowledgements The authors thank the financial support from ConsoliderIngenio MULTICAT CSD2009-00050, MAT2014-52085-C2-1-P, and Severo Ochoa Excellence Program SEV-2012-0267. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.procbio.2015.11. 020.

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