Encapsulation of lipase using magnetic fluorescent calix[4]arene derivatives; improvement of enzyme activity and stability

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Encapsulation of lipase using magnetic

ﬂuorescent calix[4]arene

derivatives; improvement of enzyme activity and stability

Elif Ozyilmaz

a

, Sevilay Cetinguney

b

, Mustafa Yilmaz

b,

⁎

a

Department of Biochemistry, Selcuk University, 42075 Konya, Turkey

b_{Department of Chemistry, Selcuk University, 42075 Konya, Turkey}

a b s t r a c t

a r t i c l e i n f o

Article history: Received 21 January 2019

Received in revised form 26 April 2019 Accepted 27 April 2019

Available online 28 April 2019

In this study, iron magnetic nanoparticles capped withﬂuorescent calixarene derivatives (Fe3O4@Calix-2 and

Fe3O4@Calix-3) were prepared in one-step using coprecipitation of Fe(II) and Fe(III) in basic solution. Different

techniques were used to characterize the synthesized magnetic nanoparticles, such as Fourier Transform Infrared spectroscopy (FTIR), powder X-ray diffraction (XRD), vibrating sample magnetometer (VSM) and confocal mi-croscopy. Candida rugosa was encapsulated on synthesized nanoparticles following sol–gel method. It has been observed that under the optimum conditions, the activity of encapsulated lipase (Fe3O4@Calix-2E) was

119 U/g of support which is 4.1 times more that of the encapsulated lipase without calix[4]arene derivative (Fe3O4@E). Comparative study show that the encapsulated lipase on nanoparticles has higher thermal and

oper-ational stability than encapsulated lipase without calix[4]arene derivative. Among these encapsulated lipase nanoparticles, Fe3O4@Calix-2 was capable of effectively catalyze hydrolysis of racemic Flurbiprofen methyl

ester with high conversion of 49% and substrate enantiomeric excess (ees) of 85% at optimum pH and tempera-ture. The efﬁciency of these nanoparticles was assessed by their reusability, for that after ﬁve consecutive oper-ational uses these encapsulated lipase nanoparticles retained their conversion ratios up to 38% and 30% respectively, in the hydrolysis of (R,S)-Flurbiprofen methyl ester. The results showed that encapsulated lipases nanoparticle with calixarene moieties lead to increased activity, stability, reusability and enhanced stereoselectivity in kinetic resolution.

Keywords: Calixarene Fluorescent Fe3O4nanoparticles Lipase encapsulation Flurbiprofen 1. Introduction

Flurbiprofen is a non-steroidal anti-inflammatory drug used to cure osteoarthritis and rheumatoid arthritis, whose activity resides in S-enantiomer and cyclooxygenase (COX) inhibiting activity that is re-sponsible for reducing the production of inflammation-mediating pros-taglandins, thereby providing relief from in_{flammation [}1]. R-Flurbiprofen is used in treatment of Alzheimer's disease and in phase II clinical trial for colon and prostate cancer. [2–4].

Lipases as biocatalysts are very important enzyme that biocatalysts the hydrolysis of long-chain triglycerides and generally exhibit excel-lent chemo-, regio- and enantioselectivity. These enzymes have been widely applied in kinetic resolution [5]. However, lipase as a biocatalyst has some disadvantages, the difﬁculty of product recovery, poor stabil-ity under operational conditions, and the impossibilstabil-ity of multiple re-uses in industrial processes [6,7].

Calix[n]arenes can act as nanobasket structure when cast with nano-particles, they have been used to carry ions and molecules more ef ﬁ-ciently [8]. In recent years it has been observed that calixarenes are

carrier of amino acid, protein, collagens and enzymes [9,10]. The active site ofﬂap/lid of lipase (CRL) has 31 amino acids, mainly hydrophilic on its external face and hydrophobic on the internal side, directed towards the active site is the hydrophilic surface of the molecule in the open form and the inactive site is the hydrophobic surface of the molecule in the closed form. [11–13]. Calix[n]arenes interact with amino acids on the lid or with strong hydrogen bonds that leads to open up the lid and thus the activity of the enzyme is maintained for a long time [9]. In addition, the calixarenes interact with different functional groups of the enzyme to preserve the conformation [6]. In this regard, different calixarene derivative have been used previously by our group for binding of enzymes and analyzed enzyme activity, stability, and enantioselectivity.

For example, we synthesized calix[n]arenes substituted at the lower rim by carboxylic acid [14], hydrazide [15], pyridyl [16], mercapto [4], and substituted at the upper rim by N-methylglucamine [17], phos-phonic acid or iminodicarboxylic acid [6] immobilized magnetic nano-particles that were used as additives in the encapsulation process. The inﬂuence of the synthesized material on the hydrolysis and enantioselectivity of some racemic aromatic methyl esters was per-formed. However, so far, there is no study reported aboutﬂuorescent calix[4]arene derivatives for immobilization of lipase. Therefore, the

⁎ Corresponding author.

E-mail address:myilmaz42@gmail.com(M. Yilmaz).

https://doi.org/10.1016/j.ijbiomac.2019.04.182

Contents lists available atScienceDirect

International Journal of Biological Macromolecules

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aim of this work was to synthesize two lower rim-functionalized calix [4]arene derivatives carryingﬂuorescent groups and use these in mon-itoring the enzyme activity.

2. Materials and methods 2.1. Materials

Candida rugosa lipase (CRL) (Type VII), Bovine serum albumin, tetramethoxysilane (TMOS), p-nitrophenyl palmitate (p-NPP), and octyltriethoxysilane (OTES) and the other reagents were supplied from Merck, Sigma-Aldrich or Fluka. All aqueous solutions were pre-pared with deionized water that had been passed through a Millipore Milli-Q Plus water puriﬁcation system.

2.2. Instrumentation

IR spectra were recorded on a Bruker Vertex FTIR spectrometer. UV/ Vis spectra were measured with a Shimadzu UV-1700. An Agilent 1200 Series High-performance liquid chromatography (HPLC) with a diode array detector was used for enantiomeric excess of Flurbiprofen. The surface morphology of samples was examined by scanning electron mi-croscope (SEM, Jeol, JSM 5310, Japan).1HNMR spectra were recorded on a Varian 400 MHz spectrometer. A Zeiss 510 Meta Confocal Laser Scan-ning Microscope was used to be imaged forﬂuorescence magnetite nanoparticles. An X-ray diffraction (XRD) analysis was performed using by a Bruker D8 ADVANCE X ray diffractometer. Magnetic property was measured using a vibrating sample magnetometer (VSM, LDJ9600). 2.3. Synthesis

Scheme 1represent the synthesis of different derivative of calix[4] arene in which, calix[4]arene derivatives 1–4 were prepared according to literature methods [18–20] Calix-2 and Calix-3 were synthesized by published method with some modiﬁcation and their procedure is given below [21,22].

2.3.1. Dansyl derivative of 4 (Calix-2)

Dansyl chloride (0.363 mg, 1.344 mmol) was added dropwise to the stirred solution of diaminopropyl derivative of calix[4]aren (4) (0.544 g, 0.611 mmol) and Et3N (0.136 g, 1.344 mmol) in 50 mL dry

dichloro-methane. The reaction was stirred at room temperature for 6 h. After the completion of reaction, the reaction mixture was washed with 1 M HCl. The organic phase is reacted with NaHCO3and washed with

water. The organic layer was separated, dried over anhydrous Na2SO4

and distilled under reduced pressure to give Calix-2 in 90%. IRνmax (KBr pellet, cm−1) 1614 and 1574 cm−1(C=C).

1_{HNMR (CDCl}

3):δ 1.00 (s, 18H, But), 1.40 (s, 18H, But), 1.90 (bp, 4H,

–CH2-) 2.85 (s, 12H, N-CH3), 3.20 (t, 4H, CH2-N), 3.30 (d, 4H J = 12.6 Hz,

ArCH2Ar)1H NMR (CDCl3): 3.85 (s, 4H, CH2-N), 4.20 (d, 4H J = 12.6 Hz,

ArCH2Ar) 6.80 (s, 4H, ArH), 7.02 (m, 8H, ArH), 7.40–7.55 (m, 4H, ArH),

8.20–8.35 and 8.50 (m, 8H, ArH, NH). 2.3.2. Dansyl derivative of 3 (Calix-3)

Dansyl chloride (2.99 g, 11.09 mmol) was added dropwise to the stirred solution of dihydrazide derivative of p-tert-butylcalix[4]arene (3) (4 g, 5.04 mmol) and Et3N (1.12 g, 11.09 mmol) in 100 mL dry

di-chloromethane. The reaction was stirred at room temperature for 6 h. After the completion of reaction, the reaction mixture was washed with 1 M HCl. The organic phase is reacted with NaHCO3and washed

with water. The organic layer was separated, dried over anhydrous Na2SO4and distilled under reduced pressure to give Calix-3 in 88%

yield.

IRνmax (KBr pellet, cm−1_{) 1707 and 1675 cm}−1_{(C=O) and 1570}

(C=C).

1_{HNMR (CDCl}

3):δ 1.00 (s, 18H, But), 1.25 (s, 18H, But), 3.00 (s, 12H,

N-CH3), 3.25 (bd, 4H, ArCH2Ar), 3.10 (d, 4H J = 12.6 Hz, ArCH2Ar) 3.30

(bs, 4H, CH2-O), 4.15 (bd, 4H J = 12.6 Hz, ArCH2Ar) 6.65–8.90 (m, 20H,

ArH).

2.4. Preparation of Fe3O4@Calix-2 and Fe3O4@Calix-3

Iron oxide nanoparticles were prepared according to the modiﬁed literature procedure [23]. Typically, a 0.5 g of dansyl derivative of calix [4]arene (Calix-2 or Calix-3) was dissolved in 20 mL of ethanol by ultra-sonic irradiation for 20 min. The mixture was further stirred vigorously for 30 min at 50 °C. Then 177 mg of FeCl3/FeCl2salts in the mass ratio of

2:1 was added by stirring. Under inert atmosphere, the reaction was fur-ther stirred vigorously for 30 min. At the end of this period, 25 mL of 10% NH4OH solution was added drop by drop into the mixture at 60 °C

dur-ing 1 h and treated for another 2 h. Finally, the black precipitates were obtained and separated by decantation with help of external magnet, washed several times with Milli-Q water, and dried under vacuum at room temperature.

2.5. Sol–gel encapsulation of lipases and their activity assay

The encapsulated lipases were prepared according to the previous method [24]. Brieﬂy, 50 mg the encapsulated lipase was added in a 25 mL reaction vessel containing phosphate buffer (390μL; 0.05 M). The mixture was stirred in the incubator. Fe3O4@Calix-2 or Fe3O4@

Calix-3 (50 mg), 100μL of polyvinyl alcohol (PVA), 0.1 M, 50 μL of NaF, and 500μL isopropyl alcohol were mixed then homogenized on a shaker. Later, octyltriethoxysilane (2.5 mmol) and tetramethoxysilane (0.5 mmol; 120μL) were added until completely homogenous mixture was obtained. The gel-like material was lyophilized. The subsequent en-capsulated lipases were stored at 4 °C before usage. The protein concen-tration of the assay was calculated using Bradford's method [25].

To deﬁne the hydrolytic activities of the encapsulated lipase with and without additives, p-NPP was hydrolyzed in 0.05 M of buffer (pH 7.0) and the concentration of p-nitrophenol (p-NP) was measured at 410 nm by means of a spectrophotometer. The quantity of lipase, which releases 1μmol p-NP min−1_{is de}_{ﬁned as one unit enzyme}

activ-ity [26].

2.6. Effect of pH and temperature on encapsulated enzyme activity The effect of pH on the activity of encapsulated lipases was analyzed in different pH values ranges from pH 4.0 to pH 9.0. Furthermore, to see the thermal inactivation of the encapsulated lipases, experiment was performed in the temperature range of 30–60 °C by incubating both li-pases in PBS (50 mM) for 20 min.

2.7. Thermal stability

The encapsulated lipases were stored at 60 °C, for different lengths of time (ranging from 0 to 120 min), then the residual activity was assayed as above.

3. Results and discussion 3.1. Synthesis of Calix-2 and Calix-3

Dansylamide (5-dimethylaminonaphthalene-1-sulfonamide) groups asﬂuorescent probes have been used in many areas of research, including sensors for biological molecules and various metal ions [27–29]. In this work, two newﬂuorescent magnetite nanoparticles (Fe3O4@Calix-2) and (Fe3O4@Calix-3) containing dansyl derivative of

calix[4]arenes were synthesized. Preliminary, calix[4]arene was func-tionalized with dansyl moiety according to reported method (Scheme 1). In brief, calix[4]arene bearing dansyl groups (Calix-2) was prepared

1043 E. Ozyilmaz et al. / International Journal of Biological Macromolecules 133 (2019) 1042–1050

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through the reaction of dansyl chloride with corresponding diaminopropyl derivative of calix[4]arene (4), according to a published procedure [21,22]. Calix-3 was synthesized by the reaction of 1,3-dihydrazide derivative of calix[4]arene (3) and dansyl chloride. The structures of Calix-2 and Calix-3 have been characterized by1_HNMR

and FT-IR techniques. From the1HNMR data, two signals for equivalent tert-butyl group are observed (two singlets atδ 1.00 and 1.40 ppm for Calix-2 and atδ 1.00 and 1.25 ppm for Calix-3). Calix-2 and Calix-3 show a single AB spin system for bridging methylene groups [6]. These compounds were conﬁrmed to be present in the cone

conformation by detailed study of the1_{HNMR spectrum a typical AB}

pattern was observed for the splitting pattern of the ArCH2Ar methylene

protons atδ 3.30 ppm and 4.20 ppm for Calix-2; at δ 3.25 ppm and 4.15 for Calix-3 (See, Figs. S1-S5).

3.2. Preparation of Fe3O4@Calix-2 and Fe3O4@Calix-3

Fe3O4@Calix-2 and Fe3O4@Calix-3 were prepared by alkaline

co-precipitation of Fe(II) and Fe(III) precursors in water/ethanol (2:1) solu-tion of Calix-2 or Calix-3 (Fig. 1). To obtain surface modiﬁed magnetic

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nanoparticles, Molday's procedure [23] was applied. It is a well-known that coordination bonds formed between the calix[4]arene dansyl de-rivative (Calix-2 or Calix-3) and Fe(II)/Fe(III). In this process the calix [4]arene dansyl derivative (Calix-2 or Calix-3) is bound to the nanopar-ticles. The hydrophilic group of calix[4]arene cavity provides an impor-tant role in the preparation of Fe3O4nanoparticles [30,31] (Fig. 1).

Compared with the FTIR spectrum of Fe3O4@Calix-2 and Fe3O4@

Calix-3, exhibited the characteristic absorption of Fe\\O at 789, 569 cm−1for Fe3O4@Calix-2 and at 787, 573 cm−1Fe3O4@Calix-3. In

the FTIR spectrum of magnetic calix-silica composites the band around 1072 and 973 cm−1were attributed to -Si-O stretching vibrations (Fig. S5 and Fig. S6). This indicates that the surface of the encapsulated nanoparticles is covered with silica.

The Fe3O4@Calix-2 and Fe3O4@Calix-3 gave also satisfactory

analyt-ical data, consistent with the proposed formulas integrating residual molecules. According to the elemental analysis, the resulting Fe3O4@

2 contains 2.24% nitrogen, corresponding to 0.38 mmol, of Calix-2/g of support and Fe3O4@Calix-3 contains 3.26% nitrogen,

correspond-ing to 0.40 mmol of Calix-3/g of support [32].

Confocal microscopy is utilized to get information about the surface morphology offluorescent materials [33]. The study demonstrated the fluorescent calixarene labeling for investigation of interactions between the fluorescent calixarene and Fe3O4 nanoparticles and used for

calixarene based lipase immobilization imaging agent. The presence of ﬂuorescence calix[4]arene on iron oxide nanoparticles (Fe3O4@Calix-2

and Fe3O4@Calix-3) was conﬁrmed with confocal microscopy.Fig. 2

shows the confocal microscope image of Fe3O4@Calix-2 and Fe3O4@

Calix-3 nanoparticles with brightfields due to calix[4]arene fluores-cence. However, a homogeneousfluorescence irradiation before lipase encapsulation (Fig. 2A and C), but after lipase encapsulation, it was ob-served that thefluorescence mostly seen in the calixarene interacted with a part of the lipase (Fig. 2B and D).

The X-ray diffraction (XRD) patterns of calixarene and Fe3O4grafted

and calixarene, enzyme and-Fe3O4/SiOXnanoparticles are shown in

Fig. 3. It is clear that the diffraction pattern of the calixarene immobilized Fe3O4nanoparticles is close to the standard pattern for

crystalline magnetite (Fig. 3(A and B). XRD data showed the standard Fe3O4crystal with spinel structure has six diffraction peaks. They were

also observed for encapsulated lipases (Fig. 3C and D). The magnetic property of the Fe3O4@Calix-2 and the encapsulated lipase (Fe3O4@

Calix-2E) were investigated by vibrating sample magnetometer (VSM). Maximum saturation supermagnetizations Fe3O4@Calix-2, and

Fe3O4@Calix-2E are obtained at 40 and 30 emu g−1, respectively

(Fig. 4A and B)). Although the addition of the nonmagnetic portion leads to decreased saturation supermagnetizations, the obtained encap-sulated lipase still has a high saturation supermagnetization of 30 emu g−1.

3.3. Determination of hydrolytic activity of encapsulated lipases

It has been well known that disubstituted calixarene derivatives have effective binding sites for ions and molecules [7]. In view of these properties of the calixarene derivatives, two dansyl derivative of calix [4]arenes (Calix-2 or Calix-3) were used as additive for the

OHOH O O HN NH O O S NCH3 H3C O O S N H3C CH3 O O

Calix-3

OH OH O O NH HN S N H3C CH3 O O S NCH3 H3C O O

Calix-2

HN NH

Fe

₃

O

₄

or

Fig. 1. The proposed structure of Fe3O4@Calix-2 and Fe3O4@Calix-3.

Fig. 2. Confocal microscopy photos a) Fe3O4@Calix-2 b) Fe3O4@Calix-2E c) Fe3O4@Calix-3 d) Fe3O4@Calix-3E.

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encapsulation of the lipase. The speciﬁc activities, the hydrolytic activ-ity, and amounts of the protein of encapsulated lipases are found by the hydrolysis reaction of p-nitrophenyl palmitate (p-NPP) (Table 1).

Table 1shows the data generated from the encapsulated lipases by the sol–gel method for hydrolysis of p-NPP. The speciﬁc activities of en-capsulated nanoparticles were observed as 6.2 for Fe3O4@Calix-2E, 4.0

for Fe3O4@Calix-3E, while it was observed as 1.3 when no calixarene

derivatives were used. It was well known that lipase has a certain part of protein and structural activity relationship studies demonstrated that lower-rim arylation, resulting in maintaining cone conformation, is important for calixarene complexation. Calix[4]arenes interact with the amino acid building blocks and with relevant short or longer peptide sequences [34,35]. When we look at these results, it is understood that dansyl groups of Fe3O4@Calix-2E and Fe3O4@Calix-3E nanoparticles

are effective for interaction with proteins and enzymes. In addition, the Fe3O4@Calix-2E showed more efﬁciency as compared to Fe3O4@

Calix-3E and Fe3O4@E. The efﬁcacy can be attributed due to fact that

Fe3O4@Calix-2E containing hydrazide groups that make more

effective-ness and protecting the active center of the enzyme by electrostatic in-teractions and hydrogen bonds [6,36–38].

3.4. Effect of pH and temperature on encapsulated enzyme activity It has been known that lipase activity is affected with pH of the solu-tion. Residual activities of the encapsulated enzymes were measured in different pH values (4.0–9.0) (Fig. 5A). The optimum pH values for reaching efﬁcient hydrolysis of p-NPP were observed to be 7.0 for both Fe3O4@E and encapsulated lipases (Fe3O4@Calix-2E and Fe3O4@

Calix-3E). The results indicated that pH did not differ by encapsulation because there was no change in the conformation of the lipase and that

the charge density on the lipase surface did not change (at pH 7). Similar results have also been reported in the literature [39–47]. The encapsu-lated lipase with calix[4]arene derivative (Fe3O4@Calix-2E or Fe3O4@

Calix-3E) was more stable than the encapsulated lipase without calix [4]arene derivative (Fe3O4@E) between pH 4 and 5. The increased

toler-ance towards acidic pH for encapsulated lipase may be because the calix [4]arene dansyl derivative complexed with the functional groups of the lipase to provide a suitable microenvironment to reduce the deforma-tion of enzyme structure [42]. However, all encapsulated lipases lost some of their activity at alkaline condition (pH 8–9), remarkably, Fe3O4@Calix-2E lost more activity than Fe3O4@Calix-3E below pH 5.0,

while the pH tolerance of the two encapsulated lipases was comparable in the pH range of 6 to 9, possibly due to the different natures of the calix [4]arene derivatives. The encapsulated lipase without calix[4]arene de-rivative (Fe3O4@E) maintained 80% at pH 8.0 and 70% at pH 9.0 of its

ini-tial activity. Lack of improvement in stability under alkaline pH could be due to the dansyl groups carry amino groups, they do not form com-plexes with enzyme in an alkaline environment.

The optimum temperature was determined by the hydrolysis of p-nitrophenyl palmitate at pH 7.0 from 30 °C to 60 °C (Fig. 5B). The opti-mal reaction temperature of the encapsulated lipase without calixarene derivatives (Fe3O4@E) was nearly 35 °C, whereas it shifted to 45 °C for

the encapsulated lipases (Fe3O4@Calix-2E and Fe3O4@Calix-3E).

This effect can be arise due to conformational mobility of enzyme and consequently multipoint interaction between the calixarene and the enzyme or improved substrate diffusion at a high temperature [48,49]. The results suggested that the sol-gel encapsulation process with calixarene derivative might be able to increase the conformational rigid-ity of enzyme and protect the lipase against denaturation at high temperature.

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3.5. Thermal stability of encapsulated enzymes

Thermal stability of encapsulated lipases (Fe3O4@E, Fe3O4@Calix-2E

and Fe3O4@Calix-3E) was incubated at 60 °C for 2 h and analyzed

enzy-matic activity over different periods (Fig. 5C). Encapsulated lipase with-out calixarene derivative (Fe3O4@E) lost its initial activity within about

100 min, after keeping 60 °C for 120 min, while the encapsulated lipases (Fe3O4@Calix-2E and Fe3O4@Calix-3E) retained about 43% and 40% of

their initial activity, respectively.

As a result Fe3O4@Calix-2E and Fe3O4@Calix-3E were more stable

than that of Fe3O4@E. These results have enabled the preservation of

enzyme activity in Fe3O4@Calix-2E, and Fe3O4@Calix-3E nanoparticles

at high temperatures over a long period of time.

3.6. Enantioselective hydrolysis of racemic Flurbiprofen methyl ester Enzymatic resolutions of racemic mixtures are used to obtain enantiopure compounds by chiral chemical synthesis for the pharma-ceutical industry. The reaction was monitored by HPLC, the enantio-meric compositional analysis of the materials obtained and the enantiomeric excesses (% ee) determined by chiral columns in high

pressure liquid chromatography (HPLC). The results were analyzed on HPLC using a mobile phase of hexane/2-propanol/triﬂuoroacetic acid (100/0.1/0.1) at 254 nm at aﬂow rate of 1 mL/min (Scheme 2,Table 2). In our earlier study [4], we used two mercapto derivative of calix[4] arenes capped on Fe3O4nanoparticles as additives for the encapsulation

of enzyme to work the possible effects of the calix[4]arene derivatives in the enantioselective hydrolysis reaction of racemic Flurbiprofen methyl ester. It was perceived that enantioselectivity (E = 244) after 72 h was found for most enzyme preparations in this work with an ee % value of S-Flurbiprofen about 98%.

Table 1

The comparison between different encapsulated enzymes. Encap. protein (mg/g) Lipase activity (U/g support) Speciﬁc activity Fe3O4@Ea 15 29 1.3 Fe3O4@Calix-2E 19 119 6.2 Fe3O4@Calix-3E 29 116 4.0 a

Encapsulated enzyme without magnetic calixarene derivatives. Fig. 4. A) Vibrating sample magnetometer (VSM) supermagnetization curves of Fe3O4@Calix-2; (B) Encapsulated lipase Fe3O4@Calix-2E;

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Enantioselective hydrolysis of (R,S)-Flurbiprofen methyl ester was determined by incubation with encapsulated lipases at different tem-peratures (35 °C, 45 °C) at pH = 7 in the presence of different additives (Fe3O4@Calix-2E and Fe3O4@Calix-3E). Optimum temperature values

for enantioselective hydrolysis studies were best observed at 45 °C and 48 h for both encapsulated enzymes and the best enantioselectivity and conversion. As shown inFig. 6, the encapsulated enzyme with calix [4]arene derivatives (Fe3O4@Calix-2E and Fe3O4@Calix-3E) show high

enantioselectivity and more conversion at pH 7, temperature 45 °C and 48 h than the encapsulated lipase without calix[4]arene derivative (Fe3O4@E).

The reusability of encapsulated enzymes is a key factor for its cost-effective use for industrial application. Therefore, the reusability of the encapsulated lipases (Fe3O4@Calix-2E and Fe3O4@Calix-3E) for

re-peated batch biotransformation of (R,S)-Flurbiprofen methyl esters to (S)-Flurbiprofen was assessed. As shown inFig. 7, the encapsulated en-zymes (Fe3O4@Calix-2E and Fe3O4@Calix-3E) still kept 38% and 30% of

their conversion ratios for the encapsulated enzymes using (R,S)-Flurbiprofen methyl esters as substrate after the 5 cycles, respectively, making the encapsulated enzyme suitable for reuse in industrial applications.

4. Conclusions

In the present work, an encapsulated lipase was prepared using a novel approach involving co-entrapment ofﬂuorescent calix[4]arene derivative containing dansyl moiety and enzyme and magnetic nano-particles in a silica matrix using enzyme-assisted direct condensation reactions of silicic acid. It has been examined that the resulting magnetic encapsulated lipases exhibited higher operational and thermal stabili-ties than the encapsulated lipase without calix[4]arene derivative. The encapsulated enzyme could be easily recovered by applying an external

Table 2

Enantioselective hydrolysis of racemic Flurbiprofen methyl ester using encapsulated lipases.-x (%) ees(%) eep(%) E Fe3O4@E (48 h)a 36 55 N98 173 Fe3O4@E (96 h)a 20 22 N98 123 Fe3O4@E (144 h)a 14 16 N98 116 Fe3O4@Calix-2E (48 h) 49 85 N98 270 Fe3O4@Calix-2E (96 h) 34 50 N98 163 Fe3O4@Calix-2E (144 h) 19 20 N98 120 Fe3O4@Calix-3E (48 h) 47 81 N98 249 Fe3O4@Calix-3E (96 h) 32 48 N98 159 Fe3O4@Calix-3E (144 h) 10 10 N98 109 a

Encapsulated enzyme without calixarene derivatives.

Scheme 2. Schematic representation of enantioselective hydrolysis of (R,S)-Flurbiprofen methyl ester with Fe3O4@Calix-2E or Fe3O4@Calix-3E

Fig. 5. The pH activity curves (A), temperature activity curves (B) and thermal stability curves (C) for encapsulated enzymes.

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magnet. For this reason, the simple route employed can yield easily recycled biocatalyst with high activity, and this can offer a very promis-ing applications.

Acknowledgments

We would like to thank the Scientiﬁc Research Foundation of Selcuk University, Konya, Turkey (BAP Project No. 15201094) forﬁnancial sup-port of this work.

Appendix A. Supplementary data

Supplementary data to this article can be found online athttps://doi. org/10.1016/j.ijbiomac.2019.04.182.

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