Langmuir 2008, 24, 8877-8884
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Immobilization of Lipases on Hydrophobilized Zirconia Nanoparticles: Highly Enantioselective and Reusable Biocatalysts Yi Zhao Chen, Cai Ting Yang, Chi Bun Ching, and Rong Xu* DiVision of Chemical & Biomolecular Engineering, School of Chemical and Biomedical Engineering, Nanyang Technological UniVersity, 62 Nanyang DriVe, Singapore 637459 ReceiVed May 5, 2008. ReVised Manuscript ReceiVed June 9, 2008 Our study has demonstrated for the first time that zirconia nanoparticles modified by a simple carboxylic surfactant of a very long alkyl chain can significantly enhance the activity of the immobilized lipases for asymmetric synthesis in organic media. Zirconia nanoparticles of ca. 20 nm diameter were grafted with carboxylic surfactant modifiers from Tween 85 and erucic acid. The surface of nanoparticles was successfully changed from hydrophilic to hydrophobic. Lipases from Candida rugosa and Pseudomonas cepacia were immobilized on the modified zirconia nanoparticles by adsorption in aqueous solution. The immobilized lipases were used for the resolution of (R,S)-ibuprofen and (R,S)-1-phenylethanol through esterification and acylation, respectively, in isooctane organic solvent. When immobilized on erucic acid-modified zirconia, both lipases gave significantly higher activity and enantioselectivity compared with those from their corresponding crude lipase powders. The nanohybrid biocatalysts are stable and can be reused for eight cycles without loss in activity and selectivity. The interaction between the hydrophobic surface of zirconia support and lipases probably induces the conformational rearrangement of lipases into an active, stable form.
Introduction The formation of enzyme-solid support composite materials via immobilization is important for applications in biosensing and biocatalysis.1,2 Over the past decade, increasing research effort has been focused on the production of optically pure enantiomers from racemic mixtures as a result of strict regulation by governments and pressure from the public.3,4 Lipases (i.e., triacylglycerol hydrolases (EC 3.1.1.3)) can be used to catalyze many types of asymmetric syntheses. Besides catalyzing hydrolysis in aqueous media for the production of enantiopure alcohols, acids, and amines, lipases have also been found with versatile applications for enantioselective acyl transfer in organic media via aminolysis, transamidation, esterification, transesterification, and so forth.5–8 However, the catalytic activities of lipases in organic solvents are usually lower than those in water, which limits their practical applications. In addition, the recycled use of the enzymes that is crucial in industry in terms of process economy is difficult if native enzymes are used. Therefore, the development of robust biocatalysts with increased activity, enantioselectivity, stability, and reusability for organic-phase syntheses is still a major challenge. The immobilization of lipases on appropriate solid supports could potentially address these issues. Solid supports can effectively spread enzyme molecules, prevent them from aggregation, and stabilize the active forms.2 Immobilized enzymes can also be easily separated from the reaction medium and recycled for repeated use. Numerous types of matrices have been * Corresponding author. E-mail:
[email protected]. (1) Zong, S. Z.; Cao, Y.; Zhou, Y. M.; Ju, H. X. Biosens. Bioelectron. 2007, 22, 1776. (2) Mateo, C.; Palomo, J. M.; Fernandez-Lorente, G.; Guisan, J. M.; FernandezLafuente, R. Enzyme Microb. Technol. 2007, 40, 1451. (3) Testa, B.; Trager, W. F. Chirality 1990, 2, 129. (4) Rekoske, J. E. AIChE J. 2001, 47, 2. (5) Faber, K., Biotransformations in Organic Chemistry; Springer-Verlag: Berlin, 1997. (6) Kazlauskas, R. J.; Bornscheuer, U. T., Biotransformations with lipases. In Biotechnology; Rehm, H. J., Pihler, G., Stadler, A., Kelly, P. J. W., Eds.; VCH: New York, 1998; Vol. 8, p 37. (7) Jaeger, K. E.; Reetz, M. T. Curr. Opin. Chem. Biol. 2000, 4, 68. (8) Koeller, K. M.; Wong, C. H. Nature 2001, 409, 232.
employed so far for the immobilization of lipases via adsorption, cross-linking, and entrapment. Most of the supports used are polymeric in nature, such as cellulose derivatives,9–11 sepharose (cross-linked polysaccharide) derivatives,12 polyethylene,13 polyvinyl alcohol,14 polystyrene, and polymethylmethacrylate,15 which could provide a hydrophobic environment for lipase immobilization and activation. Compared to polymeric materials, inorganic supports may offer the advantages of higher mechanical strength, higher chemical stability in organic media, and lower cost. However, the use of lipases immobilized on inorganic materials for organic-phase synthesis is less frequently reported in the literature, but several types of inorganic materials including silica,12,16,17 aluminum silicate,18 magnesium silicate,19 phyllosilicate sol-gel matrixes,20 and magnetic iron oxide nanoparticles,21,22 have been investigated. The results obtained in these studies demonstrated in general that pristine inorganic supports are not suitable for attaining active biocatalysts because of their hydrophilic surfaces. For example, substantial activity loss was found when Candida rugosa lipase was immobilized on unmodified silica.17 The Toyonite 200-M (aluminum silicate) without an organic coating showed a poor binding ability for lipase.18 After modification by organic species with long (9) Ikeda, Y.; Kurokawa, Y. J. Biosci. Bioeng. 2002, 93, 98. (10) Kosaka, P. M.; Kawano, Y.; El Seoud, O. A.; Petri, D. F. S. Langmuir 2007, 23, 12167. (11) Hwang, S.; Ahn, J.; Lee, S.; Lee, T. G.; Haam, S.; Lee, K.; Ahn, I. S.; Jung, J. K. Biotechnol. Lett. 2004, 26, 603. (12) Arroyo, M.; Sanchez-Montero, J. M.; Sinisterra, J. V. Enzyme Microb. Technol. 1999, 24, 3. (13) Chen, J. C.; Tsai, S. W. Biotechnol. Prog. 2000, 16, 986. (14) Wang, Y.; Hsieh, Y. L. J. Membr. Sci. 2008, 309, 73. (15) Palocci, C.; Chronopoulou, L.; Venditti, I.; Cernia, E.; Diociaiuti, M.; Fratoddi, I.; Russo, M. V. Biomacromolecules 2007, 8, 3047. (16) Gamoh, K.; Sakata, C.; Kotsuki, H. Bunseki Kagaku 1999, 48, 1149. (17) Ujang, Z.; Husain, W. H.; Seng, M. C.; Rashid, A. H. A. Process Biochem. 2003, 38, 1483. (18) Kamori, M.; Hori, T.; Yamashita, Y.; Hirose, Y.; Naoshima, Y. J. Mol. Catal. B: Enzym. 2000, 9, 269. (19) Cho, S. W.; Rhee, J. S. Biotechnol. Bioeng. 1993, 41, 204. (20) Hsu, A. F.; Foglia, T. A.; Shen, S. Biotechnol. Appl. Biochem. 2000, 31, 179. (21) Shu, B.; Zheng, G.; Wei, L.; Yan, S. Food Chem. 2006, 96, 1. (22) Gardimalla, H. M. R.; Mandal, D.; Stevens, P. D.; Yen, M.; Gao, Y. Chem. Commun. 2005, 4432.
10.1021/la801384c CCC: $40.75 2008 American Chemical Society Published on Web 07/26/2008
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hydrophobic chains, such as octadecyl and stearate derivatives16 and 7-aminoheptanoic acid,19 the immobilized lipases exhibited much higher activity compared to those from the native lipases. Such phenomena could be attributed to the structural characteristics of lipases. It has been observed that lipases have a polypeptide chain called a “lid” that secludes the active center from the reaction medium.23 The presence of the hydrophobic surface can promote the stabilization of the open form of lipases, thus exposing the active site to the reaction medium and substrates.24 Therefore, it is possible to alter the activity by modifying the microenvironment of the lipases. For instance, it was reported that the modification of Candida rugosa lipase by dextrans (branched polysaccharide) produced more active biocatalysts than did the native enzyme (commercial crude or semipurified lipase).25 Owing to the characteristics of a large specific surface area (SSA), high thermal/mechanical/chemical resistance, and excellent biocompatibility, zirconia (ZrO2) nanoparticles have recently been considered to be a potential solid support for the immobilization of bioactive molecules for biosensor applications.1,26,27 In the past few years, both functionalized and unfunctionalized zirconia nanoparticles have also been developed as solid supports for enzyme immobilization and biocatalysis. For example, Hearn’s group reported the covalent attachment of enzymes to the surface of porous zirconia activated with NCSsilane (3-isothiocyanatopropyltriethoxy silane).28 Cipiciani and co-workers carried out the adsorption of myoglobin onto phosphate and phosphonate-grafted zirconia nanoparticles.29 Most recently, Sugunan’s group reported the adsorption of R-amylase on zirconia without modification.30 For all of the enzymes immobilized on zirconia support so far, activity was measured in aqueous solutions, and no enantioselectivity was reported. In this study, we report the generation of highly active, enantioselective, reusable biocatalysts by immobilizing lipases to the hydrophobic surface of zirconia nanoparticles via adsorption. Two relatively cheap commercial lipases from Candida rugosa (CRL) and Pseudomonas cepacia (PCL) were investigated. The resultant nanostructured biocatalysts have demonstrated excellent performance for the esterification of (R,S)ibuprofen and the acylation of (R,S)-1-phenylethanol in isooctane organic solvent, respectively (Schemes 1 and 2). Ibuprofen is an important nonsteroidal anti-inflammatory drug (NSAID) and has its pharmacological activity mainly in the S enantiomers.31 The enzymatic resolution of ibuprofen to obtain the (S)-ibuprofen ester prodrug using both native and immobilized lipase9,12,13,16,25,32–45 has been carried out by many research groups. (23) Brady, L.; Brzozowski, A. M.; Derewenda, Z. S.; Dodson, E.; Dodson, G.; Tolley, S.; Turkenburg, J. P.; Christiansen, L.; Hugejensen, B.; Norskov, L.; Thim, L.; Menge, U. Nature 1990, 343, 767. (24) Palomo, J. M.; Munoz, G.; Fernandez-Lorente, G.; Mateo, C.; FernandezLafuente, R.; Guisan, J. M. J. Mol. Catal. B: Enzym. 2002, 19, 279. (25) de la Casa, R. M.; Sanchez-Montero, J. M.; Sinisterra, J. V. Biotechnol. Lett. 1999, 21, 123. (26) Liu, B.; Cao, Y.; Chen, D.; Kong, J.; Deng, J. Anal. Chim. Acta 2003, 478, 59. (27) Zong, S. Z.; Cao, Y.; Zhou, Y. M.; Ju, H. X. Langmuir 2006, 22, 8915. (28) Huckel, M.; Wirth, H. J.; Hearn, M. T. W. J. Biochem. Biophys. Meth. 1996, 31, 165. (29) Bellezza, F.; Cipiciani, A.; Quotadamo, M. A. Langmuir 2005, 21, 11099. (30) Reshmi, R.; Sanjay, G.; Sugunan, S. Catal. Commun. 2007, 8, 393. (31) Hutt, A. J.; Caldwell, J. Clin. Pharmacokinet. 1984, 9, 371. (32) Mustranta, A. Appl. Microbiol. Biotechnol. 1992, 38, 61. (33) Carvalho, P. D.; Contesini, F. J.; Bizaco, R.; Calafatti, S. A.; Macedo, G. A. J. Ind. Microbiol. Biotechnol. 2006, 33, 713. (34) Carvalho, P. D.; Contesini, F. J.; Ikegaki, M. Braz. J. Microbiol. 2006, 37, 329. (35) Contesini, F. J.; Carvalho, P. D. Tetrahedron: Asymmetry 2006, 17, 2069. (36) Ducret, A.; Trani, M.; Lortie, R. Enzyme Microb. Technol. 1998, 22, 212. (37) Lopez, N.; Pernas, M. A.; Pastrana, L. M.; Sanchez, A.; Valero, F.; Rua, M. L. Biotechnol. Prog. 2004, 20, 65.
Chen et al. Scheme 1. Candida rugosa Lipase (CRL)-Catalyzed Resolution of (R,S)-Ibuprofen
Scheme 2. Pseudomonas cepacia Lipase (PCL)-Catalyzed Resolution of (R,S)-1-Phenylethanol
The effects of organic solvent, ionic liquid solvent, and water activity have been extensively investigated. However, high activity, enantioselectivity and reusability achieved with lipases immobilized on inorganic support materials have been seldom reported, except in work by Gamoh et al.16 Their support material, octadecyl silica gel (ODS-silica), was modified by a relatively complicated organic modifier, monostearate (sugar ester), before lipase immobilization. However, the enzymatic resolution of 1-phenylethanol, which is one of the important secondary alcohols for many organic syntheses, has been recently studied using immobilized lipases.46–49 Our study has demonstrated for the first time that zirconia nanoparticles modified by a simple carboxylic surfactant of a very long alkyl chain can significantly enhance the activity of the immobilized lipases for asymmetric synthesis in organic media. Furthermore, the immobilized lipase can be subjected to recycled use with high stability maintained.
Experimental Section Materials. Crude lipase from Candida rugosa (CRL) was purchased from Aldrich. Crude lipase from Pseudomonas cepacia (PCL) was from Amano Pharmaceuticals Co. Ltd. (Nagoya, Japan). Zirconium oxychloride octahydrate (ZrOCl2 · 8H2O, 99%), aqueous ammonia solution (28 wt %), and silver nitrate (AgNO3, 99.7%) were purchased from Kanto Chemical. Sodium hydroxide pellets (>99%), ethanol (>99.9%), and Tween 85 were purchased from Merck. (R,S)-Ibuprofen (99%) was purchased from Acros. Isooctane (38) Shang, C. S.; Hsu, C. S. Biotechnol. Lett. 2003, 25, 413. (39) Tsai, S. W.; Lin, J. J.; Chang, C. S.; Chen, J. P. Biotechnol. Prog. 1997, 13, 82. (40) Xie, Y. C.; Liu, H. Z.; Chen, J. Y. Biotechnol. Lett. 1998, 20, 455. (41) Yu, H. W.; Wu, J. C.; Bun, C. C. Chirality 2005, 17, 16. (42) Gowoun, L.; Joo, H.; Kim, J.; Lee, J. H. J. Microbiol. Biotechnol. 2008, 18, 465. (43) Lopez, N.; Perez, R.; Vazquez, F.; Valero, F.; Sanchez, A. J. Chem. Technol. Biotechnol. 2002, 77, 175. (44) Yu, H. W.; Wu, J. C.; Ching, C. B. Biotechnol. Lett. 2004, 26, 629. (45) Zhao, X. G.; Wei, D. Z.; Song, Q. X. J. Mol. Catal. B: Enzym. 2005, 36, 47. (46) Suan, C. L.; Sarmidi, M. R. J. Mol. Catal. B: Enzym. 2004, 28, 111. (47) Xue, P.; Yan, X. H.; Wang, Z. Chin. Chem. Lett. 2007, 18, 929. (48) Hara, P.; Hanefeld, U.; Kanerva, L. T. J. Mol. Catal. B: Enzym. 2008, 50, 80. (49) Zhang, Y. M.; Li, J.; Han, D. F.; Zhang, H. D.; Liu, P.; Li, C. Biochem. Biophys. Res. Commun. 2008, 365, 609.
Immobilization of Lipases on Zirconia Scheme 3. Molecular Structure of (A) Erucic Acid in Anionic Form and (b) Tween 85a
a The dotted lines indicate the locations where hydrolysis can take place to form oleic acid or oleate.
(99.0%) was from Fluka. A bicinchoninic acid kit for protein determination and buffer solutions of pH 4.0, 5.0, 6.0, and 7.0 were from Sigma. Erucic acid (technical, 85%), (R,S)-1-phenylethanol (97%), and 1-propanol (>99.5%) were from Alfa Aesar. Hexane (HPLC grade) was from Fisher. Synthesis and Surface Modification of Zirconia Nanoparticles. Briefly, 200 mL of an aqueous solution of ZrOCl2 · 8H2O (0.5 M) was first prepared and kept in a two-necked round-bottomed flask. Then, 38 mL of the aqueous ammonia solution (28 wt %) was added dropwise with rigorous stirring. The mixture was kept at 70 °C in an oil bath with a condenser mounted on the top and continuously stirred for 1 day. The precipitate was collected by filtration and washed with an excess amount of deionized water until no chloride ions were detected using an aqueous solution of AgNO3 (0.01 M). The sample was dried overnight in the oven at 70 °C. The asprepared solid sample was calcined in a furnace in static air. A ramp of 1 °C/min was used until the temperature reached 500 °C, which was then held for 12 h. The calcined sample was grinded to a fine powder for further application. During surface modification, 5 g of the surfactant modifier, either erucic acid or Tween 85, was added to 50 mL of deionized water and mixed well. The molecular structures of these two surfactants are shown in Scheme 3. To this mixture, 1.5 g of as-prepared zirconia nanoparticles was added. In the case of erucic acid, the pH of the mixture was adjusted to about 10.0 by adding an aqueous solution of NaOH (2.0 M). The resultant mixture was vigorously stirred at 90 °C for 1 day. The samples were centrifuged and washed thoroughly with ethanol to remove the excess surfactants and dried overnight in the oven at 70 °C. The modified supports were named TweenZrO2 and erucic-ZrO2. Characterization of Zirconia Nanoparticles. The chemicophysical properties of zirconia nanoparticles before and after surface modification were characterized by various methods. Chemical bonding information was studied with Fourier transform infrared (FTIR) spectroscopy on a Biorad FTS 3100 using a standard KBr pellet technique. The weight percentages of carbon and hydrogen were measured in a Elementarvario CHN elemental analyzer. Thermogravimetric analysis (TGA) was carried out by heating the dry powder samples at a rate of 10 °C/min with a flow of air at 200 mL/min over 25-600 °C in a TA Instrument SDT Q600. The particle size and morphology of the nanoparticles were observed with a transmission electron microscope (TEM, JEOL 3010) operated at an accelerating voltage of 200 kV. Lipase Immobilization on Zirconia Nanoparticles. The immobilization of both Candida rugosa lipase (CRL) and Pseudomonas
Langmuir, Vol. 24, No. 16, 2008 8879 cepacia lipase follows the same procedure. An aqueous solution of lipase (1.2 mg protein/mL for CRL, 0.8 and 1.2 mg protein/mL for PCL) was first prepared by dissolving the lipase from the crude powder in a buffer solution of a certain pH value. After being stirred for 15 min, the mixture was centrifuged to remove the undissolved residues. The zirconia support (modified or unmodified) in powder form (50 mg) was first mixed with 5 mL of the buffer solution of the same pH, followed by the addition of 2 mL of 1-propanol. The resultant mixture was sonicated for 5 min to disperse the solid particles well. The mixture was then added to 15 mL of the as-prepared lipase solution, and the final mixture was shaken at 180 rpm for 1 day at room temperature. The lipase-immobilized solid particles were collected by centrifugation, followed by washing with 10 mL of buffer solution and freeze drying. The protein concentration in the lipase solution before and after immobilization was determined using a BCA assay kit. The difference in protein concentrations was used to calculate the loading of lipase on the support materials. The pH of the buffer solution for the immobilization of CRL was 4.0, 5.0, 6.0, and 7.0. For PCL, a pH of 5.0 was used. Resolution of (R,S)-Ibuprofen by Immobilized, Crude, and Freeze-Dried CRL. (R,S)-Ibuprofen (4 mg, 0.019 mmol) was dissolved in 4 mL of isooctane. Then, 4.5 µL of 1-propanol (0.060 mmol) and 40-50 mg of CRL-immobilized ZrO2 nanoparticles were added. The mixture was incubated at 30 °C with continuous shaking at 180 rpm. At a certain interval, 200 µL of the liquid sample was withdrawn, mixed with 800 µL of hexane, and filtered for analysis. The conversion and enantiomeric excess (ee) of ibuprofen were determined using HPLC (Agilent 1100) with a Chiracel OD-H column (4.6 × 250 mm2, 5 µm). The samples were eluted with a mixture of n-hexane/2-propanol/trifluoroacetic acid (98:2:0.2 by volume) at 0.8 mL/min for 20 min and detected at 220 nm. The reaction was also carried out using the as-received crude CRL powder or the freeze-dried CRL pellets containing approximately an equivalent amount of protein for comparison. The latter was obtained from the lipase solution (1.6 mg protein/mL) prepared by dissolving the crude lipase powder in either pH 5 buffer solution or deionized water, followed by centrifugation to remove the undissovled residuals. The supernatant lipase solution was freeze dried to obtain the lipase pellets. The reaction was also carried out at higher substrate concentrations by dissolving 40 mg (0.19 mmol) and 80 mg (0.38 mmol) of (R,S)-ibuprofen in 4 mL of isooctane, respectively. The reaction was catalyzed by crude CRL powder and CRL immobilized on erucic acid-modified ZrO2 nanoparticles. Other reaction conditions were kept the same, such as the reaction volume, the ratio of (R,S)ibuprofen to 1-propanol, the amount of protein, the reaction temperature, and so forth. Resolution of (R,S)-1-Phenylethanol by Immobilized and Crude PCL. (R,S)-1-Phenylethanol (20 µL, 0.16 mmol) was dissolved in 4 mL of isooctane. Then, 25 µL of vinyl acetate (0.27 mmol) and 40-50 mg of PCL-immobilized ZrO2 nanoparticles were added. The mixture was incubated at 30 °C with continuous shaking at 180 rpm. At a certain interval, 200 µL of the liquid sample was withdrawn, mixed with 800 µL of hexane, and filtered for analysis. The conversion and ee of 1-phenylethanol were determined using HPLC (Agilent 1100) with a Chiracel OD-H column (4.6 × 250 mm2, 5 µm). The samples were eluted by a mixture of n-hexane/ 2-propanol (90:10 by volume) at 0.7 mL/min for 12 min and detected at 220 nm. The reaction was also carried out using the as-received crude PCL powder containing approximately an equivalent amount of protein for comparison. The reaction was also carried out at higher substrate concentrations by dissolving 200 µL (1.6 mmol) and 400 µL (3.2 mmol) of (R,S)-1-phenylethanol in 4 mL of isooctane. The reaction was catalyzed by crude PCL powder and PCL immobilized on erucic acid-modified ZrO2 nanoparticles. Other reaction conditions were kept the same, such as the reaction volume, the ratio of (R,S)1-phenylethanol to vinyl acetate, the amount of protein, the reaction temperature, and so forth. Reuse Study for Immobilized CRL. After each batch of reaction, the immobilized CRL on erucic acid-modified zirconia nanoparticles (CRL-erucic-ZrO2) were allowed to settle and the supernatant solution was removed. Then, 4 mL of isooctane was mixed with the solid
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particles, and the mixture was votex mixed in order to wash away the remaining substrate and product species. After the supernatant solution was removed, the solid particles were ready for the next run. The reaction time for each run was kept at 3 days. Calculations. The conversion and enantiomeric excess (ee) of the substrate were calculated from the data obtained from HPLC analysis using eqs 1 and 2,
area(R, t) - area(S, t) (1) area(R, t) + area(S, t) area(S, t) + area(R, t) apparent conversion(Capp) ) 1 area(S, 0) + area(R, 0) (2) ees )
where the area(R, t) and area(S, t) are the areas under the curves for R and S enantiomers of ibuprofen, respectively, at time t. During our experiment, it was found that ibuprofen, which has a carboxylic acid group, was partially adsorbed on the ZrO2 support as a result of the formation of the metal-carboxylate complex. (R)- and (S)-ibuprofen were adsorbed to approximately the same extent according to the adsorption study using bare ZrO2 and erucic-ZrO2 without lipase (Results and Discussion). Because CRL selectively transforms (S)ibuprofen,50 the extent of adsorption was obtained by comparing the area of (R)-ibuprofen before and after reaction using eq 3. The actual conversion for the reaction with a substrate concentration of approximately 1 g/L was obtained by subtracting the adsorption from the apparent conversion using eq 4. However, at higher substrate concentrations, the percentage of adsorption was generally found to be less than 5%. As a result, the apparent conversion data were presented and used for further calculations.
area(R, t) area(R, 0) conversion(C) ) Capp - Ads
adsorption(Ads) ) 1 -
(3) (4)
The calculations for the resolution of 1-phenylethanol are similar. During this reaction, no adsorption of the substrate was found on the supports. Because PCL selectively catalyzes the transformation of (R)-1-phenylethanol, the ee of the substrate was calculated from the data obtained from HPLC analysis using eq 5,
ees )
area(S, t) - area(R, t) area(S, t) + area(R, t)
(5)
The enantioselectivity (E) for both lipases was calculated using eq 6
E)
ln[(1 - C)(1 - ees] ln[(1 - C)(1 + ees]
(6)
Results and Discussion Physicochemical Properties of Surface-Modified Zirconia Nanoparticles. Erucic acid and Tween 85 were used to modify ZrO2 nanoparticles in order to obtain inorganic support materials but with a hydrophobic surface. During the modification by erucic acid, an alkaline condition was maintained. Therefore, the grafted species on the ZrO2 surface should be in the form of a carboxylate anion (Scheme 3A). Although Tween 85 (Scheme 3B) is a nonionic surfactant, it was previously reported by us that on the surface of transition-metal oxides the carbonyl ester group of Tween 85 could be cleaved (indicated by the dotted lines in Scheme 3B) and the resultant carboxylate anion (oleate) can attach to and form an irreversible binding with the metal oxide surfaces.51 The FTIR spectra of ZrO2 nanoparticles before and after surfactant modification as shown in Figure 1 provide direct (50) Hernaiz, M. J.; SanchezMontero, J. M.; Sinisterra, J. V. Biotechnol. Lett. 1997, 19, 303. (51) Xu, R.; Zeng, H. C. Langmuir 2004, 20, 9780.
Figure 1. FTIR spectra of (A) ZrO2, (B) Tween-ZrO2, and (C) erucicZrO2.
evidence for the attachment of organic modifiers on the ZrO2 surface. In both cases of Tween-ZrO2 and erucic-ZrO2, the absorption peaks in the range of 2850-2950 cm-1 can be attributed to C-H stretches of the hydrophobic chains of the surfactants.52 The two weaker adsorption bands between 1400 and 1600 cm-1 can be assigned to stretching vibrations of the -COO- group of the surfactant.53 The absence of the adsorption band at about 1110 cm-1 (C-O-C stretching)52 for TweenZrO2 confirms that only the R-COO- (oleate) portions of this surfactant are grafted onto the ZrO2 surface instead of the whole nonionic molecule. It is also observed that the adsorption at about 1640 cm-1 due to water bending54 is greatly reduced after surfactant modification. Therefore, it can be concluded that the surfaces of ZrO2 nanoparticles have been coated with carboxylic surfactant anions. Our observation that the modified ZrO2 samples can no longer be dispersed well in aqueous solutions has consistently indicated that the modification by surfactants has changed their surface from hydrophilic to hydrophobic. TEM images show that modification with Tween 85 and erucic acid does not alter the size or shape of ZrO2 nanoparticles (Figure 2). The average diameter of ZrO2 nanoparticles is about 20 nm. Table 1 shows the carbon and hydrogen weight percentages obtained from CHN elemental analysis. On the basis of the carbon results, the calculated weight percentages of carboxylate anions are 6.5 and 12.3% when using Tween 85 and erucic acid, respectively. These results are close to the measured weight loss percentages, 7.3 and 11.6%, respectively, from the TGA method in the temperature range of 200-500 °C, which account for the decomposition and combustion of organic modifiers on the ZrO2 surface. The lower weight percentage of the organic modifier in Tween-ZrO2 compared to that in erucic-ZrO2 is expectedly due to the shorter alkyl chain of the carboxylate anion (oleate) from Tween 85 compared to that from erucic acid. For comparison, the weight percentage of carbon and the weight loss from the unmodified ZrO2 sample are very low. Influence of Surface Modification of ZrO2 Nanoparticles on the Catalytic Performance of Immobilized Lipases. Table 2 displays the activity and ee data using various CRL biocatalysts for the resolution of (R,S)-ibuprofen. The results demonstrate that the activity of CRL strongly depends on the surface properties of the ZrO2 nanoparticles. CRL immobilized on the pristine ZrO2 (52) Dean, J. A. Analytical Chemistry Handbook; McGraw-Hill: New York, 1995; p 61. (53) Arenas, J. F.; Marcos, J. Spectrachim. Acta Part A 1979, 35, 355. (54) Xu, R.; Zeng, H. C. Chem. Mater. 2003, 15, 2040.
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Figure 2. TEM images of (A) ZrO2, (B) Tween-ZrO2, and (C) erucic-ZrO2. Table 1. Elemental and Thermal Analysis Results of ZrO2 Modified with Surfactants sample ZrO2 Tween-ZrO2 Erucic-ZrO2
C (wt %) H (wt %) 0.13 5.29 9.64
0.52 0.91 1.39
thermal cald carboxylate loss (wt%)b (wt%)a 6.5 12.3
0.67 7.3 11.6
a Calculated weight percentage of the carboxylate anions on the surface of ZrO2 nanoparticles. b Weight loss data from TGA analysis in the range of 200-500 °C due to the decomposition and combustion of organic modifiers.
nanoparticles are almost inactive for this reaction, although the nanocomposite exhibits good activity for the esterification of lauric acid (data not shown). CRL immobilized on surfactantmodified ZrO2 nanoparticles shows enhanced activity. The specific activity obtained from CRL-erucic-ZrO2 is approximately 214% of that from the crude CRL powder at a reaction time of about 1 day. The ee of the (R)-ibuprofen substrate gradually increased, and it reached 96.3% for CRL-erucic-ZrO2 after 3 days but only 80% for the crude CRL powder. The E value (43) of CRL-erucic-ZrO2 obtained after 3 days was 1.9 times as much as that of the crude CRL powder (23). The higher activity of CRL supported on erucic-ZrO2 could be due to a high surface area of ZrO2 nanoparticles for effective dispersion of the lipase. CRL immobilized on Tween-ZrO2 (ZrO2 modified by the carboxylate anion with a shorter hydrophobic chain) is not as active as that of crude CRL powder with 71% of the relative activity and less than 50% of the ee obtained at 58 h. For comparison, the lyophilized CRL pellets obtained by freeze drying the aqueous solutions (both deionized water and buffer solution of pH 5.0) were used under the same reaction condition. The reaction did not take place at all in the presence of these pellets even when the time was extended to 5 days. In fact, lipases can dissolve in aqueous media but not organic media. Therefore, it is not surprising that the lyophilized pellets that are composed of the aggregated CRL are inactive in organic solvents. It is also indicated in Table 2 that the CRL loading on both erucic-ZrO2 and Tween-ZrO2 is higher than that on the unmodified ZrO2 support. This is likely because the surface characteristics can influence the interaction between the lipase and the support. CRL has a relatively hydrophobic surface55 whereas the surface of unmodified ZrO2 is hydrophilic. After modification, the hydrophobic surface of the support can probably accommodate the lipase better. Both literature results50 and our data using crude CRL powder indicate that CRL mainly catalyzes the esterification of (S)(55) Maeda, R.; Hino, S.; Matsumoto, M.; Kondo, K. J. Chem. Eng. Jpn. 2004, 37, 109.
ibuprofen. However, it was noted that the concentration of (R)ibuprofen also decreased with the reaction time, which should be due to the adsorption of ibuprofen on the ZrO2 surface. To verify this, both ZrO2 and erucic-ZrO2 were incubated with (R,S)ibuprofen under the reaction condition. The HPLC results showed clearly that no reaction occurred in the absence of CRL but around 50% of ibuprofen (both R and S) was adsorbed on the unmodified ZrO2. This can be explained by the structure of the ibuprofen substrate. It has a carboxylic group that can form a complex with the metal oxide. When the substrate was modified with erucic acid, the adsorption percentage was decreased to about 24%. After CRL loading, the percentage of adsorption further decreased to about 18-20% for all samples. The activity data shown in Table 2 are based on the actual conversions that are the apparent conversion minus the adsorption percentage (Experimental Section). The extent of adsorption was greatly reduced during the recycled use of CRL-erucic-ZrO2 as a result of the saturation of the ibuprofen substrate on the ZrO2 surface, which is to be discussed shortly. The data presented in Table 2 were obtained from the reaction using a substrate concentration of around 1 g/L. On the basis of our solubility test, the maximum concentration of (R,S)-ibuprofen in isooctane solvent attained at room temperature was in the range of 20-30 g/L, which is higher than the value (10 g/L) reported by Higgins et al.56 Because the use of high substrate concentrations is attractive for practical applications in the chemical and pharmaceutical industries,57,58 we further carried out our reaction with the concentrations of (R,S)-ibuprofen at ca. 10 and 20 g/L, respectively. Under these conditions, the percentage adsorption of the substrate on ZrO2 nanoparticles dropped to less than 5%, which was considered insignificant. The conversion, enantiomeric excess, and initial activity data are shown in Figure 3. The calculated initial activities increased to about 1.5 and 3.0 times those shown in Table 2 when the substrate concentration was raised from 1 to 10 and 20 g/L, respectively, for both crude CRL powder and CRL-erucic-ZrO2. It can be observed that the immobilized CRL still gave superior performance compared to the crude CRL powder at higher substrate concentrations. However, the overall low conversion and enantiomeric excess data obtained even at a long reaction period of 12 days indicate that further improvement in the activity is required to make this type of biocatalyst useful for the resolution of (R,S)-ibuprofen in the practical application. (56) Higgins, J. D.; Gilmor, T. P.; Martellucci, S. A.; Bruce, R. D. Anal. Profiles Drug Subst. Excip. 2001, 27, 265. (57) Cao, L. Q.; van Langen, L.; Sheldon, R. A. Curr. Opin. Biotechnol. 2003, 14, 387. (58) Straathof, A. J. J.; Panke, S.; Schmid, A. Curr. Opin. Biotechnol. 2002, 13, 548.
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Table 2. Activity, Enantiomeric Excess, and Enantioselectivity Data Obtained with CRL Biocatalysts for the Resolution of (R,S)-Ibuprofen with a Substrate Concentration of ca. 1 g/L ees (%) biocatalysts
protein loading (mg/g)
activityc (µmol/mg/h)
rel.activityd (%)
23 h
47 h
58 h
72 h
crude CRL powder CRL-ZrO2a CRL-erucic-ZrO2a CRL-Tween-ZrO2a freeze-dried CRLb
30.2 39.4 40.4
0.078 0.005 0.167 0.055 0
100 6 214 71 0
22.4 0.8 54.8 17.6 0
59.6 2.1 82.8 37.1 0
63.4 2.5 88.0 45.7 0
80.0 (23f) n.d.e 96.3 (43f) n.d.e 0
a CRL immobilization was carried out in the buffer solution of pH 5.0. b Obtained from purified CRL solution prepared using both deionized water and buffer solution of pH 5.0. c Calculated using the conversion (C) data obtained at 23 h, expressed as µmol of (S)-ibuprofen reacted per milligram of protein per h. d Relative activity based on that of crude CRL powder. e Not determined. f Enantioselectivity (E) calculated in the basis of the data obtained at 72 h.
Figure 3. Conversion, enantiomeric excess, and initial activity data obtained with CRL biocatalysts for the resolution of (R,S)-ibuprofen at higher substrate concentrations: (A) 10 and (B) 20 g/L. Legends: (•, 0) C and ees, respectively, for crude CRL powder and (2, ∆) C and ees, respectively, for CRL-erucic-ZrO2.
Erucic-ZrO2 can also be used as an effective support for another lipase (i.e., that from Pseudomonas cepacia (PCL)) for the resolution of (R,S)-1-phenylethanol. (R,S)-1-Phenylethanol, which is different from the ibuprofen substrate, is an alcohol and does not have an absorption problem on ZrO2 nanoparticles. Table 3 shows the results of conversion and the ee of the substrate for both crude PCL powder and PCL immobilized at pH 5.0. Similar to the previous results, the lipase immobilized on unmodified ZrO2 nanoparticles gave very low activity. After immobilization on the erucic-ZrO2 support, the activity of PCL showed even greater improvement than that of CRL. At a reaction time of 4.5 h, a conversion of 53.3% and an ee of 95.5% were achieved when PCL-erucic-ZrO2 was used, whereas only 23.1% (C) and 31.2% (ee) were obtained for crude PCL with an equivalent amount of protein (2.0 mg). Furthermore, the data obtained from the PCL-erucic-ZrO2 biocatalyst containing a lower amount of protein (1.3 mg) are still higher than those from the crude PCL with 2.0 mg of protein. The solubility of (R,S)-1-phenylethanol in isooctane is much higher than that of (R,S)-ibuprofen. When the resolution of (R,S)1-phenylethanol was determined at higher substrate concentrations of ca. 50 and 100 g/L, high conversion (54.6 and 58.1%) and enantiomeric excess (95.2 and 97.6%) can still be achieved using the immobilized PCL biocatalyst at 8 and 22 h, respectively, as shown in Figures 4 and 5. The calculated E values for PCLerucic-ZrO2 based on the last data point in Figure 4A,B are
around 32 and 24, respectively, which are 3 to 4 times higher than that of the crude PCL powder (6). Influence of pHs during Lipase Immobilization. It has been reported that the pH value of the buffer solution during immobilization has a great influence on the activity and enantioselectivity of the biocatalyst.30 In this work, the immobilization of CRL on erucic-ZrO2 was carried out at pH 4.0, 5.0, 6.0, and 7.0. As shown in Table 4, similar enzyme loadings within the range of 34.4-40.6 mg/g are obtained, but the activities are quite different with optimal results achieved at pH values of 5.0 and 6.0. Because the isoelectric point (pI) of CRL is 5 to 6,59 the protein is kept at its most stable conformation at these pH values. Although the active confirmation and the stable confirmation of a protein may not be the same, in this case, the neutral protein surface may enhance the hydrophobic interaction between the lipase and the erucic acid-modified ZrO2 surface. Interfacial Activation of Lipases by Hydrophobic Chains of Surfactants. Our study has shown that by applying the straightforward step of surfactant coating, the inorganic ZrO2 can be effectively turned to a hydrophobic support that significantly enhances the activities of immobilized Candida rugosa and Pseudomonas cepacia lipases (CRL and PCL). The surfactant used in this work, erucic acid, can be considered to be one of the simplest anionic surfactants. However, compared to other more frequently applied surfactants of the carboxylic acid type, such as oleic, linoleic, and octadecyl acids, erucic acid has a longer alkyl chain of 21 carbons (Scheme 3A). It was reported that the polypeptide “lid” of lipase is mainly hydrophobic toward the catalytic “pocket” and hydrophilic on its external surface.23,59 The hydrophobic interaction between the long hydrocarbon chain of the surfactant and the “lid” is believed to facilitate its conformational rearrangement so that the active center becomes accessible to the substrates. This kind of interfacial activation mechanism has been used to explain the enhanced activities of lipases immobilized on other hydrophobic supports, such as octadecyl-sepabeads24,60 and octyl-agarose.61 In contrast, when immobilized on unmodified zirconia, the hydrophilic interaction between the ZrO2 surface and the external surface of the lid could keep the enzyme in a closed form. Nevertheless, the catalytic mechanism of lipases can be very complicated. Further investigation of the effect of chain length and concentration of surfactant molecules in a more systematic manner is underway. Here, we use a simple schematic diagram (Scheme 4) to illustrate the bionanocomposite system developed in this work. Because the mechanism of modification by the carboxylic surfactant is associated with the formation of a metal-carboxylic (59) Benjamin, S.; Pandey, A. Yeast 1998, 14, 1069. (60) Wilson, L.; Palomo, J. M.; Fernandez-Lorente, G.; Illanes, A.; Guisan, J. M.; Fernandez-Lafuente, R. Enzyme Microb. Technol. 2006, 38, 975. (61) Fernandez-Lorente, G.; Palomo, J. M.; Cabrera, Z.; Guisan, J. M.; Fernandez-Lafuente, R. Enzyme Microb. Technol. 2007, 41, 565.
Immobilization of Lipases on Zirconia
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Table 3. Conversion and Enantiomeric Excess Data Obtained with PCL Biocatalysts for the Resolution of 1-Phenylethanol with a Substrate Concentration of ca. 5 g/L 2.0 h
4.5 h
biocatalysts
protein loading (mg/g)
protein amount (mg)
C (%)
ees (%)
C (%)
ees (%)
crude PCL powder 1.2-PCL-ZrO2a 1.2-PCL-erucic-ZrO2a 0.8-PCL-erucic-ZrO2b
43.6 46.2 28.7
2.0 2.1 2.0 1.3
12.3 0.1 44.5 38.1
14.8 6.3 76.9 64.7
23.1 2.4 53.3 48.8
31.2 10.2 95.5 91.6
a
The lipase concentration used for loading was 1.2 mg protein/mL.
b
The lipase concentration used for loading was 0.8 mg protein/mL.
Table 4. Activity and Enantiomeric Excess Data Obtained with CRL Immobilized on Erucic-ZrO2 at Different pH Values for the Resolution of (R,S)-Ibuprofen with a Substrate Concentration of ca. 1 g/L ees (%) pH
protein loading (mg/g)
activity(µmol/mg/h)a
23 h
47 h
58 h
4.0 5.0 6.0 7.0
34.4 39.4 38.6 40.6
0.094 0.167 0.194 0.049
17.6 54.8 53.7 10.3
37.8 82.8 84.8 23.7
46.8 88.0 90.3 29.1
a
Calculated using the conversion (C) data obtained at 23 h.
Scheme 4. Schematic Representation of the Nanostructured Lipase-Surfactant-Zirconia Biocatalysta
Figure 4. Conversion and enantiomeric excess data obtained with PCL biocatalysts for the resolution of (R,S)-1-phenylethanol at higher substrate concentrations: (A) 50 and (B) 100 g/L. Legends: (•, 0) C and ees, respectively, for crude PCL powder and (2, ∆) C and ees, respectively, for PCL-erucic-ZrO2.
Figure 5. Conversion and enantiomeric excess of substrate vs the recycle number during the reuse study using CRL-erucic-ZrO2 for the resolution of (R,S)-ibuprofen.
complex, the surfaces of ZrO2 nanoparticles could be grafted with a surfactant monolayer that renders the nanoparticles suitable for lipase immobilization and activation (Scheme 4A). Scheme 4B shows the bionanocomposite of the lipase-surfactant-zirconia system in which the lipase could adopt an open form as a result of its interaction with the hydrocarbon chains of the surfactant. In addition to the proposed interfacial activation mechanism, better solvation of the surfactant modified ZrO2 nanoparticles in isooctane due to the hydrophobic surface could also contribute to the improved catalytic performance.
a (A) Surfactant-modified zirconia nanoparticle and (B) surfactant-modified zirconia nanoparticle after lipase immobilization.
Activity of the CRL-erucic-ZrO2 Biocatalyst in the Recycle Study. It is important to investigate the performance of immobilized enzymes during recycled use for potential industrial applications. The advantage of the lipase-surfactant-zirconia biocatalyst prepared in this work lies not only in its improved activity due to a suitably modified large surface but also in its ease of recovery and recycling. Concerning the reuse of immobilized CRL for enantioselective reactions in organic media, several reports were found in the literature. Gao and co-workers showed that the conversion of racemic carboxylates decreased from 29 to 20% after four reaction cycles using the lipase immobilized on iron oxide nanoparticles.22 In the other two studies, the remaining activity was only 56.4 and 58.6% after seven and eight cycles, respectively.21,62 In the present work, the recycle study of lipase was carried out using CRL-erucic-ZrO2 for the resolution of ibuprofen. After each cycle, the biocatalyst was simply washed with the reaction solvent (isooctane) and then used for the next run. For each cycle, the reaction time was kept at 3 days. The conversion and ee versus recycle number are plotted in Figure 3, which shows that the immobilized lipase on erucic-ZrO2 is highly stable, which could be attributed to the hydrophobic interaction between them. There was no loss in either activity or enantioselectivity for the first eight cycles. Subsequently, the activity started to decrease slightly. Further studies are necessary to get more data in order (62) Othman, S. S.; Basri, M.; Hussein, M. Z.; Rahman, M. B. A.; Abd Rahman, R. N. Z.; Salleh, A. B.; Jasmani, H. Food Chem. 2008, 106, 437.
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to investigate the deactivation mechanism. Finally, it was noted in all recycled runs that the adsorption of the ibuprofen substrate decreased from 18.3% of the initial run to ca. 10%. This could be due to the “saturation” of the ZrO2 surface with the substrate. Further decreases in the adsorption percentage of ibuprofen could possibly be achieved by modifying the surface properties of the support.
Conclusions In summary, we have demonstrated that after surface modification by erucic acid with a long hydrophobic alkyl chain zirconia nanoparticles can be functionalized to become an effective support for lipase immobilization. The immobilized Candida rugosa and Pseudomonas cepacia lipases gave higher activity and enantioselectivity compared with their corresponding crude lipase powders. The interaction between the hydrophobic surface of zirconia support and lipases is crucial, which probably
Chen et al.
induces the conformational rearrangement of lipases into an active form. The recycle study was conducted for immobilized Candida rugosa lipase, and its catalytic performance was found stable without loss in activity up to eight cycles for resolution of (R,S)ibuprofen in isooctane. Besides achieving enhanced activities and good stability, it is believed that such a bionanocomposite system can be used for more fundamental studies of the interfacial activation mechanism because the surfactant used in this work has a very simple molecular structure. Finally, the concept and method used in this work may be extended to other types of inorganic supports and enzymes for versatile applications. Acknowledgment. We gratefully acknowledge the funding of this work by the Ministry of Education and Nanyang Technological University, Singapore (AcRF grant RG02/04). LA801384C
