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Functional Group Effects of New Calixarene
Derivatives on Catalytic and Enantioselective
Behavior of Lipase
Vildan Dogan, Serkan Sayin, Arzu Uyanik & Mustafa Yilmaz
To cite this article: Vildan Dogan, Serkan Sayin, Arzu Uyanik & Mustafa Yilmaz (2019) Functional Group Effects of New Calixarene Derivatives on Catalytic and Enantioselective Behavior of Lipase, Polycyclic Aromatic Compounds, 39:4, 318-331, DOI: 10.1080/10406638.2017.1326949
To link to this article: https://doi.org/10.1080/10406638.2017.1326949
Published online: 08 Jun 2017.
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Functional Group Effects of New Calixarene Derivatives on Catalytic
and Enantioselective Behavior of Lipase
Vildan Dogana, Serkan Sayinb, Arzu Uyanika, and Mustafa Yilmaza
aDepartment of Chemistry, Selcuk University, Konya, Turkey;bDepartment of Environmental Engineering, Giresun
University, Giresun, Turkey
ARTICLE HISTORY Received August Accepted April KEYWORDS Calixarene; enantioselectivity; FeO nanoparticles; flurbiprofen; lipase ABSTRACT
In this study, two new calixarene derivatives bearing thiourea and carba-mate moieties were synthesized and characterized. Moreover, thiourea- and carbamate-bridged calixarene derivatives with Fe3O4 magnetic
nanopar-ticle were employed for the first time as the convenient additives in the encapsulation process of lipase. The results of catalytic activity and enantios-electivity of the encapsulated lipases in the hydrolysis reaction of racemic flurbiprofen methyl ester indicate that both of the encapsulated lipases (Enc-TuC[4]@Fe3O4and Enc-CbC[4]@Fe3O4) exhibit higher conversion and
enantioselectivity compared to the free-encapsulated lipase (Enc-Lipase). However, the highest affinities result was obtained when the encapsulated lipase (Enc-CbC[4]@Fe3O4) was used in the kinetic resolution reaction of
racemic flurbiprofen methyl ester.
Introduction
Flurbiprofen [2-(2-fluoro-4-biphenyl)-propionic acid], also known as a member of the 2-arypropanoic acids, is one of the major nonsteroidal anti-inflammatory drugs that is mainly used in pain treatment and tissue injury inflammation(1–3). Because each of its enantiomers exhibit different effects, the enan-tiomeric resolution of flurbiprofen is required(4). For instance, S-enantiomer inhibits the activity of cyclooxygenase Cox-2 and cyclooxygenase Cox-1 that causes gastrointestinal side effects, while (R)-flurbiprofen is considered as an inactive isomer for not inhibiting cyclooxygenase(4). Furthermore, (S)-flurbiprofen demonstrates 30 times greater anti-inflammatory affinity than its R-enantiomer(5, 6). Therefore, producing the enantiopure (S)-flurbiprofen for the market is highly needed.
Lipases being the most widely used class of enzymes are employed as catalysts(7, 8)in the hydrolysis of 2-arypropanoate esters in order to produce optically pure isomer (mostly S-enantiomer)(9–12). Thanks to the active center of lipase, lipase having a stable conformation builds a specific environment for the enantiomeric separation(4). However, it is hard to recover and reuse from the aqueous solutions of lipases(13, 14). In this regard, to enhance both of catalytic/enantioselective affinities and their recovery and reuse issues, different kind of carriers such as cyclodextrin, Fe3O4 nanoparticles, celite, chitosan,
amberlite XAD 7, and calixarene etc. have been employed as an additive for the lipase encapsulation
(15–20).
Calixarenes produced from the condensation of p-substituted phenol and formaldehyde have played a prominent role in supramolecular chemistry(21–29). Up to now, various calixarene derivatives such as carboxylic acids, alcohols, or amines have been applied as an additive for the lipase encapsulation in order to enhance lipase’s catalytic activity and enantioselectivity(14, 15, 30–32).
CONTACT Mustafa Yilmaz myilmaz@yahoo.com Department of Chemistry, Selcuk University, -Konya, Turkey. Color versions of one or more of the figures in the article can be found online atwww.tandfonline.com/gpol.
To our best knowledge, no effort about the affinities of carbamate- or thiourea-bridged calixarene derivative on the catalytic activity and enantioselectivity of the lipase has been carried out. With that in mind, we synthesized two new p-nitro aniline-substituted calixarene derivatives, bridged with thiourea, and carbamate moieties. FTIR,1H-NMR,13C-NMR, and elemental analysis were performed to char-acterize their structures. In addition, carbamate-bridged calixarene and thiourea-bridged calixarene derivative with Fe3O4nanoparticles were used as additives for the encapsulation of lipase by applying
sol-gel process for the first time. In order to determine affinities of the encapsulated lipases, the encapsulated lipases were used as catalysts for the enantioselective hydrolysis reaction of (R/S)-flufbiprofen methyl ester.
Materials and methods
Experimental section Reagents
Merck plates (silica gel 60 F254 on aluminum) were used for Analytical Thin-Layer Chromatography
(TLC). All chemicals, such as starting materials, reagents, bovine serum albumin, p-NPP, and Candida rugosa lipase, were obtained in standard analytical grades from Sigma-Aldrich, Across and Merck. HPLC grade solvents were driven as the mobile phase solvents. All aqueous solutions were prepared with deion-ized water using a Millipore milli-Q Plus water purification system.
Instrumentation
1H and13C-NMR spectra were recorded on a Varian 400 MHz spectrometer. A Perkin-Elmer 1605
spec-trometer with ATR probe was performed for FTIR analysis. UV-vis spectra were obtained on a Shimadzu 160A UV-vis spectrophotometer. Melting points were obtained on an EZ-Melt apparatus in a sealed capillary and are uncorrected. A Leco CHNS-932 analyzer was used for elemental analyses. A Chiralcel OD-H (25 cm, 4.6 mm) column was performed for HPLC studies using Agilent 1200 HPLC instrument.
Synthesis
p-tert-butylcalix[4]arene (1), calix[4]arene (2), 25,27-dimethoxycarbonylmethoxy-26,28-dihydroxy-calix[4]arene (3), 5,11,17,23-tetra-tert-butyl-25,27-dimethoxycarbonylmethoxy-26,28-dihydroxy-calix [4]arene (4), 5,11,17,23-tetra-tert-butyl-25,27-dihydrazinamidcarbo-nylmethoxy-26,28-dihydroxy-calix[4]arene (5), and Fe3O4 nanoparticles were synthesized according to the literature
pro-cedures (15, 32–34). The syntheses of 5,11,17,23-tetra-tert-butyl-25,27-(4-(4-nitrophenyl)-1-dithiosemicarbazide) carbonylmetoxy-26,28-dihydroxy-calix[4]arene (6), 25,27-bis(3-hydroxypropyl)amidcarbonylmethoxy-26,28-dihydroxy-calix[4]arene (7) and 25,27-bis(3-(p-nitrophenylcarbamate)propyl)amidcarbonylmethoxy-26,28-dihydroxy-calix[4]arene (8) are reported here for the first time.
Synthesis of ,,,-tetra-tert-butyl-,-(-(-nitrophenyl)--dithiosemi-carbazide) carbonylmetoxy-,-dihydroxy-calix[]arene ()
To a solution of5 (0.5 g, 0.630 mmol) in 10 mL of CH2Cl2was added 4-nitrophenylisothiocyanate (0.91 g,
5.043 mmol). The mixture was stirred at ambient temperature. After 4.5 h, the formed precipitate was collected, and washed with CH2Cl2and water. Synthesized pale yellow-colored6 was yielded in 82.5%.
M.p.: 184–187°C. FTIR : νmax(ATR): 3448, 2955, 2869, 1708, 1332 cm−1;1H-NMR (400 MHz
DMSO-d6):δ 1.10 (s, 18H, But), 1.18 (s, 18H, But), 3.43 (d, 4H, J=12.8 Hz, Ar-CH2-Ar), 4.35 (d, 4H, J=12.8 Hz, Ar-CH2-Ar), 4.73 (s, 4H, OCH2), 7.12 (brs, 8H, ArH), 7.88–7.92 (m, 4H, ArH), 8.17–8.20 (m, 6H, NH
and ArH), 10.01–10.64 (m, 6H, NH and OH) (seeFigure 2). 13C-NMR (100 MHz, DMSO):δ 31.3 (– CH3, But), 31.8 (–CH3, But), 34.1 (–C, But), 34.4 (Ar–CH2–Ar), 34.4 (–C, But), 73.6 (ArO–CH2–), 112.8, 122.5, 124.3, 124.9, 125.4, 125.9, 126.3, 126.8, 127.5, 127.8, 133.0, 133.3, 141.8, 145.8, 147.7, and 150.2 (ArC), 169.0 (C=O), 181.0 (C=S). Anal. Calcd. For C62H72N8O10S2(%): C; 64.56, H; 6.29, N; 9.71,
Synthesis of ,-bis(-hydroxypropyl)amidcarbonylmethoxy-,-dihydroxy-calix[]arene ()
A reaction mixture of diester derivative 3 (1.5 g, 2.638 mmol) and 3-aminopropan-1-ol (1.59 g, 21.104 mmol) in 30 mL of toluene/MeOH (1/1, v/v) was stirred under reflux for 2 h. The volatile com-ponents were evaporated to dryness, and residue was washed with water to adjust pH 7.0. The crude product was re-crystallized from MeOH to produce white-colored product7 in 76% of yield. M.p.: 244– 246°C. FTIR (ATR) cm−1: 1661 (C=O).1H-NMR (400 MHz, CDCl3):δ 1.71 (brs, 2H, –OH), 1.83 (p,
4H, J= 6.4 Hz, –CH2–), 3.54 (d, 4H, J= 13.2 Hz, Ar–CH2–Ar), 3.62 (q, 4H, J= 6.4 Hz, –CH2–O), 3.69 (t,
4H, J= 6.0 Hz, –CH2–N), 4.17 (d, 4H, J= 13.2 Hz, Ar–CH2–Ar), 4.65 (s, 4H, ArO–CH2), 6.48 (t, 2H, J=
7.6 Hz, ArH), 6.89 (t, 2H, J= 7.2 Hz, ArH), 7.02 (d, 4H, J= 7.6 Hz, ArH), 7.14 (d, 4H, J= 7.6 Hz, ArH), 8.15 (s, 2H, Ar–OH), 9.03 (t, 2H, J= 5.6 Hz, –NH). Anal. Calcd. For C38H42N2O8: C, 69.71; H, 6.47; N,
4.28. Found (%): C, 69.77; H, 6.30; N, 4.36.
Synthesis of ,-bis(-(p-nitrophenylcarbamate) propyl)amidcarbonyl methoxy-,-dihydroxy-calix[]arene ()
To a solution of7 (0.2 g, 0.305 mmol) in 10 mL CH2Cl2 was added 4-nitrophenylisocyanade (0.5 g,
3.05 mmol). The reaction mixture was stirred under reflux for 4 h, then the volatile components were evaporated to dryness. Remaining residue was neutralized with water, and re-crystallized from MeOH. White-colored product8 was synthesized in 81% of yield. M.p.: 253–255°C. FTIR (ATR) cm−1: 1734 (C=O, carbamate), 1664 (C=O, amide), 1495 (N–O asymmetric vibration), 1297 (N–O symmetric vibration).1H-NMR (400 MHz, DMSO):δ 1.93–1.99 (m, 4H, –CH2–), 3.44–3.49 (m, 8H, Ar–CH2–
Ar and O–CH2), 4.20–4.24 (m, 8H, Ar–CH2–Ar and N–CH2), 4.57 (s, 4H, ArO–CH2), 6.59 (t, 2H, J = 7.2 Hz, ArH), 6.81 (t, 2H, J = 7.2 Hz, ArH), 7.05 (d, 4H, J = 7.6 Hz, ArH), 7.13 (d, 4H, J = 7.6 Hz, ArH), 7.64 (d, 4H, J= 9.2 Hz, ArH), 8.14 (d, 4H, J = 8.8 Hz, ArH), 8.33 (s, 2H, –NH), 8.59–8.61 (m, 2H, –NH), 9.64–9.73 (m, 2H, –OH). 13C-NMR (100 MHz, DMSO):δ 29.0 (–CH2–), 30.9 (N–CH2–), 35.8 (Ar–CH2–Ar), 63.2 (O–CH2–), 74.6 (ArO–CH2–), 120.0 (ArC), 125.4 (ArC), 125.6 (ArC), 126.2 (ArC), 126.8 (ArC), 127.8 (ArC), 129.2 (ArC), 129.6 (ArC), 134.0 (ArC), 142.0 (ArC–N), 146.1 (ArC– NO2), 146.2 (O–CAr), 152.3 (O–CAr), 153.6 (C=O, carbamate), 168.3 (C=O, amide). Anal. Calcd. For C52H50N6O14: C, 63.54; H, 5.13; N, 8.55. Found (%): C, 63.46; H, 5.07; N, 8.62.
Encapsulation studies of lipase
A modified method of Reetz(35)was carried out in the sol-gel encapsulation process of lipase with or without calixarene and Fe3O4 magnetic nanoparticles additives. Typically, a mixture of CRL-type
VII (0.245 g) in 50 mM phosphate buffer (1.56 mL, pH 7.0) was shaken. Calixarene derivative and Fe3O4magnetic nanoparticles was added to the mixture, and followed by addition of polyvinyl
alco-hol (400µL, 4% w/v), 0.1 M NaF (200 µL), isopropyl alcohol (400 µL), TEOS (460 µL, 0.5 mmol), and octyltrimethoxysilane (3.2 mL, 2.5 mmol), respectively. After 10–15 s, 15 mL of isopropyl alcohol was poured onto the produced white solid. Obtained gel was washed with 10 mL of ultra-pure water and isopropyl alcohol, and let it to be lyophilized to afford the encapsulated lipases.
Determination of the encapsulated lipases’ catalytic activity
The hydrolysis reaction of p-NPP in 0.05 M of buffer (pH 7.0) was performed to assess the activities of the encapsulated lipases with or without additives. The concentration of the corresponding hydrolysis product was determined at 400 nm using a spectrophotometer(36).
The Bradford protein assay method(37)relying on measurement of initial and final concentration of protein was used to find protein content to the encapsulated lipases.
Various pHs ranging from 4.0 to 9.0 were evaluated to elucidate the changes of free and immobilized lipases’ activities. Furthermore, the thermal inactivation behaviors of the free and immobilized lipases were evaluated at 30–60°C. Each form of enzymes was incubated in PBS (50 mm) at pH 7.0 for 20 min
at different temperatures and, after lowering the temperature down, the obtaining lipase activity was assayed by applying the standard conditions and analyzed(36, 37).
Thermal stability
Both encapsulated lipases in the presence and absence of additive were immersed into 50 mM of buffer solution adjusted at pH 7.0 and 60°C for 2 h, in order to determine their catalytic activity as mentioned above.
Enantioselective hydrolysis of (R/S)-flurbiprofen methyl ester
(R/S)-Flurbiprofen methyl ester was hydrolyzed by the encapsulated lipases with or without additives in a mixture of PBS and isooctane according to the literature procedure(15, 20). The conversion and enantioselectivity of (R/S)-Flurbiprofen methyl ester by the encapsulated lipases were expressed as the enantiomeric ratio (E), which was calculated from the conversion (x) and the enantiomeric excess of the substrate (ees) and the product (eep) by using HPLC(38).
E = In[(1− x)(1 − ees)] In[(1− x)(1 + ees)] x= ees ees+ eep ees= CR− CS CR+ CS eep = CS− CR CS+ CR
E, ees, eep, x, CR, and CSimply enantiomeric ratio for irreversible reactions, enantiomeric excess of product, enantiomeric excess of substrate, racemate conversion, concentration of R-and S-enantiomer of flurbiprofen, respectively.
Results and discussion
Synthesis and characterization of calixarene additives for the encapsulation processes
The concept of this work is not only the synthesis of new calixarene additives for the encapsulation of lipase, but also to provide insight into which functional groups of calixarene has a significant effect on the lipase activity in the enantioselective hydrolysis reaction. For this goal, di-hydrazine amine derivative 5 was interacted with 4-nitrophenylisothiocyanate to produce thiourea-bridged calixarene derivative (see
Scheme 1). FTIR,1H, and13C-NMR, and elemental analysis techniques were performed to assess the structure of all synthesized calixarene derivatives.
Two specific vibrations at 1709 cm−1 (for C=S) and 1697 cm−1 (for C=O) in the FTIR spec-tra of 5,11,17,23-tespec-tra-tert-butyl-25,27-(4-(4-nitrophenyl)-1-dithiosemi-carbazide) carbonylmetoxy-26,28-dihydroxy-calix[4]arene (6) confirm the structure of 6 (seeFigure 1)
1H-NMR spectrum of the thiourea-conjugated calixarene 6 exhibits that derivative 6 is in a cone
confirmation; it possesses a typical AX pattern for the methylene bridge protons (ArCH2Ar) at 3.43 and
4.35 ppm (J= 12.8 Hz) (seeFigure 2). Moreover, appearance of additional peaks at 169 ppm (for –C=O)
and 181 ppm (–C=S) on the13C-NMR spectrum confirm precisely the structure of 6 (seeFigure 3).
To synthesize carbamate-bridged calixarene derivative, first, 25,27-bis(3-hydroxypropyl) amidecarbonylmethoxy-26,28-dihydroxy-calix[4]arene (7) was obtained by the reaction of di-ester derivative 3 with 3-hydroxypropylamine in toluen/MeOH. Then, the reaction of 25,27-bis(3- hydroxypropyl)amidcarbonyl-methoxy-26,28-dihydroxy-calix[4]arene (7) with 4-nitrophenylisocyanade yielded 25,27-bis(3-(p-nitrophenylcarbamate)propyl)amidcarbonylmethoxy-26,28-dihydroxy-calix[4]arene (8), conjugated with carbamate moieties (seeScheme 1).
In the FTIR spectra presented in Figure 4, appearance of the additional band at 1733 cm−1 of carbamate carbonyl (C=O) confirms the structure of 5,27-bis(3-(p-nitrophenylcarbamate)propyl)amidcarbonylmethoxy-26,28-dihydroxy-calix[4]arene (8). Moreover,
Scheme .Preparation of calixarene derivatives (6 and 8). Reaction conditions: (i) HCHO, NaOH; (ii) AlCl, Phenol; (iii) KCO, methylbro-moacetate; (iv) NH.HO; (v) -nitrophenylisothiocyanate; (vi) -aminopropan--ol; and (vii) -nitrophenylisocyanade.
1H-NMR spectrum of 8 confirms its structure appearing not only all the protons of amide and
carba-mate groups, but also the characteristic peaks at 7.64 and 8.14 ppm with 9.2 and 8.8 Hz “J” values of aromatic protons of p-nitro aniline moieties (seeFigure 5). Besides the appearance of the peaks of both the amide carbonyl at 168.4 ppm and the carbamate carbonyl at 153.6 ppm on13C-NMR spectrum, it clarifies complementary information to the FTIR, 1H-NMR, and elemental analysis results (see
Figure .FTIR (ATR) spectra of ,,,-tetra-tert-butyl-,-(-(-nitrophenyl)--dithiosemicarbazide) carbonylmetoxy-,-dihydroxy-calix[]arene (6).
Two different functional groups-bridged calix[4]arene derivatives and Fe3O4additives contributed lipase encapsulation processes
In this study, two different functional groups-bridged calixarene derivatives (6 and 8) and Fe3O4
nanoparticles were used as new additives for the encapsulation of C. rugosa lipase to afford two new encapsulated lipases (Enc-TuC[4]@ Fe3O4, Enc-CbC[4]@Fe3O4, represented inFigure 7). To evaluate
their catalytic and enantioselective affinities, the encapsulated lipases were used as catalysts in the hydrolysis reaction of (R/S)-flurbiprofen methyl ester. The Bradford method using bovine serum albumin (BSA) as a standard was performed to assess the protein amount in both the enzyme solution and the elution solute(37). According to a previously published procedure(36), the catalytic resolution activity of the encapsulated lipases with or without calixarene and Fe3O4 magnetic nanoparticles
additives was estimated, as well.
Figure .H-NMR ( MHz DMSO-d
) spectrum of ,,,-tetra-tert-butyl-,-(-(-nitrophenyl)--dithiosemicarbazide)
Figure .C-NMR ( MHz DMSO-d
) spectrum of ,,,-tetra-tert-butyl-,-(-(-nitrophenyl)--dithiosemicarbazide)
carbonylmetoxy-,-dihydroxy-calix[]arene (6).
The micrograph of the nanoparticles in the free-encapsulated lipases (Figure 8a) were compared with the micrograph of scanning electron microscopy in order to prove the presence of calixarene into the Enc-TuC[4]@ Fe3O4(Figure 8b). The differences on SEM images indicated that calixarene was
success-fully immersed into the encapsulated lipase.
The results of the initial attempt on the investigation of the catalytic activity of the encapsulated lipases (Enc-TuC[4]@ Fe3O4, Enc-CbC[4]@ Fe3O4, and Enc-lipase that produced in the absence of
additives) were given inTable 1. It can be shown that the encapsulated lipase Enc-TuC[4]@ Fe3O4
exhibits the highest affinity in terms of specific activity among others encapsulated lipases (Enc-lipase and Enc-CbC[4]@Fe3O4). However, Enc-CbC[4]@Fe3O4 demonstrates higher specific activity than Enc-lipase. This finding clearly attributes that the functional groups of calixarenes play critical role to form complex such as hydrogen bonding, host-guest and dipole/ion-dipole interactions with lipase into sol-gel matrix. The complex strength of thiourea-bridged calixarene and lipase may hold the protein in a stable conformation reducing the overall flexibility besides enhancing the lipase activity.
Figure .H-NMR ( MHz DMSO-d
) spectrum of ,-bis(-(p-nitrophenylcarbamade)propyl)
amidcarbonylmethoxy-,-dihydroxy-calix[]arene (8).
Effect of temperature and pH on the lipase activity
Analytical parameters, such as pH and temperature, indeed, are known to be influential parameters that guide different catalytic activities by conformational changing of the enzyme. In this work, different pHs (4.0–9.0) and temperatures (25–50°C) were applied in the hydrolysis process of p-nitro phenyl palmitate (p-NPP) by the encapsulated lipases.
Figure .C-NMR ( MHz DMSO-d
) spectrum of ,-bis(-(p-nitrophenylcarbamade)propyl)
Figure .Encapsulation of lipase. Reaction conditions: (i) phosphate buffer at pH ., tetramethoxysilane, isopropyl alcohol, NaF, polyvinyl alcohol, octyltri-methoxysilane.
Figure 9indicates that free-encapsulated lipase (Enc-lipase) demonstrates maximum enzymatic activ-ity at pH 7.0, whereas the observed efficient hydrolysis capacactiv-ity for both encapsulated lipases (Enc-TuC[4]@ Fe3O4and Enc-CbC[4]@ Fe3O4) was found at pH 8.0.
Figure 10 depicts the results of relative activities of the encapsulated lipases at various tempera-tures (25–50°C) and optimal pHs. It was observed that the encapsulated lipase with carbamate-bridged
Figure .SEM images of (a) the nanoparticles in the free encapsulated lipases, (b) the nanoparticles in the encapsulated lipases ( Enc-TuC[4]@Fe3O4).
calixarene in the presence of Fe3O4nanoparticles (Enc-CbC[4]@Fe3O4) showed high catalytic
activ-ity at 40°C, whereas the encapsulated lipase with thiourea-bridged calixarene in the presence of Fe3O4
nanoparticles (Enc-TuC[4]@Fe3O4) had the highest activity percentage at 35°C.
Enantioselective hydrolysis of (R/S)-flurbiprofen methyl ester
In order to enhance the lipase activity and enantioselectivity in the catalytic resolution reaction of racemic pro-drugs, up to now, several calixarene derivatives have been designed as an additive, which mostly complexes with lipase into sol-gel media by means of electrostatic, hydrogen bonding, dipole– dipole, and ion–dipole interactions etc.(15, 18–20). However, in none of them, the functional groups effects have been addressed. In order to understand the functional groups effects of additives on the lipase activity and enantioselectivity in the catalytic resolution reaction of racemic flurbiprofen methyl ester, herein, two different calixarene derivatives (6 and 8) were employed as an additive together with Fe3O4nanoparticles, which makes the separation easy and quick task, in the encapsulation processes of
Table .Enantioselectivity and catalytic activity behaviors of the encapsulated lipases in enantioselective hydrolysis reaction of (R/S)-flurbiprofen methyl ester under optimum reaction conditions.
Protein loading Lipase activity Specific activity Conversion
Lipase (mg/g−sol gel) (Ug−sol gel) (U mg−protein) (x, %) eep(%) E
Enc-Lipasea . . >
Enc-TuC[4]@Fe3O4 . . >
Enc-CbC[4]@Fe3O4 . . >
aEncapsulated lipase without additive.
To evaluate the conversion and enantioselectivity affinities of encapsulated lipases against (R/S)-flurbiprofen methyl ester, HPLC was performed according to the literature procedure(38). The catalytic resolution reaction of racemic flurbiprofen methyl ester by the encapsulated lipases in a mixture of aque-ous buffer solution and isooctane yielded with R-flurbiprofen methyl ester and the corresponding acid, the percentage of conversion (x) 40.0% for Enc-TuC[4]@Fe3O4and 43.0% for Enc-CbC[4]@Fe3O4(see Table 1). Moreover,Table 1also shows the encapsulated lipases’ enantioselectivities as E value of 199 for Enc-TuC[4]@Fe3O4and 224 for Enc-CbC[4]@Fe3O4, whereas the encapsulated lipase without additive
(Enc-lipase) exhibits E value of 174. These findings attribute that the encapsulation of the lipase with calixarenes led higher stereoselectivity and better conversion than the encapsulated free lipase. Besides,
Figure .Substrate pH affinity on residual catalytic activity of encapsulated lipases. Conditions: pHs (.–.).
Figure .The residual catalytic activity of encapsulated lipases against reaction temperature. Conditions: various temperature (– °C), at pH . for Enc-lipase, and at pH . for both of Enc-TuC[]@ FeOand Enc-CbC[]@ FeO.
Figure .Reusability on the conversion (x) in the hydrolysis of racemic flurbiprofen methyl ester. Conditions: at °C and optimal pHs. these results also address that the carbamate-bridged calixarene holds the protein of the enzyme in a stable conformation and consequently reduces the overall flexibility and enhances the lipase activity.
To visualize the recovery and reusability capacities of the encapsulated lipases, an effort was carried out (seeFigure 11).Figure 11depicts Enc-TuC[4]@Fe3O4and Enc-CbC[4]@Fe3O4 still retained 19%
and 17% of their conversion ratios after the fourth reuse, respectively. These results express that the encapsulated lipases with calixarene in the presence of Fe3O4nanoparticles would make them a unique
candidate in terms of effect and economical usage of enzymes.
Conclusion
In this study, we synthesized two new calixarene derivatives, substituted with different p-nitroanilin derivatives bridged with thiourea and carbamate moieties. Then, the carbamate-bridged calixarene derivative and thiourea-bridged calixarene derivative were used as additives with Fe3O4 magnetic
nanoparticles in the encapsulation process of Candida rugosa lipase to afford two different encapsu-lated lipases (Enc-TuC[4]@ Fe3O4, Enc-CbC[4]@ Fe3O4). To elucidate the influences of the functional
groups of the calixarene additives on lipase activity and enantioselectivity, two new encapsulated lipases (Enc-TuC[4]@ Fe3O4, Enc-CbC[4]@ Fe3O4) were employed as catalysts for the enantioselective hydrol-ysis reaction of (R/S)-flufbiprofen methyl ester. Enc-TuC[4]@Fe3O4exhibited higher enantioselectivity,
with an E value of 199, than the encapsulated lipase without additive (Enc-lipase), with an E value of 174; however, Enc-TuC[4]@Fe3O4had less enantioselectivity affinity for (R/S)-flufbiprofen methyl ester than
Enc-CbC[4]@Fe3O4, with an E value of 224. These findings indicate that carbamate-bridged calixarene
plays a substantial role for lipase encapsulation. In sum, the lipases encapsulated with calixarene in the presence Fe3O4nanoparticles provide an economically viable use of expensive enzymes and hence opens
a new horizon for enzymatic catalysis in biotechnology.
Funding
This work was supported by the Research Foundation of Selcuk University (BAP grant no. 15201004), Konya, Turkey.
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![Figure 9 indicates that free-encapsulated lipase (Enc-lipase) demonstrates maximum enzymatic activ- activ-ity at pH 7.0, whereas the observed efficient hydrolysis capacactiv-ity for both encapsulated lipases (Enc-TuC[4]@ Fe 3 O 4 and Enc-CbC[4]@ Fe 3 O 4](https://thumb-eu.123doks.com/thumbv2/9libnet/5386376.101319/10.756.130.633.72.838/indicates-encapsulated-demonstrates-enzymatic-efficient-hydrolysis-capacactiv-encapsulated.webp)
