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Covalent immobilization of lipase onto hydrophobic group incorporated poly(2-hydroxyethyl methacrylate) based hydrophilic membrane matrix
Journal of Food Engineering 52 (2002) 367–374 www.elsevier.com/locate/jfoodeng
Covalent immobilization of lipase onto hydrophobic group incorporated poly(2-hydroxyethyl methacrylate) based hydrophilic membrane matrix G€ ulay Bayramo glu a, Yasemin Kacßar b, Adil Denizli c, M. Yakup Arıca a
b,*
Department of Chemistry, Kırıkkale University, 71450 Yahsßihan-Kırıkkale, Turkey Department of Biology, Kırıkkale University, 71450 Yahsßihan-Kırıkkale, Turkey c Department of Chemistry, Hacettepe University, 06531 Beytepe-Ankara, Turkey
b
Received 23 April 2001; accepted 27 June 2001
Abstract In this study, a hydrophobic group containing monomer, 2-methacrylamidophenyalanine (MAPA) was prepared by using methacrylochloride and phenylalanine. Then, poly(2-hydroxyethyl methacrylate-co-methacrylamido-phenlyalanine) (pHEMAMAPA) membranes were prepared by UV-initiated photopolymerization of HEMA and MAPA in the presence of an initiator a-a0 -azobisisobutyronitrile (AIBN). The lipase was immobilized onto these membranes by covalent bonding through carbodiimide activation. The amount of enzyme loading on the membranes was increased as the MAPA ratio increased in the membrane structure. Immobilization improved the pH stability of the enzyme as well as its temperature stability. Thermal stability was found to increase with immobilization and at 60 °C the thermal stability constants were 1:1 101 min for free enzyme and 1:2 102 min for the immobilized enzyme. The immobilized enzyme activity was found to be quite stable in repeated experiments. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Poly(2-hydroxyethyl methacrylate) membrane; Phenylalanine; Covalent bonding; Enzyme immobilization; Lipase
1. Introduction Immobilization confers additional stability to a variety of enzymes against several forms of denaturation. Enzymes have been immobilized on different shapes of supports (i.e., membranes or beads) either by covalent binding, entrapment, or adsorption. Membrane-immobilized enzymes may serve as model systems for enzymes, naturally bound to membranes, or may find practical application in biosensors and enzyme reactors as less expensive, more stable and reusable alternatives to free enzymes (Arıca, Denizli, Salih, Pisßkin, & Hasırcı, 1997; Ivanov & Schneider, 1997; Zhou & Chen, 2001). Lipase (triacylglycerol acyl ester hydrolyses, EC 3.1.1.3) is an enzyme possessing an intrinsic capacity to
*
Corresponding author. Tel.: +90-318-357-2477; fax: +90-318-3572329. E-mail address: [email protected] (M. Yakup Arıca).
catalyse cleavage of carboxy ester bonds in tri-, di-, and monoacylglycerols to glycerol and fatty acids. The enzyme is distributed among higher animals, plants and micro-organisms in which it plays a key role in the lipid metabolism. The enzyme has been widely used for biotechnological applications in dairy industry, oil processing, production of surfactants and preparation of enantiomerically pure pharmaceuticals (Balcao, Paiva, & Malcata, 1996; Pavia, Balcao, & Malcata, 2000). The immobilized lipase could be used in the dairy industry for the controlled hydrolysis of milk fat, a process which is useful for acceleration of cheese ripening, flavour enhancement of butter, manufacture of cheese-like products, and preparation of enzyme-modified cheese for use as ingredients in dressings, and sauces (Balcao et al., 1996). The immobilized lipase could also be applied to the production of x3-polyunsaturated fatty acid concentrates from fish liver oils (which have been claimed to provide beneficial health effects via prevention of coronary heart diseases) for use as
0260-8774/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 0 - 8 7 7 4 ( 0 1 ) 0 0 1 2 8 - 5
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nutropharmaceutical food supplements. Lipase immobilization was carried out by covalent attachment, entrapment, and adsorption to hydrophobic or hydrophilic surface (Al-Duri & Yong, 2000; Arıca, Kacßar, Ergene, & Denizli, 2001; Balcao et al., 1996; Demir, Acar, Sarıo glu, & Mutlu, 2001; de Oliveira, Alves, & de Castro, 2000). After immobilization of lipase, changes were observed in the enzyme activity, optimum pH, affinity to substrate and stability (Arıca et al., 2001; Itoyama, Tokura, & Hayashi, 1994; Reetz, Zonta, & Simpelkapt, 1996). The extent of these changes depended on the source of enzyme, the type of support and the method of immobilization. It is, thus, important that the choice of proper support material and immobilization method over the free bioactive agent should be well € ktem, O € ktem, & Tuncel, 1999; de justified (Arıca, O Oliveira et al., 2000; Gandhi, Vijayalakshmi, Sawant, & Joshi, 1996). Poly(2-hydroxyethyl methacrylate) (pHEMA) is a hydrophilic polymer and it is described as a well biocompatible material. The properties of pHEMA can be modified in a wide range, such as by co-polymerization or additives. Hydrophobic groups could be introduced on the pHEMA structure either attachment through functional –OH group or co-polymerization of HEMA with a hydrophobic group carrying co-monomer. In this way, a pHEMA based hydrophilic/hydrophobic support can be obtained and would be used in the enzyme and protein immobilization studies as a hydrophobic group carrying support (Arıca et al., 1997; Arıca et al., 1999; Basri, Ampon, Yunus, Razak, & Salleh, 1994a,b). In the present study, a hydrophobic group containing porous pHEMA based matrix was prepared in the membrane form by co-polymerization of 2-hydroxyethyl methacrylate (HEMA) monomer with a hydrophobic group introduced co-monomer (2-methacrylamidophenylalanine, MAPA). This method is effective in that the hydrophobic group could be easily introduced into the membrane matrix at a desired density by adjusting the concentration of hydrophobic group carrying co-monomer in the polymerization mixture. A series of pHEMA-MAPA membranes with different HEMA/ MAPA ratios were prepared by UV-initiated photopolymerization in the presence of an initiator (a; a0 -azobisisobutyronitrile, AIBN). These membrane matrices were used for the immobilization of Candida rugosa lipase after activation of carbodiimide via covalent attachment. The system parameters such as the effect of phenylalanine concentration of support on the lipase loading capacity and the effect of loading on the activity yield of immobilized lipase were determined at various lipase loadings. The influence of several parameters was also characterized such as activity retention, catalytic properties, thermal and operational stability aspects were compared.
2. Experimental section 2.1. Materials Lipase (Type VII, from Candida rugosa, lyophilized powder) was supplied from Sigma chemicals (St Louis, MO, USA) and used as-received. 2-hydroxyethyl methacrylate (HEMA) was obtained from Fluka AG (Switzerland), distilled under reduced pressure in the presence of hydroquinone and stored at 4 °C until use. Phenylalanine, methacrylochloride, a-a0 -azoisobisbutyronitrile (AIBN), gum arabic, carbodiimide (1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride) (EDC) and bovine serum albumin (BSA) were obtained from Sigma chemicals. All other chemicals were of analyticalgrade and were purchased from Merck AG (Darmstadt, Germany). 2.2. Preparation of 2-methacrylamidophenylalanin monomer Phenylalanine (5.0 g) and sodium nitrite ðNaNO2 ; 0:2 gÞ were dissolved in a potassium carbonate solution (K2 CO3 ; 30 ml, 5%, w/v). The mixture was transferred in a 100 ml round-bottomed three-necked flask fitted with a dropping funnel. The reaction chamber was cooled down to 0 °C in an ice-water bath and stirred magnetically under a nitrogen atmosphere. A 6.0 ml of methacrylochloride was placed into the dropping funnel and was introduced dropwise to the reaction mixture in 10 min. The reaction chamber was then removed from the ice-water bath and the reaction was maintained at room temperature for 2 h. After reaction, the pH of the mixture was adjusted to 3.0 and the product was extracted with chloroform (50 ml). After phase separation, the organic phase was dried with magnesium sulphate and chloroform was evaporated with a rotary evaporator. The product (i.e., 2-methacrylamidophenylalanine monomer, MAPA) was crystallized in an ether–cyclohexane mixture (5:95%, v/v). 2.3. Preparation of poly(2-hydroxyethylmethacrylate-comethacrylamido-phenylalanine) membranes The poly(2-hydroxyethylmethacrylate-co-methacrylamido-phenylalanine) (pHEMA-MAPA) hydrogel in the membrane form was prepared by a UV-initiated photopolymerization, as previously described (Arıca et al., 1997). The polymerization was carried out in a round glass mould (diameter: 9.0 cm) at 25 °C under nitrogen atmosphere for 1 h. To check the effect of monomer ratio on the lipase loading capacity of the membrane, in the initial polymerization mixture five different HEMA/MAPA ratios were used (Table 1). The membrane preparation mixture (5 ml) contained 2 ml HEMA, 25–200 mg (or 107–856 lmol) methacrylamido-
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Table 1 Properties of the pHEMA-MAPA-1–5 membranes Membrane type
HEMA/MAPA (mol ratio)
Phenylalanine density ðlmol g1 Þ
Enzyme loading (mg g1 membrane)
Recovered activity (%)
Enzyme activity (U g1 membrane)
pHEMA-MAPA-1 pHEMA-MAPA-2 pHEMA-MAPA-3 pHEMA-MAPA-4 pHEMA-MAPA-5
154 77 52 39 26
52 104 156 208 312
1.53 2.62 3.24 3.84 4.23
78 73 66 59 55
841 1348 1507 1597 1670
phenylalanine (MAPA), 10 mg AIBN as polymerization initiator and 3 ml phosphate buffer (0.1 M, pH 7.0). The resulting mixture was equilibrated at 25 °C for 30 min in a thermostatic water bath. The mixture was then poured into the mould and exposed to long-wave ultraviolet radiation for 20 min. The pHEMA-MAPA membranes (their chemical structure being shown in Fig. 1) were washed several times with distilled water and cut into circular pieces (diameter: 1.0 cm) with a perforator. The degree of MAPA incorporation in the synthesized pHEMA-MAPA membranes was determined by measuring the in C, H, N and O contents with a Laco (Model CHNSO-932) elemental analyzer. The scanning electron micrograph (SEM) of the dried pHEMA-MAPA membranes was obtained by using a JEOL 5600 scanning electron microscope, after coating with gold under reduced pressure. 2.4. Activation of pHEMA-MAPA membrane with carbodiimide The activation of carboxyl groups of phenylalanine in the co-polymer structure was achieved by reaction with carbodiimide (EDC) (Fig. 2). pHEMA-MAPA membrane discs (about 20 g) were transferred into carbodiimide solution (50 ml, 2 mg carbodiimide ml1 ), and the pH of the solution was adjusted to 5.0 with 0.1 M HCl. The container was closed and the reaction was
Fig. 2. Schematic representation of lipase immobilization on to pHEMA-MAPA membrane.
carried out at 25 °C for 24 h, while continuously stirring the medium. At the end of this period, the activated membrane discs were removed and washed several times with methanol and then dried in a vacuum oven for 6 h. They were then stored at 4 °C until use. 2.5. Immobilization of lipase on membranes through covalent bonding
Fig. 1. Chemical structure of the polymeric pHEMA-MAPA membrane.
EDC-activated pHEMA-MAPA membrane discs (10 g, diameter 1.0 cm) were equilibrated in phosphate buffer (50 mM, pH 7.0) for 2 h, and transferred to the same fresh medium containing lipase (50 mg, 2 mg ml1 ). Immobilization of Candida rugosa lipase on the pHEMA-MAPA membrane discs was carried out at 4 °C for 18 h, while continuously stirring the reaction
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medium. After this period, the pHEMA-MAPA membrane discs were removed from medium and washed with 1 M NaCl and then phosphate buffer (0.1 M, pH 7.0). The amount of immobilized lipase on the pHEMAMAPA membrane discs was determined by measuring the initial and final concentrations of protein within the immobilization medium using Coomassie Brilliant Blue (Bradford, 1976). A calibration curve was constructed with BSA solution of known concentration (0:05– 0:50 mg ml1 ) and was used in the calculation of protein in the enzyme and wash solutions. 2.6. Activity assays of free and immobilized lipase The activity of free and immobilized lipase was determined by olive oil hydrolysis. A 100 ml olive oil emulsion was prepared by mixing olive oil (50 ml) with a gum arabic solution (50 ml) while stirring for 30 min. The assay mixture consisted of emulsion (5 ml), phosphate buffer (2.0 ml, 0.1 M, pH 7.0) and free enzyme (0.5 ml, 1 mg ml1 ) or immobilized enzyme (5 membrane discs). The oil hydrolysis was carried out at 35 °C for 30 min in a shaker water bath. The reaction was stopped by adding 10 ml of acetone–ethanol solution (1:1 v/v). The resulting fatty acids in the medium were determined by titration with 25 mM NaOH solution. These activity assays which were carried out by varying the pH range and temperature in 4–8 and 20–50 °C, respectively, would involve effects of pH and temperature on free and immobilized enzymes. One lipase unit was expressed as release of one lmol fatty acid per min in the assay conditions, while the specific activity the number of lipase units per mg protein. 2.7. Batch use of immobilized lipase Retention of immobilized lipase activity was tested as described above in activity assays section. After any run, the enzyme-membrane discs were washed with phosphate buffer (0.1 M, pH 7.0) and reintroduced into a fresh medium, this being repeated up to 20 cycles. 2.8. Thermal stability The thermal stability of free and immobilized lipase was determined by measuring the residual enzymatic activity at two different temperatures (50 and 60 °C) in a phosphate buffer (0.1 M, pH 7.0) for 2 h. After every 15 min time interval, a sample was removed and assayed for enzymatic activity as described above. The firstorder inactivation rate constants ki were calculated from ln A ¼ ln A0 ki t;
ð1Þ
where A0 is the initial activity and A is the activity after time t (min).
3. Results and discussion 3.1. Properties of the pHEMA-MAPA In enzyme technology, suitable matrices include hydrogels that are highly compatible for immobilization of enzymes and cells due to their hydrophilic nature and high water content to provide the enzyme with a micro-environment similar to that in vivo. The copolymerization of 2-hydroxyethyl methacrylate with 2methacrylamido-phenylalanine as a hydrophobic groups carrying co-monomer improves the bio-compatible properties of the hydrogel and also creates a hydrophilic/ hydrophobic micro-environment for the immobilized biological macro-molecules. This method is effective in that the hydrophobic group is readily introduced into the porous membrane matrix at a required density by selecting the co-monomer ratio in the polymer preparation mixture. For the preparation of hydrophobic group containing membrane matrix, MAPA was synthesized from 2methacrylochloride and L -phenylalanine. It was then used as a co-monomer in the synthesis of pHEMAMAPA copolymer. The chemical structure of the p(HEMA-MAPA) is presented in Fig. 1. The SEM micrographs presented in Figs. 3(A) and (B) show the surface and cross-sectional structure of pHEMA-MAPA membrane, respectively. The surface and cross-sectional structures are very porous, and this should lead to a large external surface area for the immobilization of the proteins. 3.2. Immobilization of lipase onto pHEMA-MAPA membrane A two-step process was carried out for the covalent immobilization of lipase on the pHEMA-MAPA membrane. In the first step, the carboxyl groups of the phenylalanine in the pHEMA-MAPA structure were activated with carbodiimide. The second step consisted of the condensation reaction of the amino groups of the enzyme with the activated carboxyl groups of the support. During the condensation reaction, amide bonds were formed between amino groups of the enzyme and carboxyl groups of the support (Fig. 2). The amount of phenylalanine in the pHEMA-MAPA membranes was determined by elemental analysis. The phenylalanine content of the membranes was calculated from nitrogen stoichiometry and found to be between 52 and 312 lmol g1 dry membrane (Table 1). The effect of HEMA/MAPA mole ratio on the enzyme loading and retention of lipase activity is presented in Table 1. As observed in Fig. 4, an increase in the phenylalanine co-monomer ratio (carrying carboxyl group for coupling of enzyme onto membrane) led to an increase in enzyme loading but the activity retention
G. Bayramo glu et al. / Journal of Food Engineering 52 (2002) 367–374
Fig. 3. SEM micrographs of pHEMA-MAPA membrane: (A) surface; (B) cross-section.
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decreased with increasing phenylalanine density on the membrane. Thus, a maximum enzyme loading of 4:23 mg g1 was observed (Table 1). As seen in Fig. 4, the highest retention of enzyme activity was obtained with the lowest enzyme content (1:53 mg g1 membrane). As the enzyme content increased (from 1.53 to 4:23 mg g1 membrane), retention of activity decreased, dropping to a minimum of 56%. A high enzyme load on the support generally leads to a low retained activity. This is brought either by over-saturation of the pore space of the membrane with the enzyme, as a result of which relatively large substrate (olive oil) diffusion limitations occur or the presence of protein–protein interactions becomes more important and these hinder the substrate conversions. By increasing the lipase content from 1.53 to 4:23 mg g1 , its specific activity also increased from 841 to 1670 U g1 pHEMA-MAPA membrane, but this was not at the same rate, because of the loss in retained activity with increased load (Fig. 4). Lipases have a higher level of hydrophobicity than conventional proteins, and the total percentage of hydrophobic amino acid residues (i.e., Ile, Leu, Val, Met, Tyr, Phe) in lipases isolated from various microbial sources is varied between 28% and 33% (Hiol et al., 2000). The lipases have inherent affinity toward hydrophobic media, and the hydrophilic/hydrophobic nature of the pHEMA-MAPA membrane supports could provide a proper micro-environment for lipase, and thus, reasonable retained immobilized lipase activities were obtained with the hydrophobic group carrying supports (Xu, Li, & He, 1995). The covalent immobilization of Candida rugosa lipase on to two different supports agarose and SiO2 was studied and the retained lipase activity was varied between 33% and 82%, these activities were dependent on the type of support (Moreno, Arroyo, Hernaiz, & Sinestra, 1997; Moreno et al., 1997). Lipase from Candida rugosa was immobilized on synthetic polymer beads and the retained activity of the immobilized enzyme was varied between 15% and 32% (Basri et al., 1996). Thus, the retained lipase activity was obtained up to 78% in this study, and is comparable with the related literature. Hereafter, the rest of the study was carried out with pHEMA-MAPA-3 hydrogel composition. 3.3. Effect of pH and temperature on the catalytic activity
Fig. 4. Effect of enzyme loading on the activity retention and enzyme hydrogel activity.
The effect of pH on the activity of free and immobilized lipase in olive oil hydrolysis was assayed in the range 4.0–8.0 (Fig. 5). The maximum activity was obtained at pH 7.5 for the free enzyme and in the pH range 6.5–8.0 for the covalently bound lipase, this variation in pH optima upon immobilization has been reported by several researchers, and could depend on the method of immobilization, as well as the secondary interactions
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Fig. 5. pH profile of free and immobilized lipase.
(hydrophilic and hydrophobic) between the enzyme and matrix (Arıca et al., 1997; Basri et al., 1994a,b; Bryjak, Bachmann, Melizewska, Trochimczuk, & Kolarz, 1997). For the immobilized the pH profile was also much broader than that of free counterpart, as probably due to the product fatty acid, forming layers and causing external diffusional limitations on the enzyme membrane surface. The effect of temperature on enzyme activity was investigated in phosphate buffer (0.1 M, pH 7.0) in the range 20–50 °C. The apparent temperature optimum of the free lipase was about 35 °C, whereas the optimum temperature of the immobilized lipase was around 45 °C (Fig. 6). Thus, for lipase immobilized on the pHEMAMAPA membrane, the immobilization promoted a shift toward higher optimal temperature values. These results suggested that the immobilization enhanced the lipase thermal stability. This could be explained by either creation of conformational limitations on the enzyme movements as a result of multi-point attachment and hydrophobic interaction between the enzyme and the support or a low restriction in the diffusion of the substrate and products at a higher reaction temperature (Arıca, 2000). 3.4. Thermal stability The effect of temperature on the stability of the free and immobilized lipase is shown in Fig. 7. The pattern of heat stability indicated that a smaller rate of thermal inactivation was observed for the immobilized lipase on the pHEMA-MAPA-3 membrane than that of the free enzyme. At 50 °C, the free enzyme lost all its initial
Fig. 6. Effect of temperature on the activity of free and immobilized lipase.
Fig. 7. Influence of temperature on the stability of the free and immobilized lipase.
activity after a 120 min of heat treatment, while the immobilized enzyme showed significant resistance to thermal inactivation (retaining about 63% of its initial activity after the same period). At 60 °C, the free lipase lost all its initial activity after 45 min heat treatment. Under the same conditions, the immobilized counterpart still retained 45% of its initial activity. The thermal inactivation rate constants for free and immobilized
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preparation at 60 °C were calculated as kiðfreeÞ ; 1:1 101 min1 and kiðimmÞ ; 1:3 102 min1 , respectively. These results suggest that the thermostability of immobilized lipase becomes significantly higher at higher temperature. If the heat stability of enzymes increased upon immobilization, the potential application of these enzymes would be extended. Increased thermal stability has been reported for a number of immobilized enzymes, and the polymer network and multi-point attachment in covalent immobilization method are supposed to preserve the tertiary structure of enzyme. In addition, it has also been reported that hydrogel carriers such as Sephadex, Sepharose, poly(hydroxyethyl methacrylate) and polyacrylamide protect enzymes from thermal inactivation and yield higher thermal stabilities. On the basis of these observations, pHEMA-MAPA is a hydrophobic group carrying hydrophilic network which induces higher thermal stability compared to that of its free counterpart. 3.5. Stability in batch operation The operational stability of immobilized enzyme systems is very important economically, and an increased stability could make the immobilized enzyme more advantageous than its free counterparts. Operational stability of the immobilized lipase was determined for successive batch reactions at 35 °C. The results presented in Fig. 8 show that the immobilized enzyme activity remained almost the same as the original activity after seven cycles. After this, a steady decrease in
Fig. 8. Operational stability of immobilized lipase in a batch system.
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the oil hydrolysis rate was observed, and this loss reached 37% after 20 cycles of batch operation, possibly resulting from the inactivation of lipase upon use.
4. Conclusion One of the most important aims of the enzyme technology is to enhance the conformational stability of the enzyme. The extent of stabilization depends on the enzyme structure, the immobilization methods, and type of support. In the present study, a new hydrophobic amino acid phenylalanine containing pHEMA-MAPA membrane support was prepared by co-polymerization of 2-hydroxyethylmethacrylate with 2-methacrylamidophenylalanin and was used in the covalent immobilization of lipase. The hydrophobic amino acid carrying hydrophilic support may provide a artificial natural micro-environment for the enzyme. The phenylalanine containing pHEMA-MAPA membrane revealed good properties as a membrane support and will be useful in the enzyme immobilization technology.
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Bradford, M. M. (1976). A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–252. Bryjak, J., Bachmann, P. B., Melizewska, I., Trochimczuk, A., & Kolarz, M. N. (1997). Immobilization of lipase on various acrylic copolymers. Chemical Engineering Journal, 65, 249–256. Demir, N., Acar, J., Sarıoglu, K., & Mutlu, M. (2001). The use of commercial pectinase in fruit juice industry. Part 3: Immobilized pectinase for mash treatment. Journal of Food Engineering, 47, 275– 280. de Oliveira, P. C., Alves, G. M., & de Castro, H. F. (2000). Immobilization studies and catalytic properties of microbial lipase onto stryrenedivinylbenzene copolymer. Biochemical Engineering Journal, 5, 63–71. Gandhi, N. N., Vijayalakshmi, V., Sawant, B., & Joshi, J. B. (1996). Immobilization of Mucor miehei lipase on ion exchange resins. The Chemical Engineering Journal, 61, 149–156. Hiol, A., Jonzo, M. D., Rugani, N., Druet, D., Sarda, L., & Comeau, L. C. (2000). Purification and characterization of an extracellular lipase from a thermophilic Rhizopus arise strain isolated from palm fruit. Enzyme Microbial Technology, 26, 421–430. Itoyama, K., Tokura, S., & Hayashi, T. (1994). Lipoprotein lipase immobilization onto porous chitosan beads. Biotechnology Progress, 10, 225–229.
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Covalent immobilization of lipase onto hydrophobic group incorporated poly(2-hydroxyethyl methacrylate) based hydrophilic membrane matrix G€ ulay Bayramo glu a, Yasemin Kacßar b, Adil Denizli c, M. Yakup Arıca a
b,*
Department of Chemistry, Kırıkkale University, 71450 Yahsßihan-Kırıkkale, Turkey Department of Biology, Kırıkkale University, 71450 Yahsßihan-Kırıkkale, Turkey c Department of Chemistry, Hacettepe University, 06531 Beytepe-Ankara, Turkey
b
Received 23 April 2001; accepted 27 June 2001
Abstract In this study, a hydrophobic group containing monomer, 2-methacrylamidophenyalanine (MAPA) was prepared by using methacrylochloride and phenylalanine. Then, poly(2-hydroxyethyl methacrylate-co-methacrylamido-phenlyalanine) (pHEMAMAPA) membranes were prepared by UV-initiated photopolymerization of HEMA and MAPA in the presence of an initiator a-a0 -azobisisobutyronitrile (AIBN). The lipase was immobilized onto these membranes by covalent bonding through carbodiimide activation. The amount of enzyme loading on the membranes was increased as the MAPA ratio increased in the membrane structure. Immobilization improved the pH stability of the enzyme as well as its temperature stability. Thermal stability was found to increase with immobilization and at 60 °C the thermal stability constants were 1:1 101 min for free enzyme and 1:2 102 min for the immobilized enzyme. The immobilized enzyme activity was found to be quite stable in repeated experiments. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Poly(2-hydroxyethyl methacrylate) membrane; Phenylalanine; Covalent bonding; Enzyme immobilization; Lipase
1. Introduction Immobilization confers additional stability to a variety of enzymes against several forms of denaturation. Enzymes have been immobilized on different shapes of supports (i.e., membranes or beads) either by covalent binding, entrapment, or adsorption. Membrane-immobilized enzymes may serve as model systems for enzymes, naturally bound to membranes, or may find practical application in biosensors and enzyme reactors as less expensive, more stable and reusable alternatives to free enzymes (Arıca, Denizli, Salih, Pisßkin, & Hasırcı, 1997; Ivanov & Schneider, 1997; Zhou & Chen, 2001). Lipase (triacylglycerol acyl ester hydrolyses, EC 3.1.1.3) is an enzyme possessing an intrinsic capacity to
*
Corresponding author. Tel.: +90-318-357-2477; fax: +90-318-3572329. E-mail address: [email protected] (M. Yakup Arıca).
catalyse cleavage of carboxy ester bonds in tri-, di-, and monoacylglycerols to glycerol and fatty acids. The enzyme is distributed among higher animals, plants and micro-organisms in which it plays a key role in the lipid metabolism. The enzyme has been widely used for biotechnological applications in dairy industry, oil processing, production of surfactants and preparation of enantiomerically pure pharmaceuticals (Balcao, Paiva, & Malcata, 1996; Pavia, Balcao, & Malcata, 2000). The immobilized lipase could be used in the dairy industry for the controlled hydrolysis of milk fat, a process which is useful for acceleration of cheese ripening, flavour enhancement of butter, manufacture of cheese-like products, and preparation of enzyme-modified cheese for use as ingredients in dressings, and sauces (Balcao et al., 1996). The immobilized lipase could also be applied to the production of x3-polyunsaturated fatty acid concentrates from fish liver oils (which have been claimed to provide beneficial health effects via prevention of coronary heart diseases) for use as
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nutropharmaceutical food supplements. Lipase immobilization was carried out by covalent attachment, entrapment, and adsorption to hydrophobic or hydrophilic surface (Al-Duri & Yong, 2000; Arıca, Kacßar, Ergene, & Denizli, 2001; Balcao et al., 1996; Demir, Acar, Sarıo glu, & Mutlu, 2001; de Oliveira, Alves, & de Castro, 2000). After immobilization of lipase, changes were observed in the enzyme activity, optimum pH, affinity to substrate and stability (Arıca et al., 2001; Itoyama, Tokura, & Hayashi, 1994; Reetz, Zonta, & Simpelkapt, 1996). The extent of these changes depended on the source of enzyme, the type of support and the method of immobilization. It is, thus, important that the choice of proper support material and immobilization method over the free bioactive agent should be well € ktem, O € ktem, & Tuncel, 1999; de justified (Arıca, O Oliveira et al., 2000; Gandhi, Vijayalakshmi, Sawant, & Joshi, 1996). Poly(2-hydroxyethyl methacrylate) (pHEMA) is a hydrophilic polymer and it is described as a well biocompatible material. The properties of pHEMA can be modified in a wide range, such as by co-polymerization or additives. Hydrophobic groups could be introduced on the pHEMA structure either attachment through functional –OH group or co-polymerization of HEMA with a hydrophobic group carrying co-monomer. In this way, a pHEMA based hydrophilic/hydrophobic support can be obtained and would be used in the enzyme and protein immobilization studies as a hydrophobic group carrying support (Arıca et al., 1997; Arıca et al., 1999; Basri, Ampon, Yunus, Razak, & Salleh, 1994a,b). In the present study, a hydrophobic group containing porous pHEMA based matrix was prepared in the membrane form by co-polymerization of 2-hydroxyethyl methacrylate (HEMA) monomer with a hydrophobic group introduced co-monomer (2-methacrylamidophenylalanine, MAPA). This method is effective in that the hydrophobic group could be easily introduced into the membrane matrix at a desired density by adjusting the concentration of hydrophobic group carrying co-monomer in the polymerization mixture. A series of pHEMA-MAPA membranes with different HEMA/ MAPA ratios were prepared by UV-initiated photopolymerization in the presence of an initiator (a; a0 -azobisisobutyronitrile, AIBN). These membrane matrices were used for the immobilization of Candida rugosa lipase after activation of carbodiimide via covalent attachment. The system parameters such as the effect of phenylalanine concentration of support on the lipase loading capacity and the effect of loading on the activity yield of immobilized lipase were determined at various lipase loadings. The influence of several parameters was also characterized such as activity retention, catalytic properties, thermal and operational stability aspects were compared.
2. Experimental section 2.1. Materials Lipase (Type VII, from Candida rugosa, lyophilized powder) was supplied from Sigma chemicals (St Louis, MO, USA) and used as-received. 2-hydroxyethyl methacrylate (HEMA) was obtained from Fluka AG (Switzerland), distilled under reduced pressure in the presence of hydroquinone and stored at 4 °C until use. Phenylalanine, methacrylochloride, a-a0 -azoisobisbutyronitrile (AIBN), gum arabic, carbodiimide (1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride) (EDC) and bovine serum albumin (BSA) were obtained from Sigma chemicals. All other chemicals were of analyticalgrade and were purchased from Merck AG (Darmstadt, Germany). 2.2. Preparation of 2-methacrylamidophenylalanin monomer Phenylalanine (5.0 g) and sodium nitrite ðNaNO2 ; 0:2 gÞ were dissolved in a potassium carbonate solution (K2 CO3 ; 30 ml, 5%, w/v). The mixture was transferred in a 100 ml round-bottomed three-necked flask fitted with a dropping funnel. The reaction chamber was cooled down to 0 °C in an ice-water bath and stirred magnetically under a nitrogen atmosphere. A 6.0 ml of methacrylochloride was placed into the dropping funnel and was introduced dropwise to the reaction mixture in 10 min. The reaction chamber was then removed from the ice-water bath and the reaction was maintained at room temperature for 2 h. After reaction, the pH of the mixture was adjusted to 3.0 and the product was extracted with chloroform (50 ml). After phase separation, the organic phase was dried with magnesium sulphate and chloroform was evaporated with a rotary evaporator. The product (i.e., 2-methacrylamidophenylalanine monomer, MAPA) was crystallized in an ether–cyclohexane mixture (5:95%, v/v). 2.3. Preparation of poly(2-hydroxyethylmethacrylate-comethacrylamido-phenylalanine) membranes The poly(2-hydroxyethylmethacrylate-co-methacrylamido-phenylalanine) (pHEMA-MAPA) hydrogel in the membrane form was prepared by a UV-initiated photopolymerization, as previously described (Arıca et al., 1997). The polymerization was carried out in a round glass mould (diameter: 9.0 cm) at 25 °C under nitrogen atmosphere for 1 h. To check the effect of monomer ratio on the lipase loading capacity of the membrane, in the initial polymerization mixture five different HEMA/MAPA ratios were used (Table 1). The membrane preparation mixture (5 ml) contained 2 ml HEMA, 25–200 mg (or 107–856 lmol) methacrylamido-
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Table 1 Properties of the pHEMA-MAPA-1–5 membranes Membrane type
HEMA/MAPA (mol ratio)
Phenylalanine density ðlmol g1 Þ
Enzyme loading (mg g1 membrane)
Recovered activity (%)
Enzyme activity (U g1 membrane)
pHEMA-MAPA-1 pHEMA-MAPA-2 pHEMA-MAPA-3 pHEMA-MAPA-4 pHEMA-MAPA-5
154 77 52 39 26
52 104 156 208 312
1.53 2.62 3.24 3.84 4.23
78 73 66 59 55
841 1348 1507 1597 1670
phenylalanine (MAPA), 10 mg AIBN as polymerization initiator and 3 ml phosphate buffer (0.1 M, pH 7.0). The resulting mixture was equilibrated at 25 °C for 30 min in a thermostatic water bath. The mixture was then poured into the mould and exposed to long-wave ultraviolet radiation for 20 min. The pHEMA-MAPA membranes (their chemical structure being shown in Fig. 1) were washed several times with distilled water and cut into circular pieces (diameter: 1.0 cm) with a perforator. The degree of MAPA incorporation in the synthesized pHEMA-MAPA membranes was determined by measuring the in C, H, N and O contents with a Laco (Model CHNSO-932) elemental analyzer. The scanning electron micrograph (SEM) of the dried pHEMA-MAPA membranes was obtained by using a JEOL 5600 scanning electron microscope, after coating with gold under reduced pressure. 2.4. Activation of pHEMA-MAPA membrane with carbodiimide The activation of carboxyl groups of phenylalanine in the co-polymer structure was achieved by reaction with carbodiimide (EDC) (Fig. 2). pHEMA-MAPA membrane discs (about 20 g) were transferred into carbodiimide solution (50 ml, 2 mg carbodiimide ml1 ), and the pH of the solution was adjusted to 5.0 with 0.1 M HCl. The container was closed and the reaction was
Fig. 2. Schematic representation of lipase immobilization on to pHEMA-MAPA membrane.
carried out at 25 °C for 24 h, while continuously stirring the medium. At the end of this period, the activated membrane discs were removed and washed several times with methanol and then dried in a vacuum oven for 6 h. They were then stored at 4 °C until use. 2.5. Immobilization of lipase on membranes through covalent bonding
Fig. 1. Chemical structure of the polymeric pHEMA-MAPA membrane.
EDC-activated pHEMA-MAPA membrane discs (10 g, diameter 1.0 cm) were equilibrated in phosphate buffer (50 mM, pH 7.0) for 2 h, and transferred to the same fresh medium containing lipase (50 mg, 2 mg ml1 ). Immobilization of Candida rugosa lipase on the pHEMA-MAPA membrane discs was carried out at 4 °C for 18 h, while continuously stirring the reaction
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medium. After this period, the pHEMA-MAPA membrane discs were removed from medium and washed with 1 M NaCl and then phosphate buffer (0.1 M, pH 7.0). The amount of immobilized lipase on the pHEMAMAPA membrane discs was determined by measuring the initial and final concentrations of protein within the immobilization medium using Coomassie Brilliant Blue (Bradford, 1976). A calibration curve was constructed with BSA solution of known concentration (0:05– 0:50 mg ml1 ) and was used in the calculation of protein in the enzyme and wash solutions. 2.6. Activity assays of free and immobilized lipase The activity of free and immobilized lipase was determined by olive oil hydrolysis. A 100 ml olive oil emulsion was prepared by mixing olive oil (50 ml) with a gum arabic solution (50 ml) while stirring for 30 min. The assay mixture consisted of emulsion (5 ml), phosphate buffer (2.0 ml, 0.1 M, pH 7.0) and free enzyme (0.5 ml, 1 mg ml1 ) or immobilized enzyme (5 membrane discs). The oil hydrolysis was carried out at 35 °C for 30 min in a shaker water bath. The reaction was stopped by adding 10 ml of acetone–ethanol solution (1:1 v/v). The resulting fatty acids in the medium were determined by titration with 25 mM NaOH solution. These activity assays which were carried out by varying the pH range and temperature in 4–8 and 20–50 °C, respectively, would involve effects of pH and temperature on free and immobilized enzymes. One lipase unit was expressed as release of one lmol fatty acid per min in the assay conditions, while the specific activity the number of lipase units per mg protein. 2.7. Batch use of immobilized lipase Retention of immobilized lipase activity was tested as described above in activity assays section. After any run, the enzyme-membrane discs were washed with phosphate buffer (0.1 M, pH 7.0) and reintroduced into a fresh medium, this being repeated up to 20 cycles. 2.8. Thermal stability The thermal stability of free and immobilized lipase was determined by measuring the residual enzymatic activity at two different temperatures (50 and 60 °C) in a phosphate buffer (0.1 M, pH 7.0) for 2 h. After every 15 min time interval, a sample was removed and assayed for enzymatic activity as described above. The firstorder inactivation rate constants ki were calculated from ln A ¼ ln A0 ki t;
ð1Þ
where A0 is the initial activity and A is the activity after time t (min).
3. Results and discussion 3.1. Properties of the pHEMA-MAPA In enzyme technology, suitable matrices include hydrogels that are highly compatible for immobilization of enzymes and cells due to their hydrophilic nature and high water content to provide the enzyme with a micro-environment similar to that in vivo. The copolymerization of 2-hydroxyethyl methacrylate with 2methacrylamido-phenylalanine as a hydrophobic groups carrying co-monomer improves the bio-compatible properties of the hydrogel and also creates a hydrophilic/ hydrophobic micro-environment for the immobilized biological macro-molecules. This method is effective in that the hydrophobic group is readily introduced into the porous membrane matrix at a required density by selecting the co-monomer ratio in the polymer preparation mixture. For the preparation of hydrophobic group containing membrane matrix, MAPA was synthesized from 2methacrylochloride and L -phenylalanine. It was then used as a co-monomer in the synthesis of pHEMAMAPA copolymer. The chemical structure of the p(HEMA-MAPA) is presented in Fig. 1. The SEM micrographs presented in Figs. 3(A) and (B) show the surface and cross-sectional structure of pHEMA-MAPA membrane, respectively. The surface and cross-sectional structures are very porous, and this should lead to a large external surface area for the immobilization of the proteins. 3.2. Immobilization of lipase onto pHEMA-MAPA membrane A two-step process was carried out for the covalent immobilization of lipase on the pHEMA-MAPA membrane. In the first step, the carboxyl groups of the phenylalanine in the pHEMA-MAPA structure were activated with carbodiimide. The second step consisted of the condensation reaction of the amino groups of the enzyme with the activated carboxyl groups of the support. During the condensation reaction, amide bonds were formed between amino groups of the enzyme and carboxyl groups of the support (Fig. 2). The amount of phenylalanine in the pHEMA-MAPA membranes was determined by elemental analysis. The phenylalanine content of the membranes was calculated from nitrogen stoichiometry and found to be between 52 and 312 lmol g1 dry membrane (Table 1). The effect of HEMA/MAPA mole ratio on the enzyme loading and retention of lipase activity is presented in Table 1. As observed in Fig. 4, an increase in the phenylalanine co-monomer ratio (carrying carboxyl group for coupling of enzyme onto membrane) led to an increase in enzyme loading but the activity retention
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Fig. 3. SEM micrographs of pHEMA-MAPA membrane: (A) surface; (B) cross-section.
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decreased with increasing phenylalanine density on the membrane. Thus, a maximum enzyme loading of 4:23 mg g1 was observed (Table 1). As seen in Fig. 4, the highest retention of enzyme activity was obtained with the lowest enzyme content (1:53 mg g1 membrane). As the enzyme content increased (from 1.53 to 4:23 mg g1 membrane), retention of activity decreased, dropping to a minimum of 56%. A high enzyme load on the support generally leads to a low retained activity. This is brought either by over-saturation of the pore space of the membrane with the enzyme, as a result of which relatively large substrate (olive oil) diffusion limitations occur or the presence of protein–protein interactions becomes more important and these hinder the substrate conversions. By increasing the lipase content from 1.53 to 4:23 mg g1 , its specific activity also increased from 841 to 1670 U g1 pHEMA-MAPA membrane, but this was not at the same rate, because of the loss in retained activity with increased load (Fig. 4). Lipases have a higher level of hydrophobicity than conventional proteins, and the total percentage of hydrophobic amino acid residues (i.e., Ile, Leu, Val, Met, Tyr, Phe) in lipases isolated from various microbial sources is varied between 28% and 33% (Hiol et al., 2000). The lipases have inherent affinity toward hydrophobic media, and the hydrophilic/hydrophobic nature of the pHEMA-MAPA membrane supports could provide a proper micro-environment for lipase, and thus, reasonable retained immobilized lipase activities were obtained with the hydrophobic group carrying supports (Xu, Li, & He, 1995). The covalent immobilization of Candida rugosa lipase on to two different supports agarose and SiO2 was studied and the retained lipase activity was varied between 33% and 82%, these activities were dependent on the type of support (Moreno, Arroyo, Hernaiz, & Sinestra, 1997; Moreno et al., 1997). Lipase from Candida rugosa was immobilized on synthetic polymer beads and the retained activity of the immobilized enzyme was varied between 15% and 32% (Basri et al., 1996). Thus, the retained lipase activity was obtained up to 78% in this study, and is comparable with the related literature. Hereafter, the rest of the study was carried out with pHEMA-MAPA-3 hydrogel composition. 3.3. Effect of pH and temperature on the catalytic activity
Fig. 4. Effect of enzyme loading on the activity retention and enzyme hydrogel activity.
The effect of pH on the activity of free and immobilized lipase in olive oil hydrolysis was assayed in the range 4.0–8.0 (Fig. 5). The maximum activity was obtained at pH 7.5 for the free enzyme and in the pH range 6.5–8.0 for the covalently bound lipase, this variation in pH optima upon immobilization has been reported by several researchers, and could depend on the method of immobilization, as well as the secondary interactions
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Fig. 5. pH profile of free and immobilized lipase.
(hydrophilic and hydrophobic) between the enzyme and matrix (Arıca et al., 1997; Basri et al., 1994a,b; Bryjak, Bachmann, Melizewska, Trochimczuk, & Kolarz, 1997). For the immobilized the pH profile was also much broader than that of free counterpart, as probably due to the product fatty acid, forming layers and causing external diffusional limitations on the enzyme membrane surface. The effect of temperature on enzyme activity was investigated in phosphate buffer (0.1 M, pH 7.0) in the range 20–50 °C. The apparent temperature optimum of the free lipase was about 35 °C, whereas the optimum temperature of the immobilized lipase was around 45 °C (Fig. 6). Thus, for lipase immobilized on the pHEMAMAPA membrane, the immobilization promoted a shift toward higher optimal temperature values. These results suggested that the immobilization enhanced the lipase thermal stability. This could be explained by either creation of conformational limitations on the enzyme movements as a result of multi-point attachment and hydrophobic interaction between the enzyme and the support or a low restriction in the diffusion of the substrate and products at a higher reaction temperature (Arıca, 2000). 3.4. Thermal stability The effect of temperature on the stability of the free and immobilized lipase is shown in Fig. 7. The pattern of heat stability indicated that a smaller rate of thermal inactivation was observed for the immobilized lipase on the pHEMA-MAPA-3 membrane than that of the free enzyme. At 50 °C, the free enzyme lost all its initial
Fig. 6. Effect of temperature on the activity of free and immobilized lipase.
Fig. 7. Influence of temperature on the stability of the free and immobilized lipase.
activity after a 120 min of heat treatment, while the immobilized enzyme showed significant resistance to thermal inactivation (retaining about 63% of its initial activity after the same period). At 60 °C, the free lipase lost all its initial activity after 45 min heat treatment. Under the same conditions, the immobilized counterpart still retained 45% of its initial activity. The thermal inactivation rate constants for free and immobilized
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preparation at 60 °C were calculated as kiðfreeÞ ; 1:1 101 min1 and kiðimmÞ ; 1:3 102 min1 , respectively. These results suggest that the thermostability of immobilized lipase becomes significantly higher at higher temperature. If the heat stability of enzymes increased upon immobilization, the potential application of these enzymes would be extended. Increased thermal stability has been reported for a number of immobilized enzymes, and the polymer network and multi-point attachment in covalent immobilization method are supposed to preserve the tertiary structure of enzyme. In addition, it has also been reported that hydrogel carriers such as Sephadex, Sepharose, poly(hydroxyethyl methacrylate) and polyacrylamide protect enzymes from thermal inactivation and yield higher thermal stabilities. On the basis of these observations, pHEMA-MAPA is a hydrophobic group carrying hydrophilic network which induces higher thermal stability compared to that of its free counterpart. 3.5. Stability in batch operation The operational stability of immobilized enzyme systems is very important economically, and an increased stability could make the immobilized enzyme more advantageous than its free counterparts. Operational stability of the immobilized lipase was determined for successive batch reactions at 35 °C. The results presented in Fig. 8 show that the immobilized enzyme activity remained almost the same as the original activity after seven cycles. After this, a steady decrease in
Fig. 8. Operational stability of immobilized lipase in a batch system.
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the oil hydrolysis rate was observed, and this loss reached 37% after 20 cycles of batch operation, possibly resulting from the inactivation of lipase upon use.
4. Conclusion One of the most important aims of the enzyme technology is to enhance the conformational stability of the enzyme. The extent of stabilization depends on the enzyme structure, the immobilization methods, and type of support. In the present study, a new hydrophobic amino acid phenylalanine containing pHEMA-MAPA membrane support was prepared by co-polymerization of 2-hydroxyethylmethacrylate with 2-methacrylamidophenylalanin and was used in the covalent immobilization of lipase. The hydrophobic amino acid carrying hydrophilic support may provide a artificial natural micro-environment for the enzyme. The phenylalanine containing pHEMA-MAPA membrane revealed good properties as a membrane support and will be useful in the enzyme immobilization technology.
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