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M. Yousefi et al. / Journal of Molecular Catalysis B: Enzymatic 104 (2014) 87–94
2. Materials and methods
ple multipoint covalent immobilization of a monomeric enzyme
resulted to generate a favorable environments surrounding the
have been stabilized by immobilizing all enzyme subunits, thus
preventing subunit dissociation [16]. In both cases, immobilization
yield a more stable biocatalyst [17].
2.1. Materials
Ibuprofen was extracted from the readily marketed tablets
according to literature procedure [40]. p-Nitrophenyl butyrate
´
˚
(p-NPB), Octyl-sepharose TM, molecular sieves (4 A, 4–8 mesh), (S)-
(+)-Ibuprofen (purity 99%), Lipase from C. rugosa and lipase from
R. oryzae were obtained from Sigma (Steinhiem, Germany). Other
reagents and solvents were of analytical or HPLC grade.
ical character of these enzymes [18]. A characteristic feature of
lipases is their activation in the presence of hydrophobic interfaces
[19]. This lipase activation at interfaces was first reported by Sarda
and Desnuelle [20] and now through X-ray structural studies of a
number of lipases, it is recognized as a common feature [21–29].
two structural forms, the closed one; where a polypeptide chain
(lid or flat) isolates the active center from the medium and the
open form; where this lid moves and the active center is exposed
changes take place yielding the open structure of lipases result-
ing in a significant increase in activity. In this way, lipases seem
to become strongly adsorbed to hydrophobic interfaces through a
large hydrophobic surface which surrounds the internal face of the
lipases may cause some difficulties in understanding and control-
ling the behavior of lipases in organic synthesis at both laboratory
and industrial scales. However this mechanism could also be used
[32].
Among different types of lipases, Candida rugosa lipase (CRL)
Rhizopus oryzae lipase (ROL) and Rhizomucor miehei lipase (RML)
ular sizes of CRL, ROL and RML are 57, 32 and 31.6 kDa, respectively
[26,35]. RML is probably the most used lipase obtained from fungi,
dimensional structure [36]. It is commercially available enzyme in
both soluble and immobilized form with very high activity and
good stability under diverse conditions (anhydrous organic sol-
vents, supercritical fluids, etc.) [37]. Among these lipases, ROL is a
high cost lipase which needs to bring the cost down for its industrial
applications. In this case, improving the lipase performance using
immobilization techniques and increasing the expression level of
ROL can be the powerful ways to lower operating costs of the
enzyme [38].
In our previous work we have reported immobilization of
R. miehei lipase (RML) via different protocols such as physical
adsorption and covalent attachment to catalyze hydrolysis of var-
ious ibuprofen esters. The results showed that RML immobilized
on octyl-sepharose had high activity and selectivity and butyl
ester was the most interesting ester for carrying out hydrolysis
[39].
The aim of the present investigation is development of an enzy-
matic method for the production of the S-enantiomer of ibuprofen.
For this purpose CRL and ROL were immobilized on octyl-sepharose
via physical adsorption and their application was examined in
two distinct reactions; (1) esterification of (R,S)-ibuprofen by n-
propanol in presence of two ionic liquid and isooctane and (2)
resolution of various (R,S)-ibuprofen esters by hydrolysis. The opti-
mization of hydrolysis reaction was performed regarding to the
amount and activity of the immobilized lipase. Enantioselectivity
of reused immobilized lipases has also been studied.
2.2. Immobilization of the lipases on octyl-sepharose
One gram of o ctyl-sepharose was suspended in 10 mL of enzyme
solution of ROL, and CRL (0.5 mg/ml) in 10 mM sodium phosphate
buffer at pH 7.0, and the mixture was shaken at 25 ◦C and 250 rpm
for 3 h. Thereafter, the immobilized enzyme was washed with dis-
tilled water 20 mL (three times) and stored at 4 ◦C. Suspension
sample and the supernatants were withdrawn periodically, and the
hydrolytic activity was measured using p-NPB as substrate.
2.3. Synthesis
2.3.1. General method for the chemical synthesis of ibuprofen
esters (1–5)
To a solution of 0.1 mol of the racemic acid in 100 ml toluene,
0.5 mol of corresponding alcohol (methanol, ethanol, propanol,
n-butanol and iso-butanol) was added followed by few drops of
sulphuric acid (98%) [41]. The mixture was stirred under reflux
over night and the solvent was evaporated under vacuum and the
residue was neutralized with 10% sodium hydrogen carbonate. The
ester was extracted twice with 50 ml chloroform and then dried
over anhydrous sodium sulphate. After filtration, the solvent was
evaporated under vacuum to afford the racemic ibuprofen esters.
2.3.1.1. Ibuprofen methyl ester (1). Pale yellowish oil (100%): 1H
NMR (CDCl3) ı 7.1–7.3 (dd, 4H, aromatic), 3.7 (q, 1H, CHCH3), 3.6
(s, 3H, OCH3), 2.4 (d, 2H, CH2CH(CH3)2), 1.9 (m, 1H, CH(CH3)2), 1.5
(d, 3H, CHCH3), 0.9 (d, 6H, CH(CH3)2).
2.3.1.2. Ibuprofen ethyl ester (2). Pale yellowish oil (100%): 1H NMR
(CDCl3) ı 7.1–7.2 (dd, 4H, aromatic), 4.1 (q, 2H, OCH2 CH3), 3.7 (q,
1H, CHCH3), 2.4 (d, 2H, CH2CH(CH3)2), 1.8 (m, 1H, CH(CH3)2), 1.5
(d, 3H, CHCH3), 1.2 (t, 3H, OCH2CH3), 0.9 (d, 6H, CH(CH3)2).
2.3.1.3. Ibuprofen propyl ester (3). Pale yellowish oil (100%): 1H
NMR (CDCl3) ı 7.1–7.2 (dd, 4H, aromatic), 4.0 (q, 2H, OCH2 CH3), 3.7
(q, 1H, CHCH3), 2.5 (d, 2H, CH2CH(CH3)2), 1.8 (m, 1H, CH(CH3)2), 1.6
(m, 2H, OCH2CH2CH3), 1.5 (d, 3H, CHCH3), 0.9 (t, 3H, OCH2CH2CH3),
0.8 (d, 6H, CH(CH3)2).
2.3.1.4. Ibuprofen butyl ester (4). Pale yellowish oil (100%): 1H NMR
(CDCl3) ı 7.1–7.3 (dd, 4H, aromatic), 4.0 (t, 2H, OCH2CH2CH2CH3),
3.7 (q, 1H, CHCH3), 2.4 (d, 2H, CH2CH(CH3)2), 1.8 (m, 1H,
CH(CH3)2), 1.5 (m, 2H, OCH2CH2CH2CH3), 1.4 (d, 3H, CHCH3),
0.9 (m, 2H, OCH2CH2CH2CH3), 0.8 (d, 6H, CH(CH3)2), 0.8 (t, 3H,
OCH2CH2CH2CH3).
2.3.1.5. Ibuprofen isobutyl ester (5). Pale yellowish oil (100%): 1H
NMR (CDCl3) ı 7.1–7.3 (dd, 4H, aromatic), 3.9 (t, 2H, OCH2
CH(CH3)2), 3.7 (q, 1H, CHCH3), 2.4 (d, 2H, CH2CH(CH3)2), 1.9 (m, 1H,
CH(CH3)2), 1.5 (m, 2H, OCH2 CH(CH3)2 and CH2CH(CH3)2), 1.4 (d,
1H, 3H, CHCH3), 0.9 (d, 6H, CH(CH3)2), 0.8 (d, 6H, OCH2 CH(CH3)2).