• Immobilization of Candida rugosa lipase for resolution of racimic ibuprofen
  • Saeid Ghofrani1 & Abdolamir Allameh2 & Parichehreh Yaghmaei1 & Dariush Norouzian3,1 Saeid Ghofrani 1, Abdolamir Allameh 2, Parichehreh Yaghmaei 1, Dariush Norouzian 3,2 Saeid Ghofrani1 & Abdolamir Allameh2 & Parichehreh Yaghmaei1 & Dariush Norouzian3,3,*
    1. 1Science and Research Branch, Islamic Azad University, Tehran, Iran. 2Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran. 3Nano-Biotechnology Department, New Technologies Research Group, Pasteur Institute of Iran, Tehran, Iran. dnsa@pasteur.ac.
    2. 1Science and Research Branch, Islamic Azad University, Tehran, Iran. 2Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran. 3Nano-Biotechnology Department, New Technologies Research Group, Pasteur Institute of Iran, Tehran, Iran. dnsa@pasteur.ac.
    3. 1Science and Research Branch, Islamic Azad University, Tehran, Iran. 2Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran. 3Nano-Biotechnology Department, New Technologies Research Group, Pasteur Institute of Iran, Tehran, Iran. dnsa@pasteur.ac.


  • Introduction: Aim Due to lipases’ regio-selectivity and ability to catalyze different reactions such as hydrolysis, esterification, andrecently attracted much attention because of its regio-selectivity in various types of reactions, such as trans-esterification, hydrolysis, and esterification [1, 2]. The enzyme’s ability to catalyzing different reactions has recently made it possible for investigators to develop novel resolution methods based on lipase catalytic activity and specificity. The resolution of racemic mixtures based on the enzymatic reaction is the most recent novel method with high efficiency and the ability to enter the commercial scales [3]. Principally, lipase’s specific molecular structure with an active center can discriminate between a racemic mixture’s enantiomers and the enantiomeric resolution [4]. According to close (inactive) and open (active) molecular structures of lipase and the effect of the nature of reaction medium on the enzyme structure, the amount of water in lipase-catalyzed media has a remarkable impact on the enzyme catalytic enantioselectivity. From the mechanistic view, the small number of water molecules can significantly affect the 3D molecular structure of lipase, molecular stability, and the active site’s polarity, all of which play vital roles in the catalyzing process [5, 6]. Many reports indicated that lipase could catalyze a broad range of reactions in organic media for industrial applications [7–10]. Using lipase, various types of enantiomeric resolutions have been reported. The enantiomeric resolution of the profen family of anti-inflammatory drugs has attracted much attention because of such drugs’ broad application [11–14]. Considering the presence of chiral carbon atom in profen molecular structure, the lipase-based enzymatic resolution has been recently proposed for the resolution of the (S)-enantiomer of ibuprofen. Pharmacologically, the activity of the (S)-enantiomer of ibuprofen is nearly 160 times
  • Methods: Lipase of Candida rugosa, racemic ibuprofen, 4-nitrophenyl palmitate, and silicon dioxide nanoparticles was obtained from Sigma Aldrich. A chiral column from Phenomenex (USA)was used. Other reagents used were of analytical grade. Immobilization of lipase on silica nanoparticles For immobilization of Candida rugosa lipase on silica nanoparticles, 2 mg of lipase powder was dissolved in 10mL phosphate buffer (0.2MNa2HPO4 and 0.2MNaH2PO4) at various pHs (6.0–9.5). The prepared lipase solution at different pHs was then added to a series of beakers, each containing 20 mg of silica nanoparticles, and was stirred at 4 °C for 30 min. Subsequently, the immobilized lipase on silica nanoparticles was separated using centrifugation (4 °C, 6000 rpm, 10 min). After all, the immobilized lipase was washed with phosphate buffer and ethanol, respectively. The total protein on the supernatant was measured by the Bradford method [27, 28]. Lipase activity The enzyme activity was determined using p-nitrophenol palmitate (p-NPP) as a substrate. One mL of p-NPP (16.5 mM) in 2-propanol was added to 9 mL Tris-HCl (50 mM) buffer (pH 8) containing 0.4% (v/v) Triton X-100 and 0.1% (w/v) gum Arabica. Ten mg of lipase powder was weighed out and diluted with Tris-HCl buffer. Following this, 1.35 mL of the prepared substrate was incubated at 37 °C with 0.1 mL of the enzyme solution, and the absorbance was read at λ 410 nm within 3–5 min against blank devoid of the enzyme. One lipase activity unit was defined as 1 mL of enzyme solution that could liberate 1 μmol of p-nitrophenol (p-NP) as a product in one minute under assay condition [29]. Resolution of racemic ibuprofen In non-aqueous media, the enantiomeric resolution of racemic ibuprofen was made using the esterification reaction. For initiation of the esterification reaction, 40 mg of immobilized lipase with 20 mL of isooctane containing 20 μL of water were added to ibuprofen and n-propanol (0.025M). The reaction was performed at 37 °C under shaking. Conversion for esterification reaction (C) and the enantiomeric excess (ees) for products was calculated as per [30]. The analysis of racemic ibuprofen was studied through the Knauer HPLC system. The mobile phase contained n-hexane:2-propanol: acetic acid (99.2:0.6:0.2, v/v/v%) at a flow rate of 0.1 ml/min using Lux® 5 μm Cellulose-3, LC Column 250 × 4.6 mm, Ea. Results and discussion Adsorption of lipase on the silica nanoparticles The maximum adsorption of the enzyme onto nanosilica was observed at alkaline pH (pH 8.0). At an optimum pH of adsorption, the time of the immobilization process was another Fig. 1 The adsorption of Candida rugosa lipase on the silica nanoparticles
  • Results: Scanning electron microscopy of immobilized silica nanoparticles showed that the lipase agglomeration on the silica nanoparticles formed and the mentioned hybrid structures were appropriate for ibuprofen’s enzymatic resolution. Figure 2AC shows the 2, 10, and 20 μm scales of the immobilized silica nanoparticles. The size distribution analysis (DSL, Melvern, UK) of silica nanoparticles at room temperature revealed that the nanoparticles’ size distribution was in the range of 10 to 300 nm. The maximum amount of the particles were almost 100 nm in diameter. After the immobilization process, the immobilized silica nanoparticles’ size distribution was changed to be at the range of 20 to 500 nm. The immobilized particle distribution showed that the particles’ maximum amount was 200 nm in
  • Conclusion: In the present study, the silica nanoparticles as the most promising support for Candida rugosa lipase’s immobilization were applied. The immobilized lipase was used as an enantioselective catalyst for ibuprofen’s resolution during the esterification process using isooctane as an organic solvent. The results indicated that lipase’s immobilization on silica nanoparticles can enhance the optimum catalytic condition of enantioselective lipase resolution and increase conversion efficiency and enantiomeric excess (S) enantiomer of ibuprofen during the esterification reaction under the abovementioned conditions. Both time and pH of reactions affect the ees and C. Therefore, as compared to free lipase, the immobilized lipase showed better catalytic properties. Acknowledgments The authors acknowledge the technical support of the Department of Biochemistry, Tehran University of Medical Sciences. Declarations Conflict of interest The authors declare no conflict of interest. References 1. Xie Y-C, Liu H-Z, Chen J-Y. Candida rugosa lipase catalyzed esterification of racemic ibuprofen with butanol: racemization of R-ibuprofen and chemical hydrolysis of S-ester formed. Biotechnology Letters. 1998;20:455–8. 2. Shoda SI, Uyama H, Kadokawa JI, Kimura S, Kobayashi S. Enzymes as green catalysts for precisionmacromolecular synthesis. Chemical Reviews. 2016;116:2307–413. 3. Sie Yon L, Gonawan FN, Kamaruddin AH, Uzir MH. Enzymatic deracemization of (R, S)-ibuprofen ester via lipase-catalyzed membrane reactor. Ind Eng Chem Res. 2013;52:9441–53. 4. Muralidhar RV, Marchant R, Nigam P. Lipases in racemic resolutions. Journal of Chemical Technology & Biotechnology: International Research in Process, Environmental & Clean Technology. 2001;76:3–8. 5 . Bayramoğ l u G, Ar ıc a MY. Pr e p a r a t i o n of p o l y (glycidylmethacrylate–methylmethacrylate) magnetic beads: application in lipase immobilization. J Mol Catal B Enzym. 2008;55:76–83. 6. Yahya AR, Anderson WA, Moo-Young M. Ester synthesis in lipase-catalyzed reactions. Enzyme and Microbial Technology. 1998;23:438–50.
  • Keywords: Immobilization -Candida rugosa 0lipase =f racimic ibuprofen