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Preparation and characterization of sol–gel hybrid coating films for covalent immobilization of lipase enzyme
Accepted Manuscript Title: Preparation and characterization of sol-gel hybrid coating films for covalent immobilization of lipase enzyme Author: Basak Yuce-Dursun Asli Beyler Cigil Dilek Dongez M.Vezir Kahraman Ayse Ogan Serap Demir PII: DOI: Reference:
S1381-1177(16)30022-4 http://dx.doi.org/doi:10.1016/j.molcatb.2016.02.007 MOLCAB 3325
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
19-11-2015 15-1-2016 14-2-2016
Please cite this article as: Basak Yuce-Dursun, Asli Beyler Cigil, Dilek Dongez, M.Vezir Kahraman, Ayse Ogan, Serap Demir, Preparation and characterization of sol-gel hybrid coating films for covalent immobilization of lipase enzyme, Journal of Molecular Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2016.02.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Preparation and Characterization of Sol-Gel Hybrid Coating Films for Covalent Immobilization of Lipase Enzyme Basak Yuce-Dursun, Asli Beyler Cigil, Dilek Dongez, M. Vezir Kahraman, Ayse Ogan, Serap Demir* Marmara University, Faculty of Arts and Sciences, Department of Chemistry, Istanbul-Turkey *
Corresponding Author: Serap Demir
Address: Marmara University, Goztepe Campus, Faculty of Arts and Sciences, Chemistry Department, 34722 Kadikoy, Istanbul/TURKEY E-mail: [email protected] Phone: +90 216 3464553 Fax: +90 216 3478783
Graphical abstract
Highlgihts
UV-curable hybrid epoxy-silica polymer films were prepared via sol–gel method.
Lipase from Candida rugosa was immobilized on polymer through covalent binding.
The hydrolytic and synthetic activities of lipase were investigated.
Kinetic properties of immobilized and free lipase were characterized.
Immobilization improved the thermal stability, storage stability and reusability.
Abstract In this study UV-curable hybrid epoxy-silica polymer films were prepared via sol–gel method. Lipase (EC 3.1.1.3) from Candida rugosa was covalently immobilized onto hybrid epoxy-silica polymer films and immobilization capacity of polymer films was found 7.22 mg g-1. The morphology of the polymeric support was characterized by scanning electron microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR). Immobilized and free enzymes were used in two different reaction systems: hydrolysis of p-nitrophenyl palmitate in aqueous medium and synthesis of p-nitrophenyl linoleate (from p-nitrophenol and linoleic acid) in n-hexane medium. The effect of temperature on hydrolytic and synthetic activities was investigated and observed maximum activities at 50 °C and 45 °C for immobilized enzyme, orderly. Km values for free enzyme were determined 0.71 and 1.12 mM by hydrolytic and synthetic activity assays, respectively, while these values were observed as 0.91 mM and 1.19 mM for immobilized enzyme. At the end of 30 repeated cycles, 56% and 59% of initial activities remained for hydrolytic and synthetic assays, respectively. Native enzyme lost its activity completely within 20 days, whereas the immobilized enzyme retained for hydrolytic and synthetic activities was approximately 82% and 72%, respectively, under the same storage time. Keywords: Lipase; immobilization; sol-gel; hydrolytic activity; synthetic activity.
1. Introduction Lipases (triacylglycerol ester hydrolases, EC 3.1.1.3) are atypical enzymes that catalyze both hydrolysis and formation of the ester bond between glycerol and long-chain fatty acids [1]. Lipases also catalyze various bioconversion reactions in a wide variety of organic solvents such as the hydrolysis, esterification, transesterification, aminolysis, and acidolysis [2]. In the last decade, there has been an increasing interest in the use of lipases for their properties such as high specificity, efficient reaction rate, non-toxicity and biodegradability, reproducibility under normal laboratory conditions. Also they are able to recognize very different substrates [3]. Lipases are used as versatile industrial applications, including cleanser, biosensor and biodiesel production, bio-catalytic determination of pharmaceuticals, nourishment and flavor businesses, cosmetics and perfumery production [4,5]. Lipases are produced in almost every living cell, but due to a number of advantages, microbial lipases are the most widely used group for practical applications because of low costs of production and easy modification of properties. Among the lipases from various sources, Candida rugosa lipase (CRL) received much attention due to its high activity and broad specificity [5,6]. Immobilization of enzymes is one of the most essential facets of modern biotechnology. This process frequently overcome the structural instability and allow repeated use, native enzymes have been successfully immobilized on different supports using various techniques and enables long-term stability/recyclability of the biocatalyst [5,7,8]. Because of the environmental conditions, free lipases are easily inactivated and hard to be recover for reuse. The separation of products in presence of free lipases is tedious. Hence, further industrial uses of free lipases are restricted because of high costs and complex downstream processing [4]. These drawbacks of the free lipases are overcome through immobilization techniques. As a very promising strategy, immobilization of lipases on solid supports is economically advantageous in order to facilitate the stability and reusability of these enzymes. Immobilized enzymes are preferable to free enzymes because of their easy separation from the reaction mixture makes them suitable for constant use. A variety of support materials have been used in immobilization of lipases like silica [9], magnetic poly(glycidyl methacrylate-methyl methacrylate) beads [10], polypropylene hollow fiber membrane modified with hydrophobic polypeptide [11], polysulfone ion exchange
membranes [12], mesoporous silica [13], poly(hydroxyethyl methacrylate)-based membranes [14], calcium alginate gels [15], celite [16], nylon-6 [17], chitosan and agarose [18]. In this study UV-curable hybrid epoxy-silica polymer films were prepared via sol–gel process. The most generally utilized system for preparing hybrid materials is the sol–gel technique, which permits acquiring cross-linked inorganic silica into the polymer network to improve the properties of the material. Hybrid materials can be prepared by a radiation-curing technique such as using a UV curable binder system [19]. In this study, lipase from Candida rugosa (CRL) was covalently immobilized onto organic/inorganic hybrid SiO2–matrix films. Polymeric support material was characterized by using FTIR and SEM. Enzyme activity of free and immobilized lipase was examined in two different reaction systems: 1. hydrolysis of p-nitrophenyl palmitate in aqueous medium, 2. synthesis of p-nitrophenyl linoleate (from pnitrophenol and linoleic acid) in organic medium. Temperature, kinetic parameters, and storage stabilities for free and immobilized enzymes were evaluated for both hydrolytic and synthetic activities. 2. Materials and methods 2.1. Reagents Lipase from C. rugosa (700 U mg−1), p-nitrophenol (pNP), linoleic acid (LA), p-nitrophenyl palmitate (pNPP), 3-methacryloxypropyltrimethoxysilane (MEMO), p-toluene sulfonic acid, poly(ethylene glycol) diacrylate, (3-glycidyloxypropyl) trimethoxysilane were purchased from Sigma Aldrich Chemical Co. (St. Louis, USA). Bradford reagent and ovalbumin were commercial product of BioRad (BioRad Laboratories, Hercules CA, USA). All other chemicals were of analytical grade and used without further purification. 2.2. Preparation of polymeric support material A hybrid epoxy-silica polymer film was prepared through sol–gel process. The sol–gel precursor was prepared by using 3-(Methacryloxy)propyl trimethoxysilane (MEMO) as the precursor alkoxide, ethanol as solvent, distilled water for hydrolysis and p-toluene sulfonic acid (p-TSA) (0.05%) as a catalyst. Initially, MEMO, and ethanol were charged into a vessel and then water, which had been acidified by slow addition of p-TSA into the vessel while stirring continuous at room temperature. The whole mixture was then kept stirring for 12 h to obtain a silane sol. UV-curable polymeric support material was prepared by mixing various amount of poly(ethylene glycol) diacrylate (1.6 g), hydrolyzed MEMO (0.2 g), (3-glycidyloxypropyl)
trimethoxysilane (0.1 g) and 2-hydroxy-2-methyl-1-phenyl-propan-1-one as photoinitiator (0.057 g). A hybrid epoxy-silica polymer was prepared in a beaker with adequate stirring until homogenization. Before UV-curing process, the solution was purged with nitrogen gas for 15 min to eliminate dissolved oxygen in the system. Then, UV-curable liquid formulations into a Teflon® mold (10 mm × 5 mm × 1 mm). In order to prevent the inhibiting effect of oxygen, the mixture in the mold was covered by a transparent 100 μm thick Teflon® film before irradition with a high pressure UV-lamp (OSRAM, 300W). The Teflon® film was also used to obtain a smooth surface. After 300 s irradition under UV-lamp, 100 μm thick polymeric support was obtained. 2.3. Characterization of polymeric support Infrared spectrum was recorded on Perkin–Elmer Spectrum 100 FTIR (Waltham, MA, USA) equipped with a universal attenuated total reflectance (ATR) sampling accessory (ZnSe cell) with a diamond window. Spectral analyses were performed at a resolution of 2 cm-1 in the range of 400-4000 cm-1, scanned 4 times, at room temperature and humidity. SEM analysis was performed to study the surface morphology of the hybrid epoxy-silica polymer film at an accelerating voltage of 10 kV by using Philips XL30SEM FEG (Amsterdam, Netherlands). The average diameter and the diameter distribution were obtained by using a custom code image analysis program to analyze the SEM images. A small section of the material was placed on the SEM sample holder and sputter coated with Platine. 2.4. Enzyme preparation and immobilization Lipase isolated from Candida rugosa (700 Units/mg enzyme) was purchased from Sigma Aldrich Chemical Co. (St. Louis, USA) and used without purification. C. Rugosa lipase 6.1 mg (4270 U), was suspended in 50 ml 10 mM phosphate buffer (pH 7.0) and transferred to hybrid epoxy-silica polymer film. The reaction mixture was incubated in a water bath constantly shaken for 8 h at 25 °C. This immobilization process is represented in Fig.1. Subsequently, the enzyme bounded support was taken out from the solution and washed three times with phosphate buffer (10 mM, pH 7.0) to remove the unbounded enzyme. The washing solutions were kept for measuring the amount of covalently bounded enzyme on the support. Enzyme bounded hybrid epoxy-silica polymer film was then dried with lyophilization and stored at 4 °C until use. The amount of immobilized lipase on hybrid epoxy-silica polymer film was determined within the washing solutions by UV-spectrometer at 595 nm using
Bradford's dye binding assay [20]. The standard curve was developed using a series of ovalbumin. The amount of bounded enzyme was calculated per gram of polymeric support. 2.5. Hydrolytic activity assay The hydrolytic activity of pNPP of free and immobilized CRL was determined by measuring the release of pNP. In a test vial, a known amount of hybrid epoxy-silica polymer film containing CRL or native CRL was placed. Then 1.0 mL of ethanol (containing 14.4 mM pNPP) and 1.0 mL of phosphate buffer (50 mM, pH 7.0) were added to the test vial. The reaction mixture was incubated in a water bath for 15 min at 37 °C under constant shaking. And then the reaction was stopped by addition of 2.0 mL sodium carbonate solution (0.5 M). Due to low solubility of pNPP in aqueous medium, the mixture was centrifuged (6000 g for 10 min) and the release of pNP in homogeny supernatant was measured at 410 nm [9]. The standard curve was prepared by using a series of pNP solutions in different concentrations. One unit of CRL corresponded to liberate 1.0 µmol pNP per minute under assay conditions. 2.6. Synthetic activity assay 2.0 mL of n-hexane containing 1 mM pNP and 50 mM LA was added of a known amount of polymeric support containing CRL or native CRL and the mixture was incubated at 37 °C for exactly 15 min under constant shaking. At the end of the reaction, 1.0 ml solution was discarded from the reaction mixture and dropped into 3.5 mL of 25 mM NaOH. Unreacted pNP was recovered by vortexing and measured at 410 nm. Concentration of pNP was calculated by preparation standard solutions of pNP in n-hexane and extraction with 3.5 mL of NaOH solution [9]. 2.7. Thermal stability The effect of temperature on free and immobilized CRL activities was investigated for both hydrolytic activity and synthetic activity at the range of 25-65°C under the assay conditions. 2.8. The effect of pH on hydrolitic activity The effect of pH on the activity of free and immobilized CRL was investigated in the phosphate buffer (50 mM) of a pH ranging from 5.0 to 8.0 by using the hydrolytic activity assay.
2.9. Determination of kinetic parameters Kinetic constants of free and immobilized CRL were determined by using different concentration of pNPP (0.125 - 1.0 mM) in aqueous medium. In the case of synthetic activity, 0.1–1.0 mM pNP and 50 mM LA were used in n-hexane solution. Michaelis constant (Km) and the maximum reaction velocity (Vmax) values were calculated from Lineweaver-Burk plot under the corresponding pH and temperature. 2.10. Reusability of immobilized CRL The reusability of immobilized CRL was examined by directed the hydrolytic activity and synthetic activity assay at optimal conditions. At the end of the each cycle, immobilized CRL on hybrid epoxy-silica polymer film was removed from the reaction tube and washed three times (for each assay, 10 mM phosphate buffer pH 6.0 or n-hexane, respectively) to remove any substrate or product retained in the matrix. Then the film was introduced into a fresh medium and the enzyme activities were determined at the end of each cycle. The remaining activities were expressed as a percentage of the initial activity. 2.11. Storage stability The effect of storage time of free and the immobilized CRL on lipase activity was determined for both hydrolytic and synthetic activities for a period of 30 days by the spectrophotometric assay under optimum experimental conditions. 3. Results and discussion 3.1. Characterization of the polymeric support materials The FTIR spectra of polymeric support before and after enzyme immobilization are presented in Fig. 2A and 2B. As can be seen in Fig. 2A, the epoxide group displays bands at 1250 and 950 cm−1 because of the symmetric and asymmetric ring stretching [21]. Fig. 2B shows that, after enzyme immobilization, the intensity of epoxy vibration bands at 1250 and 950 cm−1 decreased sharply. On the other hand, increase of the peak intensity at 1690 cm−1 (amide band I) is due to amide bonds of the enzyme. The most obvious difference between a spectrum of free carrier and immobilized enzyme is observed in the 1200–900 cm−1 regions, and arises from the amino acid moiety of the enzyme. This evidence proves that the covalent immobilization of CRL was achieved successfully [22].
Fig. 3 shows the SEM micrographs of the fracture surface morphology of polymeric support before and after enzyme immobilization. It can be seen from these micrographs that the polymeric film has a uniform and crack-free surface before enzyme immobilization (Fig. 3A). On the other hand, it’s clearly seen that the surface morphology of the polymeric support changed after immobilization (Fig. 3B). Also these SEM micrographs show the attachment of the enzyme on the surface of hybrid epoxy-silica polymer film. 3.2. Immobilization Efficiency The amount of covalently immobilized CRL on hybrid epoxy-silica polymer film was found as 7.22 mg per gram of polymeric support. In the literature, there are many immobilization studies for CRL onto various polymeric supports. Zhu and Sun [23] investigated immobilization of CRL onto glutaraldehyde activated poly(vinyl alcohol-co-ethylene) nanofibrous membrane. They reported the amount of CRL as 39.52 mg g-1 of the polymeric support. Bayramoglu and Arica [10] reported the amount of immobilized enzyme as 23.44 mg g-1 magnetic poly(glycidyl methacrylate-methyl methacrylate) beads that were prepared via suspension polymerization. Ozyilmaz [9] who immobilized CRL onto silica gel via glutaraldehyde and via 1,6-diaminohexane spacer arm, she found the amount of bound lipase as 9.0 and 9.8 mg g-1, respectively. 3.3. Effect of pH on activity The effect of pH on pNPP hydrolysis was investigated and the results are given in Fig. 4. The optimum pH of the free and immobilized CRL was found as 7.0 and 6.0, respectively. This shift relies on upon the enzyme reaction and in addition on the structure of the hybrid epoxysilica polymer film. Such changes in the optimal pH range have been reported by other researchers. Ye and colleagues [24] and Wang and colleagues [12] found that the optimum pH was shifted to an acidic range after enzyme immobilization. Conversely, Huang and colleagues [25] and Bayramoglu and Arica [10] found that the optimal pH shifted to a basic range after immobilization. On the other hand, same value of optimum pH for free and immobilized CRL was reported Hou and colleagues [26] and Zhu and Sun [23]. 3.4. Thermal stability The effect of reaction temperature on the catalytic activities of free and immobilized lipases was investigated.
Fig. 5A shows the maximum catalytic activity on the hydrolysis activity of free and immobilized lipase for pNPP was obtained at 37 °C for free CRL and 50 °C for immobilized CRL; however, as the temperature increased, the stability of the free enzyme decreased when compared to the immobilized form. In the study of Hou and colleagues in which CRL was covalently immobilized on core–shell magnetic polydopamine/alginate biocomposite, the immobilized enzyme showed preferable thermostability over the free one [21]. Huang and colleagues [25], Zhu and Sun [23], Vaidya and colleagues [27] and Ye and colleagues [24] expressed a similar increase in optimum temperature for immobilized CRL. As seen in Fig. 5B, maximum activity for free and immobilized enzyme for pNPL synthesis was found to be at 37 °C and 45 °C, respectively. Shang and colleagues [28] investigated of CRL onto on ZnO nanowires/macroporous silica composites. They reported the optimum temperature as 50 °C for biocatalytic synthesis of phytosterol esters. Zheng and colleagues [29] immobilized of CRL onto magnetic poly (allyl glycidyl ether-co-ethylene glycol dimethacrylate) polymer microsphere. They found that the optimum temperature as 55 °C for synthesis of phytosterol esters of unsaturated fatty acids. Expanded thermal stability has been reported for various immobilized catalysts, and the polymeric material should protect the tertiary structure of enzyme’s [30]. This is probably owes to the covalent attachment through epoxy groups so the immobilized enzyme becomes more resistant to heat inactivation. 3.5. Kinetic parameters Effect of kinetic parameter (Km and Vmax) was determined for the free and immobilized CRL for the hydrolytic activity under optimum assay conditions. Km and Vmax values were found as 0.71 mM, 0.83 mM min-1 and 0.91 mM, 0.23 mM min-1 for free and immobilized CRL, respectively. Kinetic values of free and immobilized CRL were measured for synthetic activity assay at various concentrations of pNP (0.1 – 1 mM) and 50 mM LA. Respective Km and Vmax parameters for the free and immobilized CRL were calculated as 1.12 mM, 0.64 mM min-1 and 1.19 mM, 0.11 mM min-1. After enzyme immobilization, the Km values were generally increased and Vmax values were decreased. Increased Km value is indicated lower affinity for the substrate caused by steric effects, diffusional limitations, and ionic strength [9]. The results have shown that the affinity of the immobilized CRL to its substrate was decreased the immobilization process. Similar to our results, Monier et al. [31] and Bayramoglu et al. [32] reported an increase in Km value of immobilized lipase as compared to free enzyme.
3.6. Reusability and storage stability Repeated uses of the immobilized CRL samples were calculated by using the optimal conditions for hydrolytic and synthetic activity assays. The measured activities are shown in Fig. 6. The residual activity of the immobilized CRL lessened with the increasing number of washes. At the end of the 30th cycle, the residual activity retained was approximately for hydrolytic and synthetic activity 56% and 59%, respectively. The reuse stability was previously found for the covalently immobilized lipases, where about 80-90% activity was retained after 5 to 12 cycles [32-34]. In similar way, Yucel investigated operational stability of lipase from Thermomyces lanuginosus immobilized onto olive pomace and found that the immobilized lipase was stable retaining more than 80% residual activity after 10 cycles of batch operation [35]. Huang and colleagues found that the remaining activity of lipase immobilized on the cellulose fibrous membrane was about 30% after being used repeatedly for 8 consecutive batches [25]. In general, enzymes are not stable in solutions and lose their activities gradually by time. So immobilization of enzyme is favored to enhance enzyme stability. The free and immobilized CRL were stored at 4°C and their activities were tested by the hydrolytic and the synthetic activity assays for 30 days. As shown in Fig. 7, native enzyme lost its activity completely within 20 days, whereas the immobilized enzyme retained was approximately for hydrolytic and synthetic activities 82% and 72%, respectively, under the same storage time. Results showed that, by immobilization, the enzyme gained a more stable character compared with the free CRL. It has been reported that the immobilized lipase retained its full activity under all storage conditions, whereas the free lipase gradually lost its activity as the storage time increased [23,36,37]. 4. Conclusions In this study UV-curable hybrid epoxy-silica polymer films were successfully prepared and the immobilization of CRL onto these films was obtained by covalently bonding and characterized by FTIR and SEM techniques to understand immobilization of enzyme on support as well as the enzyme-support interaction. The immobilized CRL indicated that a higher hydrolysis and synthesis activities than free CRL. It also exhibited better thermal stability, storage stability and reusability compared with the free one. These results demonstrated the hybrid epoxy-silica polymer films could be used for biotechnological applications for immobilizing several bioactive molecules.
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Figure Captions
Figure. 1 Preperation of hybrid epoxy-silica polymer film via sol–gel process and enzyme immobilization on functionalized support (MEMO: 3-methacryloxypropyltrimethoxysilane pTSA: para-toluenesulfonic acid)
Figure. 2 Fourier transform infrared spectra (FTIR) of polymeric support (A) free hybrid epoxy-silica polymer (B) immobilized enyzme on hybrid epoxy-silica polymer
Figure. 3 Scanning electron micrographs of the polymeric support (A) free hybrid epoxysilica polymer (B) immobilized enyzme on hybrid epoxy-silica polymer
Figure. 4 pH stability of free and immobilized CRL.
(A)
(B)
Figure. 5 The effect of reaction temperature on the (A) hydrolysis of pNPP and (B) synthesis of pNPL
Figure. 6 The reusability of immobilized CRL under optimum conditions.
Figure. 7 Storage stabilities of free and immobilized CRL.
S1381-1177(16)30022-4 http://dx.doi.org/doi:10.1016/j.molcatb.2016.02.007 MOLCAB 3325
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
19-11-2015 15-1-2016 14-2-2016
Please cite this article as: Basak Yuce-Dursun, Asli Beyler Cigil, Dilek Dongez, M.Vezir Kahraman, Ayse Ogan, Serap Demir, Preparation and characterization of sol-gel hybrid coating films for covalent immobilization of lipase enzyme, Journal of Molecular Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2016.02.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Preparation and Characterization of Sol-Gel Hybrid Coating Films for Covalent Immobilization of Lipase Enzyme Basak Yuce-Dursun, Asli Beyler Cigil, Dilek Dongez, M. Vezir Kahraman, Ayse Ogan, Serap Demir* Marmara University, Faculty of Arts and Sciences, Department of Chemistry, Istanbul-Turkey *
Corresponding Author: Serap Demir
Address: Marmara University, Goztepe Campus, Faculty of Arts and Sciences, Chemistry Department, 34722 Kadikoy, Istanbul/TURKEY E-mail: [email protected] Phone: +90 216 3464553 Fax: +90 216 3478783
Graphical abstract
Highlgihts
UV-curable hybrid epoxy-silica polymer films were prepared via sol–gel method.
Lipase from Candida rugosa was immobilized on polymer through covalent binding.
The hydrolytic and synthetic activities of lipase were investigated.
Kinetic properties of immobilized and free lipase were characterized.
Immobilization improved the thermal stability, storage stability and reusability.
Abstract In this study UV-curable hybrid epoxy-silica polymer films were prepared via sol–gel method. Lipase (EC 3.1.1.3) from Candida rugosa was covalently immobilized onto hybrid epoxy-silica polymer films and immobilization capacity of polymer films was found 7.22 mg g-1. The morphology of the polymeric support was characterized by scanning electron microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR). Immobilized and free enzymes were used in two different reaction systems: hydrolysis of p-nitrophenyl palmitate in aqueous medium and synthesis of p-nitrophenyl linoleate (from p-nitrophenol and linoleic acid) in n-hexane medium. The effect of temperature on hydrolytic and synthetic activities was investigated and observed maximum activities at 50 °C and 45 °C for immobilized enzyme, orderly. Km values for free enzyme were determined 0.71 and 1.12 mM by hydrolytic and synthetic activity assays, respectively, while these values were observed as 0.91 mM and 1.19 mM for immobilized enzyme. At the end of 30 repeated cycles, 56% and 59% of initial activities remained for hydrolytic and synthetic assays, respectively. Native enzyme lost its activity completely within 20 days, whereas the immobilized enzyme retained for hydrolytic and synthetic activities was approximately 82% and 72%, respectively, under the same storage time. Keywords: Lipase; immobilization; sol-gel; hydrolytic activity; synthetic activity.
1. Introduction Lipases (triacylglycerol ester hydrolases, EC 3.1.1.3) are atypical enzymes that catalyze both hydrolysis and formation of the ester bond between glycerol and long-chain fatty acids [1]. Lipases also catalyze various bioconversion reactions in a wide variety of organic solvents such as the hydrolysis, esterification, transesterification, aminolysis, and acidolysis [2]. In the last decade, there has been an increasing interest in the use of lipases for their properties such as high specificity, efficient reaction rate, non-toxicity and biodegradability, reproducibility under normal laboratory conditions. Also they are able to recognize very different substrates [3]. Lipases are used as versatile industrial applications, including cleanser, biosensor and biodiesel production, bio-catalytic determination of pharmaceuticals, nourishment and flavor businesses, cosmetics and perfumery production [4,5]. Lipases are produced in almost every living cell, but due to a number of advantages, microbial lipases are the most widely used group for practical applications because of low costs of production and easy modification of properties. Among the lipases from various sources, Candida rugosa lipase (CRL) received much attention due to its high activity and broad specificity [5,6]. Immobilization of enzymes is one of the most essential facets of modern biotechnology. This process frequently overcome the structural instability and allow repeated use, native enzymes have been successfully immobilized on different supports using various techniques and enables long-term stability/recyclability of the biocatalyst [5,7,8]. Because of the environmental conditions, free lipases are easily inactivated and hard to be recover for reuse. The separation of products in presence of free lipases is tedious. Hence, further industrial uses of free lipases are restricted because of high costs and complex downstream processing [4]. These drawbacks of the free lipases are overcome through immobilization techniques. As a very promising strategy, immobilization of lipases on solid supports is economically advantageous in order to facilitate the stability and reusability of these enzymes. Immobilized enzymes are preferable to free enzymes because of their easy separation from the reaction mixture makes them suitable for constant use. A variety of support materials have been used in immobilization of lipases like silica [9], magnetic poly(glycidyl methacrylate-methyl methacrylate) beads [10], polypropylene hollow fiber membrane modified with hydrophobic polypeptide [11], polysulfone ion exchange
membranes [12], mesoporous silica [13], poly(hydroxyethyl methacrylate)-based membranes [14], calcium alginate gels [15], celite [16], nylon-6 [17], chitosan and agarose [18]. In this study UV-curable hybrid epoxy-silica polymer films were prepared via sol–gel process. The most generally utilized system for preparing hybrid materials is the sol–gel technique, which permits acquiring cross-linked inorganic silica into the polymer network to improve the properties of the material. Hybrid materials can be prepared by a radiation-curing technique such as using a UV curable binder system [19]. In this study, lipase from Candida rugosa (CRL) was covalently immobilized onto organic/inorganic hybrid SiO2–matrix films. Polymeric support material was characterized by using FTIR and SEM. Enzyme activity of free and immobilized lipase was examined in two different reaction systems: 1. hydrolysis of p-nitrophenyl palmitate in aqueous medium, 2. synthesis of p-nitrophenyl linoleate (from pnitrophenol and linoleic acid) in organic medium. Temperature, kinetic parameters, and storage stabilities for free and immobilized enzymes were evaluated for both hydrolytic and synthetic activities. 2. Materials and methods 2.1. Reagents Lipase from C. rugosa (700 U mg−1), p-nitrophenol (pNP), linoleic acid (LA), p-nitrophenyl palmitate (pNPP), 3-methacryloxypropyltrimethoxysilane (MEMO), p-toluene sulfonic acid, poly(ethylene glycol) diacrylate, (3-glycidyloxypropyl) trimethoxysilane were purchased from Sigma Aldrich Chemical Co. (St. Louis, USA). Bradford reagent and ovalbumin were commercial product of BioRad (BioRad Laboratories, Hercules CA, USA). All other chemicals were of analytical grade and used without further purification. 2.2. Preparation of polymeric support material A hybrid epoxy-silica polymer film was prepared through sol–gel process. The sol–gel precursor was prepared by using 3-(Methacryloxy)propyl trimethoxysilane (MEMO) as the precursor alkoxide, ethanol as solvent, distilled water for hydrolysis and p-toluene sulfonic acid (p-TSA) (0.05%) as a catalyst. Initially, MEMO, and ethanol were charged into a vessel and then water, which had been acidified by slow addition of p-TSA into the vessel while stirring continuous at room temperature. The whole mixture was then kept stirring for 12 h to obtain a silane sol. UV-curable polymeric support material was prepared by mixing various amount of poly(ethylene glycol) diacrylate (1.6 g), hydrolyzed MEMO (0.2 g), (3-glycidyloxypropyl)
trimethoxysilane (0.1 g) and 2-hydroxy-2-methyl-1-phenyl-propan-1-one as photoinitiator (0.057 g). A hybrid epoxy-silica polymer was prepared in a beaker with adequate stirring until homogenization. Before UV-curing process, the solution was purged with nitrogen gas for 15 min to eliminate dissolved oxygen in the system. Then, UV-curable liquid formulations into a Teflon® mold (10 mm × 5 mm × 1 mm). In order to prevent the inhibiting effect of oxygen, the mixture in the mold was covered by a transparent 100 μm thick Teflon® film before irradition with a high pressure UV-lamp (OSRAM, 300W). The Teflon® film was also used to obtain a smooth surface. After 300 s irradition under UV-lamp, 100 μm thick polymeric support was obtained. 2.3. Characterization of polymeric support Infrared spectrum was recorded on Perkin–Elmer Spectrum 100 FTIR (Waltham, MA, USA) equipped with a universal attenuated total reflectance (ATR) sampling accessory (ZnSe cell) with a diamond window. Spectral analyses were performed at a resolution of 2 cm-1 in the range of 400-4000 cm-1, scanned 4 times, at room temperature and humidity. SEM analysis was performed to study the surface morphology of the hybrid epoxy-silica polymer film at an accelerating voltage of 10 kV by using Philips XL30SEM FEG (Amsterdam, Netherlands). The average diameter and the diameter distribution were obtained by using a custom code image analysis program to analyze the SEM images. A small section of the material was placed on the SEM sample holder and sputter coated with Platine. 2.4. Enzyme preparation and immobilization Lipase isolated from Candida rugosa (700 Units/mg enzyme) was purchased from Sigma Aldrich Chemical Co. (St. Louis, USA) and used without purification. C. Rugosa lipase 6.1 mg (4270 U), was suspended in 50 ml 10 mM phosphate buffer (pH 7.0) and transferred to hybrid epoxy-silica polymer film. The reaction mixture was incubated in a water bath constantly shaken for 8 h at 25 °C. This immobilization process is represented in Fig.1. Subsequently, the enzyme bounded support was taken out from the solution and washed three times with phosphate buffer (10 mM, pH 7.0) to remove the unbounded enzyme. The washing solutions were kept for measuring the amount of covalently bounded enzyme on the support. Enzyme bounded hybrid epoxy-silica polymer film was then dried with lyophilization and stored at 4 °C until use. The amount of immobilized lipase on hybrid epoxy-silica polymer film was determined within the washing solutions by UV-spectrometer at 595 nm using
Bradford's dye binding assay [20]. The standard curve was developed using a series of ovalbumin. The amount of bounded enzyme was calculated per gram of polymeric support. 2.5. Hydrolytic activity assay The hydrolytic activity of pNPP of free and immobilized CRL was determined by measuring the release of pNP. In a test vial, a known amount of hybrid epoxy-silica polymer film containing CRL or native CRL was placed. Then 1.0 mL of ethanol (containing 14.4 mM pNPP) and 1.0 mL of phosphate buffer (50 mM, pH 7.0) were added to the test vial. The reaction mixture was incubated in a water bath for 15 min at 37 °C under constant shaking. And then the reaction was stopped by addition of 2.0 mL sodium carbonate solution (0.5 M). Due to low solubility of pNPP in aqueous medium, the mixture was centrifuged (6000 g for 10 min) and the release of pNP in homogeny supernatant was measured at 410 nm [9]. The standard curve was prepared by using a series of pNP solutions in different concentrations. One unit of CRL corresponded to liberate 1.0 µmol pNP per minute under assay conditions. 2.6. Synthetic activity assay 2.0 mL of n-hexane containing 1 mM pNP and 50 mM LA was added of a known amount of polymeric support containing CRL or native CRL and the mixture was incubated at 37 °C for exactly 15 min under constant shaking. At the end of the reaction, 1.0 ml solution was discarded from the reaction mixture and dropped into 3.5 mL of 25 mM NaOH. Unreacted pNP was recovered by vortexing and measured at 410 nm. Concentration of pNP was calculated by preparation standard solutions of pNP in n-hexane and extraction with 3.5 mL of NaOH solution [9]. 2.7. Thermal stability The effect of temperature on free and immobilized CRL activities was investigated for both hydrolytic activity and synthetic activity at the range of 25-65°C under the assay conditions. 2.8. The effect of pH on hydrolitic activity The effect of pH on the activity of free and immobilized CRL was investigated in the phosphate buffer (50 mM) of a pH ranging from 5.0 to 8.0 by using the hydrolytic activity assay.
2.9. Determination of kinetic parameters Kinetic constants of free and immobilized CRL were determined by using different concentration of pNPP (0.125 - 1.0 mM) in aqueous medium. In the case of synthetic activity, 0.1–1.0 mM pNP and 50 mM LA were used in n-hexane solution. Michaelis constant (Km) and the maximum reaction velocity (Vmax) values were calculated from Lineweaver-Burk plot under the corresponding pH and temperature. 2.10. Reusability of immobilized CRL The reusability of immobilized CRL was examined by directed the hydrolytic activity and synthetic activity assay at optimal conditions. At the end of the each cycle, immobilized CRL on hybrid epoxy-silica polymer film was removed from the reaction tube and washed three times (for each assay, 10 mM phosphate buffer pH 6.0 or n-hexane, respectively) to remove any substrate or product retained in the matrix. Then the film was introduced into a fresh medium and the enzyme activities were determined at the end of each cycle. The remaining activities were expressed as a percentage of the initial activity. 2.11. Storage stability The effect of storage time of free and the immobilized CRL on lipase activity was determined for both hydrolytic and synthetic activities for a period of 30 days by the spectrophotometric assay under optimum experimental conditions. 3. Results and discussion 3.1. Characterization of the polymeric support materials The FTIR spectra of polymeric support before and after enzyme immobilization are presented in Fig. 2A and 2B. As can be seen in Fig. 2A, the epoxide group displays bands at 1250 and 950 cm−1 because of the symmetric and asymmetric ring stretching [21]. Fig. 2B shows that, after enzyme immobilization, the intensity of epoxy vibration bands at 1250 and 950 cm−1 decreased sharply. On the other hand, increase of the peak intensity at 1690 cm−1 (amide band I) is due to amide bonds of the enzyme. The most obvious difference between a spectrum of free carrier and immobilized enzyme is observed in the 1200–900 cm−1 regions, and arises from the amino acid moiety of the enzyme. This evidence proves that the covalent immobilization of CRL was achieved successfully [22].
Fig. 3 shows the SEM micrographs of the fracture surface morphology of polymeric support before and after enzyme immobilization. It can be seen from these micrographs that the polymeric film has a uniform and crack-free surface before enzyme immobilization (Fig. 3A). On the other hand, it’s clearly seen that the surface morphology of the polymeric support changed after immobilization (Fig. 3B). Also these SEM micrographs show the attachment of the enzyme on the surface of hybrid epoxy-silica polymer film. 3.2. Immobilization Efficiency The amount of covalently immobilized CRL on hybrid epoxy-silica polymer film was found as 7.22 mg per gram of polymeric support. In the literature, there are many immobilization studies for CRL onto various polymeric supports. Zhu and Sun [23] investigated immobilization of CRL onto glutaraldehyde activated poly(vinyl alcohol-co-ethylene) nanofibrous membrane. They reported the amount of CRL as 39.52 mg g-1 of the polymeric support. Bayramoglu and Arica [10] reported the amount of immobilized enzyme as 23.44 mg g-1 magnetic poly(glycidyl methacrylate-methyl methacrylate) beads that were prepared via suspension polymerization. Ozyilmaz [9] who immobilized CRL onto silica gel via glutaraldehyde and via 1,6-diaminohexane spacer arm, she found the amount of bound lipase as 9.0 and 9.8 mg g-1, respectively. 3.3. Effect of pH on activity The effect of pH on pNPP hydrolysis was investigated and the results are given in Fig. 4. The optimum pH of the free and immobilized CRL was found as 7.0 and 6.0, respectively. This shift relies on upon the enzyme reaction and in addition on the structure of the hybrid epoxysilica polymer film. Such changes in the optimal pH range have been reported by other researchers. Ye and colleagues [24] and Wang and colleagues [12] found that the optimum pH was shifted to an acidic range after enzyme immobilization. Conversely, Huang and colleagues [25] and Bayramoglu and Arica [10] found that the optimal pH shifted to a basic range after immobilization. On the other hand, same value of optimum pH for free and immobilized CRL was reported Hou and colleagues [26] and Zhu and Sun [23]. 3.4. Thermal stability The effect of reaction temperature on the catalytic activities of free and immobilized lipases was investigated.
Fig. 5A shows the maximum catalytic activity on the hydrolysis activity of free and immobilized lipase for pNPP was obtained at 37 °C for free CRL and 50 °C for immobilized CRL; however, as the temperature increased, the stability of the free enzyme decreased when compared to the immobilized form. In the study of Hou and colleagues in which CRL was covalently immobilized on core–shell magnetic polydopamine/alginate biocomposite, the immobilized enzyme showed preferable thermostability over the free one [21]. Huang and colleagues [25], Zhu and Sun [23], Vaidya and colleagues [27] and Ye and colleagues [24] expressed a similar increase in optimum temperature for immobilized CRL. As seen in Fig. 5B, maximum activity for free and immobilized enzyme for pNPL synthesis was found to be at 37 °C and 45 °C, respectively. Shang and colleagues [28] investigated of CRL onto on ZnO nanowires/macroporous silica composites. They reported the optimum temperature as 50 °C for biocatalytic synthesis of phytosterol esters. Zheng and colleagues [29] immobilized of CRL onto magnetic poly (allyl glycidyl ether-co-ethylene glycol dimethacrylate) polymer microsphere. They found that the optimum temperature as 55 °C for synthesis of phytosterol esters of unsaturated fatty acids. Expanded thermal stability has been reported for various immobilized catalysts, and the polymeric material should protect the tertiary structure of enzyme’s [30]. This is probably owes to the covalent attachment through epoxy groups so the immobilized enzyme becomes more resistant to heat inactivation. 3.5. Kinetic parameters Effect of kinetic parameter (Km and Vmax) was determined for the free and immobilized CRL for the hydrolytic activity under optimum assay conditions. Km and Vmax values were found as 0.71 mM, 0.83 mM min-1 and 0.91 mM, 0.23 mM min-1 for free and immobilized CRL, respectively. Kinetic values of free and immobilized CRL were measured for synthetic activity assay at various concentrations of pNP (0.1 – 1 mM) and 50 mM LA. Respective Km and Vmax parameters for the free and immobilized CRL were calculated as 1.12 mM, 0.64 mM min-1 and 1.19 mM, 0.11 mM min-1. After enzyme immobilization, the Km values were generally increased and Vmax values were decreased. Increased Km value is indicated lower affinity for the substrate caused by steric effects, diffusional limitations, and ionic strength [9]. The results have shown that the affinity of the immobilized CRL to its substrate was decreased the immobilization process. Similar to our results, Monier et al. [31] and Bayramoglu et al. [32] reported an increase in Km value of immobilized lipase as compared to free enzyme.
3.6. Reusability and storage stability Repeated uses of the immobilized CRL samples were calculated by using the optimal conditions for hydrolytic and synthetic activity assays. The measured activities are shown in Fig. 6. The residual activity of the immobilized CRL lessened with the increasing number of washes. At the end of the 30th cycle, the residual activity retained was approximately for hydrolytic and synthetic activity 56% and 59%, respectively. The reuse stability was previously found for the covalently immobilized lipases, where about 80-90% activity was retained after 5 to 12 cycles [32-34]. In similar way, Yucel investigated operational stability of lipase from Thermomyces lanuginosus immobilized onto olive pomace and found that the immobilized lipase was stable retaining more than 80% residual activity after 10 cycles of batch operation [35]. Huang and colleagues found that the remaining activity of lipase immobilized on the cellulose fibrous membrane was about 30% after being used repeatedly for 8 consecutive batches [25]. In general, enzymes are not stable in solutions and lose their activities gradually by time. So immobilization of enzyme is favored to enhance enzyme stability. The free and immobilized CRL were stored at 4°C and their activities were tested by the hydrolytic and the synthetic activity assays for 30 days. As shown in Fig. 7, native enzyme lost its activity completely within 20 days, whereas the immobilized enzyme retained was approximately for hydrolytic and synthetic activities 82% and 72%, respectively, under the same storage time. Results showed that, by immobilization, the enzyme gained a more stable character compared with the free CRL. It has been reported that the immobilized lipase retained its full activity under all storage conditions, whereas the free lipase gradually lost its activity as the storage time increased [23,36,37]. 4. Conclusions In this study UV-curable hybrid epoxy-silica polymer films were successfully prepared and the immobilization of CRL onto these films was obtained by covalently bonding and characterized by FTIR and SEM techniques to understand immobilization of enzyme on support as well as the enzyme-support interaction. The immobilized CRL indicated that a higher hydrolysis and synthesis activities than free CRL. It also exhibited better thermal stability, storage stability and reusability compared with the free one. These results demonstrated the hybrid epoxy-silica polymer films could be used for biotechnological applications for immobilizing several bioactive molecules.
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Figure Captions
Figure. 1 Preperation of hybrid epoxy-silica polymer film via sol–gel process and enzyme immobilization on functionalized support (MEMO: 3-methacryloxypropyltrimethoxysilane pTSA: para-toluenesulfonic acid)
Figure. 2 Fourier transform infrared spectra (FTIR) of polymeric support (A) free hybrid epoxy-silica polymer (B) immobilized enyzme on hybrid epoxy-silica polymer
Figure. 3 Scanning electron micrographs of the polymeric support (A) free hybrid epoxysilica polymer (B) immobilized enyzme on hybrid epoxy-silica polymer
Figure. 4 pH stability of free and immobilized CRL.
(A)
(B)
Figure. 5 The effect of reaction temperature on the (A) hydrolysis of pNPP and (B) synthesis of pNPL
Figure. 6 The reusability of immobilized CRL under optimum conditions.
Figure. 7 Storage stabilities of free and immobilized CRL.