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Process Biochemistry
j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / p r o c b i o
Covalent immobilization of lipase onto amine functionalized polypropylene
membrane and its application in green apple flavor (ethyl valerate) synthesis
Gülay Bayramo˘glu
a,∗, Baki Hazer
b, Begum Altıntas¸
a, M. Yakup Arıca
aaBiochemical Processing and Biomaterial Research Laboratory, Faculty of Arts and Sciences, Gazi University, 06500 Teknikokullar, Ankara, Turkey bDepartment of Chemistry, Zonguldak Karaelmas University, 67100 Zonguldak, Turkey
a r t i c l e i n f o
Article history:Received 22 June 2010 Received in revised form 18 September 2010 Accepted 20 September 2010 Keywords: Polypropylene membrane Immobilized enzyme Enzyme stability Enzyme kinetics Lipase Ethyl valerate
a b s t r a c t
In this study, a functionalized hydrophobic polypropylene chloride membrane (PPC) was prepared by the amination of chlorinated polypropylene with hexamethylene diamine (APP). The PPC and APP mem-branes were characterized using SEM, FTIR and contact angle studies. The aminated polypropylene (APP) membrane was used for covalent immobilization of Candida rugosa lipase via glutaraldehyde coupling. The retained activity of the immobilized lipase was 76%. Kinetic analysis shows that the dependence of lipolytic activity of both free and immobilized lipase on tributyrin substrate concentration can be described by Michaelis–Menten model. The estimated apparent Kmvalues for the free and immobilized lipase were 2.9 and 8.4 mM, respectively. The Vmaxvalues of free and immobilized enzymes were calcu-lated as 926 and 741 U/mg enzyme, respectively. Optimal temperature was 5◦C higher for immobilized enzyme than that of the free enzyme. Thermal and storage stabilities were found to be increased upon immobilization. Finally, the immobilized lipase was used for the production of green apple flavor (i.e., ethyl valerate) in hexane medium.
© 2010 Elsevier Ltd. All rights reserved.
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[1–4]. Enzyme immobilized on membrane may find practical application in biosensors and enzyme reactors as less expensive, more stable and reusable alternatives to free enzymes[5,6]. A variety of support materials have been used for lipase immobilization for example silica[7]magnetic poly(GMA-MMA) beads[8], polypropylene hol-low fiber membrane modified with hydrophobic polypeptide[9], polysulfone ion exchange membranes[10], mesoporous silica[11], poly(hydroxyethyl methacrylate)-based membranes[12], calcium alginate gels[13]and recently developed nano-structured mem-brane matrices[14]. After immobilization of lipase, changes were observed in enzyme activity, optimum pH, affinity to substrate and stability. 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 justified[1,15,16]. Polypropylene (PP) is a hydrophobic polymer. It is chemically inert and stable to many chemical and
biologi-∗ Corresponding author. Tel.: +90 312 202 1533; fax: +90 312 212 2279. E-mail address:g bayramoglu@hotmail.com(G. Bayramo˘glu).
cal reaction conditions. Hydrophilic groups could be introduced on the PP structure by covalent attachment of functional –NH2
group[17,18]. Hydrophilic functional groups containing hydropho-bic support could be obtained and would be used in enzyme immobilization studies. When considering inherent properties of lipases, they are activated on interface, and hence, the immobiliza-tion of lipases on the hydrophobic membrane surface seems to be the best solution[9,12]. In a previous study, polypropylene micro-filtration membrane was modified by grafting with hydrophilic hydrophobic groups containing polymers. Lipases from Candida rugosa were immobilized via adsorption on these modified mem-branes surface[6].
Lipases (E.C. 3.1.1.3) from different sources are currently used in various biochemical reactions including triacylglycerols hydrolysis, esterification between fatty acids and alcohols. The rising interest in lipase mainly lies on its wide industrial applications, including detergent formulation, oils/fats degradation, pharmaceuticals syn-thesis, and cosmetics production[19–21]. There are several studies about ester synthesis in hexane medium using immobilized lipase. Ethyl valerate is one of the most important ester compound (green apple flavor) used in the cosmetic and food industries[22,23].
In the present study, amino functionalized non-porous polypropylene membrane was prepared using PPC and hexamethy-lene diamine. The membrane matrix was used for the covalent immobilization of Candida rugosa lipase after activation of glu-taraldehyde. The influence of several parameters was characterized such as activity retention, as well as kinetic properties, thermal and
1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.09.014
operational stability aspects were investigated and compared with free enzyme. Finally, immobilized lipase was used for ethyl valerate synthesis in both solvent free and hexane medium.
2. Materials and methods
2.1. Materials
Lipase (from Candida rugosa, Type VII,≥700 units/mg solid, (specific activity: 917 units/mg protein. The specific activity was based on protein content of the solid and the protein content was determined by the Bradford method) and polypropy-lene chloride (PPC; MW 150,000, chlorinated about each three repeating unit) were supplied from the Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Hexamethy-lene diamine (HMDA), valeric acid (pentanoic acid), and n-hexane were obtained from Fluka AG (Switzerland). Glutaraldehyde, toluene, anhydride magnesium sul-fate, tributyrin, gum Arabic and sodium cholate were obtained from Sigma Chem. Co. (St. Louis, MO, USA). All other chemicals were of analytical grade and were purchased from Merck AG (Darmstadt, Germany).
2.2. Preparation of polypropylene membrane for enzyme immobilization
The preparation of aminated polypropylene membrane was carried out as reported by Hong et al.[18]. Polypropylene chloride (3 g, MW 150,000) was dissolved in a flask containing toluene (40 mL). Hexamethylene diamine (1.0 g) was dissolved in acetone–isopropyl alcohol mixture (1:1 ratio (v/v), 50 mL). After dissolving each chemical, these solutions were dried with anhydride magnesium sulfate, and then were mixed in a round bottom two neck flask. It was equipped with a coiled con-densing tube and with an argon gas inlet adaptor. The flask was transferred in a water-bath and it was stirred magnetically at 50◦C for 48 h. Argon atmosphere was maintained during the reaction period. After reaction, the amino group function-alized membrane was obtained by the casting of aminated polymer solution on a Teflon plate. The membranes were dried at room temperature for 24 h and were peeled from Teflon plate. They were washed several times with purified water, and then were dried sequentially at 60◦C and 105◦C for 24 h and 12 h, respectively.
The activation of amino groups of the APP membranes was achieved by reaction with glutaraldehyde. APP membranes (about 2 g) were transferred into glutaralde-hyde solution (50 ml) and the concentration of glutaraldeglutaralde-hyde was varied in the range of 0.2–2.0% (v/v). The pH of the solution was adjusted to 7.0 with 0.1 M NaOH. After closing flask, the reaction was carried out at 25◦C for 12 h, while continuously stirring the reaction medium. At the end of this period, the activated membranes 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.
2.3. Immobilization of lipase on APP membranes
Activated APP membranes (1.0 g, 1.0 cm2) 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/ml, <3500 units). Immobilization of Candida rugosa lipase on the APP membrane was carried out at 4◦C for 20 h, while continuously stirring the reaction
medium. After this period, the enzyme-immobilized APP membrane was imme-diately transferred to ethylene diamine solution (5.0 mg/ml ethylene diamine) in same buffer solution to block the free reactive groups on the membranes. Schematic representation of chemical route for the enzyme immobilization is presented in
Fig. 1. Physically bound enzyme from the membrane was removed by washing with a solution containing ionic detergent (sodium cholate, 50 mM) and salt (NaCl, 1.0 M) at pH 8.0 for 2 h. The amount of immobilized lipase on the APP membrane was determined by measuring the initial and final concentrations of protein within the immobilization medium and washed solutions using Coomassie Brilliant Blue
[24]. A calibration curve constructed with lipase solution of known concentration (0.05–0.50 mg/mL) was used in the calculation of protein in the enzyme and in wash solutions.
2.4. Activity assays of lipase
The activity of free and immobilized lipase was determined by olive oil hydrol-ysis as described previously[12]. A 100 ml olive oil emulsion was prepared by mixing olive oil (50 mL) and gum Arabic solution (50 ml, 7%, w/v). The assay mix-ture consisted of emulsion (5 mL), phosphate buffer (2.0 mL, 100 mM, pH 7.0) and free enzyme (0.5 mL, 1.0 mg/mL) or immobilized enzyme (10 cm2membrane). Oil hydrolysis was carried out at 35◦C for 30 min in a shaking water-bath at 150 rpm. The reaction was stopped by the addition of 10 ml of acetone–ethanol solution (1:1, v/v). The liberated fatty acid in the medium was determined by titration with 50 mM NaOH solution. These activity assays were carried out over the pH range 4.0–9.0 and temperature range 20–60◦C to determine the pH and temperature profiles for the free and the immobilized enzymes.
2.5. Determination of the kinetic parameters enzyme preparation
The kinetic constants were determined using tributyrin as substrate in the con-centrations range (5–50 mM) using free and immobilized lipase and titrating the butyric acids produced with 50 mM NaOH as described above. The experiments were conducted under the optimized assay conditions. The apparent Kmand Vmax values for the free and immobilized lipase were calculated from Lineweaver–Burk plots by using the initial rate of the enzymatic reaction.
2.6. Thermal stability of the immobilized lipase
The thermal stabilities of the free and immobilized lipase were determined by incubation in substrate-free phosphate buffer solution (0.1 M, pH 7.0) at two differ-ent temperatures (55◦C and 65◦C) under continuous shaking at 150 rpm. At 15 min time intervals, the remaining activities of the free and immobilized lipase were measured as described above.
2.7. Operational stability of immobilized lipase
The operational stability of the immobilized lipase was examined under batch operation mode at 35◦C at intervals of 30 min. After each activity measurement, the immobilized lipase was separated from medium and washed three times with
phosphate buffer (5.0 ml, 0.1 M, pH 7.0) and then fresh reaction medium was intro-duced onto the immobilized lipase. By this way, the next activity measurement was carried out.
2.8. Storage stability
The storage stability of free and immobilized lipase were determined after stor-age in phosphate buffer (0.1 M, pH 7.0) at 4◦C during 8 weeks. The residual activities were then determined as described above and activity of the each enzyme was expressed as percentages of its residual activity compared to the initial activity. 2.9. Enzymatic synthesis of green apple flavor and repeated use of immobilized lipase
An industrially important ester (i.e., ethyl valerate; green apple flavor) was synthesized in screw-capped flasks (20 mL) using immobilized lipase on the APP membranes (10 cm2) for both solvent-free medium and hexane medium. For solvent free system, the enzymatic esterification reactions were carried out with various valeric acid/ethyl alcohol molar ratios (i.e., 0.5:1.0, 1.0:1.0, 2.0:1.0 and 3.0:1.0 con-taining of 20L distilled water) on an orbital shaker at 150 rpm and at 40◦C for 24 h[25]. For hexane medium, ester synthesis reaction was carried out in n-hexane (15 mL) containing 0.1 mol valeric acid and 0.1 mol ethyl alcohol with shaking at 150 rpm at 40◦C. Along the 24 h reaction, the amount of remaining acid was determined by titrimetric method using 50 mM NaOH and phenolphthalein as an indicator. The conversion (%) of ester synthesis was calculated based on the con-version of the valeric acid to ester after a given time. The initial reaction rate was determined in the linear region. All of the experiments were carried out in tripli-cate. The control experiments (no enzyme) were performed and it was observed that ester yield is less than 2%. After any run, the enzyme-membranes were washed with phosphate buffer (0.1 M, pH 6.5) and reintroduced into a fresh medium, and was repeated up to 5 cycles for ester synthesis.
2.10. Characterization of polypropylene based membrane
The degree of HMDA incorporation in the synthesized aminated membrane was determined by measuring the in C, H and N contents with a Laco (Model CHNSO-932) elemental analyzer. The available amino group’s content of the APP was determined by potentiometric titration. The APP membrane about 0.1 g was transferred in HCl solution (0.1 M, 10 ml) and it was then incubated in a shaking water-bath at 35◦C for 6 h. After this reaction period, the final HCl concentration in the solution was determined by a potentiometric titration with 50 mM NaOH solution. The FTIR spec-tra of the membrane samples were obtained by using an FTIR spectrophotometer (Shimadzu, FTIR 8000 Series, Japan).
The surface morphology of the APP membrane was observed by scanning elec-tron microscopy (SEM). The dried APP membrane was coated with gold under reduced pressure and their scanning electron micrographs were obtained using a JEOL (Model JSM 5600; Japan).
Contact angles to water of the PPC, APP and APP-lipase membranes were mea-sured using sessile drop method at ambient temperature by using a digital optical contact angle meter Phoneix 150 (Surface Electro Optics, Korea).
3. Results and discussion 3.1. Characterization studies
In this work, reactive polypropylene based membrane was pre-pared sequentially in two steps: (i) in the first step, polypropylene chloride membrane was aminated by using hexamethylenedi-amine; (ii) then, the aminated membrane was prepared by the conventional casting method. The total amino group content of the aminated polypropylene membrane was determined by ele-mental analysis, and was found to be 6.72 mmol/g. The level of available surface amino groups of membrane was determined by using potentiometric titration, and was found to be 5.28 mmol/g membrane.
The chemical structure of polypropylene chloride (PPC) and aminated polypropylene (APP) samples were analyzed using a FTIR (Fig. 2). Among the characteristic vibrations of PPC is the adsorp-tion bands at∼724 cm−1arising from stretching vibration of C–Cl
bonds. The stretching vibration at 2925 cm−1represents C–H bands in both PPC and APP. The peaks at 1454 and 1371 cm−1are assigned as bending of C–H and stretching of C–C bonds. The FTIR spectra of APP have characteristic broad stretching vibration of –NH and –NH2bands at between 3404 and 3300 cm−1. The most important
Fig. 2. FTIR spectra of (a) polypropylene chloride and (b) aminated polypropylene.
adsorption band at 1628 cm−1representing N–H bending, is due to hexamethylene diamine bonded to the polymer backbone.
Scanning electron microscopy (SEM) micrographs of the ami-nated polypropylene membrane are presented in Fig. 3. The membrane has a rough surface and non-porous surface structure. The non-porous surface properties of the membrane would reduce diffusion limitation of the substrate and product for the enzymatic hydrolysis and esterification reactions.
The contact polypropylene membrane for immobilization angle values for water on PPC, APP and APP-lipase immobilized mem-branes are measured by using sessile drop method. The variation of the wetting force is extremely sensitive to the surface charac-teristics since it reflects the effect of functional groups on a surface layer thickness (less 10 ˚A) and indirect contact with the liquid phase [26,27]. The PPC, APP and APP-lipase membranes gave quite differ-ent surface characteristics depending on the presence of functional groups. The polypropylene membrane has a hydrophobic surface (<90◦) with a water contact angle value of 106◦. The change of the contact angles after amination of the PPC membrane shows that the hydrophilicity of the surface is increased (78◦). The improvement of the hydrophilicity of the APP membrane surface caused by the incorporation of amino groups on the membrane surfaces was used as a functional component for enzyme coupling after glutaralde-hyde activation. The change of contact angles after immobilization of lipase shows that the hydrophilicity of the surface (67◦) is fur-ther increased with respect to APP membrane surface. It should be noted that the hydrophilic nature of the APP surface induces less conformational changes on the enzyme three-dimensional
0 20 40 60 80 100 0 4 8 12 16 20 24 Time (h) Immobilized enzyme (µg / cm 2 membrane)
Fig. 4. Effect of coupling time on the immobilization efficiency of lipase on the
aminated polypropylene membrane. The reaction conditions: lipase concentration; 2 mg/ml, temperature; 4◦C; medium pH; 7.0 (0.1 M, phosphate buffer), time; 20 h.
ture than that of the PPC hydrophobic surface. For this reason, an orientation of amino acid residues, which influences the wet-ting by water is more likely in the case of a hydrophilic surface [28,29].
3.2. Effect of coupling time on the immobilization efficiency
Glutaraldehyde can react with several functional groups of pro-teins, such as amine, thiol, phenol, and imidazole because the most reactive amino acid side-chains are nucleophiles[1,30]. Most pro-teins contain many lysine residues, usually located on the protein surface (i.e., exposed to the aqueous medium) because of the polar-ity of the amine group. In addition, lysine residues are generally not involved in the catalytic site, which allows moderate covalent link-age to preserve protein conformation and thus biological activity [31].
As observed in Fig. 4, an increase in the coupling time with activated support and lipase led to increase in extent of immobilization, but this relation leveled off after 12 h. Thus, a maximum enzyme loading of 76g/cm2 was observed. No
enzyme leakage was observed during washing of the freshly prepared enzyme immobilized membrane or during batch oper-ation mode. This indicates that the immobilizoper-ation process was irreversible.
The attachment of 6-carbon and 5-carbon atom spacer-arms on the PP based membrane surface (during amination with hexam-ethylene diamine and activation with glutaraldehyde, respectively) could prevent undesirable side interactions between the large enzyme molecule and the support. In this way, all areas of the immobilized lipase could become fully accessible to its large sub-strate olive oil. The conditions for lipase activity assay were the same for the free and the immobilized preparations as described above. The immobilization of lipase through amino groups via glu-taraldehyde coupling onto on the PP based resulted in a protein loading (76g protein/cm2membrane) and with an activity yield
(69.69 U/cm2membrane). The relative activity is the effective
activ-ity after immobilization referred to the activactiv-ity that the bound protein amount would have in solution. A low relative activity indicates that much of the protein was apparently immobilized in a non-active form. In this work, the recovered activity of the immobilized lipase was around 76%.
0 20 40 60 80 100 2 4 6 8 10 pH Relativ e activity (%) Free Lipase Immobilized Lipase
Fig. 5. Effect of pH on the free and immobilized lipase activity; the relative activities
at the optimum pH were taken as 100% for free and immobilized lipase, respectively. The free and immobilized lipase activity was assayed at different pH values between 4.0 and 9.0 and at 35◦C for 30 min.
3.3. Effect of pH and temperature on the free and immobilized lipase activity
The effect of pH on the activity of free and immobilized lipase in olive oil hydrolysis was determined in the pH range 4.0–9.0. The optimum pH of the free enzyme was 7.0. The optimum pH value of the immobilized lipase was shifted 0.5 unit to the basic region (pH 7.5). This shift depended on the method of immobilization as well as the structure of the matrix (Fig. 5). The pH profile of the immobilized enzyme was much broader with respect to the free enzyme, probably due to the product fatty acid, forming lay-ers around the immobilized enzyme. It should be noted that the shift to neutral and basic region of the optimal pH upon immo-bilized could be expected as a result of the diffusional constraint of the support retaining a higher concentration of enzyme prod-uct, fatty acids, on the surface of the membrane that immobilized lipase present. Thus, the microenvironment around the immobi-lized lipase was more acidic than that of the bulk solution. These results could probably be attributed to the stabilization of immo-bilized lipase molecules resulting from multipoint attachment on the surface of the APP membrane by covalent bonds, which limited the transition of enzyme conformation against the change of pH. Other researchers have reported similar observations upon immo-bilization of lipase and other enzymes[32–34].
The effect of temperature on the free and immobilized enzyme activity was investigated in phosphate buffer (0.1 M, pH 7.0) in the temperature range 20–60◦C. The apparent temperature optimum for free enzyme was about 35◦C, while that for the immobilized enzyme and the form stabilized by immobilization was about 40◦C (Fig. 6). In the literature, most immobilized lipases exhibited higher optimum temperature values than their free counterpart[35,36]. The multipoint covalent attachment of the lipase molecule on the membrane surface via glutaraldehyde coupling could reduce the conformational flexibility of the enzyme and might result in higher activation energy for the molecule to reorganization the proper conformation for the binding to its large substrate (i.e., oil olive). Thus, the immobilized lipase showed its catalytic activity at a higher reaction temperature compared to that of the free counterpart. 3.4. Kinetic parameters
The Kmvalue is known as the criterion for the affinity of enzymes
0 20 40 60 80 100 10 20 30 40 50 60 70 Temperature (°C) Relativ e activity (%) Free Lipase Immobilized Lipase
Fig. 6. Effect of temperature on the free and immobilized lipase activity; the relative
activities at the optimum temperature were taken as 100% for free and immobilized lipase, respectively. The free and immobilized lipase activity was assayed at different temperatures between 20 and 60◦C and at pH 7.0 for 30 min.
between enzymes and substrates. In this work, kinetic constants of free and immobilized lipase, i.e., apparent Kmand Vmaxvalues
were determined by using tributyrin as substrate. The activities of the free and immobilized lipase for various concentrations of the substrate (5–50 mM) were plotted in Lineweaver–Burk plots, and apparent Kmand Vmaxvalues were calculated from the intercepts on
x- and y-axis, respectively. The kinetic data for hydrolysis of tribu-tyrin was fitted to the Michaelis–Menten equation. For the free and immobilized lipase the apparent Kmvalues were found to be 2.9 and
8.4 mM, respectively. The apparent Kmvalue of immobilized lipase
was the same order of magnitude, and was only 2.89 times higher than that of the free enzyme. Thus, the hydrophilic modification of hydrophobic polypropylene based membrane with hexamethylene diamine and glutaraldehyde seemed to be the major factor for the better affinity leading to the lower value of apparent Kmfor
immo-bilized lipase. The Vmaxvalues for the free and immobilized lipase
were calculated as 926 and 741 U/mg enzyme, respectively. The Vmaxvalue of the immobilized enzyme decreased about 1.25-fold
compared to the free enzyme.
Several reasons can account for the variations of the Vmaxvalues
of the enzyme upon immobilization[37–41]. These variations are attributed to several factors such as the covalent attachment of the enzyme molecule on the membrane surface might have induced an inactive conformation to the enzyme molecules. It should be noted that the immobilization process does not also control the proper orientation of the immobilized enzyme on the support. This improper fixation and/or the change in the property of the active sites might hinder the active site for binding of substrates (i.e., tributyrin) to the immobilized lipase molecules.
The efficiency factor can be calculated from the maximum reaction rates of the immobilized enzyme over that of the free counterpart:
=
v
immobilizedv
freewhere
v
immobilizedandv
freewas the reaction rate of theimmobi-lized and free enzyme, respectively. From this calculation, PP based membrane enzyme system provided an efficiency factor of 0.8 for the immobilized lipase. The ratio Vmax/Km defines a measure of
0 20 40 60 80 100 0 2 4 6 8 10 12 Number of cycles Residual activity (%)
Fig. 7. Operational stability of the immobilized lipase during olive oil hydrolysis
in repeated use. The free and immobilized lipase activity was assayed at 35◦C at intervals of 30 min and at pH 7.0.
the catalytic efficiency of an enzyme–substrate pair (Vmax is the
maximum enzymatic reaction rate). In this study, the catalytic effi-ciencies (Vmax/Km) of the free and immobilized lipase were found
to be 319.3 and 88.2, respectively. The catalytic efficiency of lipase was decreased about 3.6-fold upon immobilization.
Lipases have a higher level of hydrophobicity than conven-tional 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%[42]. The lipases have inherent affinity toward hydrophobic media, and the hydrophilic/hydrophobic nature of the aminated polypropylene membrane supports could provide a proper micro-environment for lipase, and thus, reasonable retained immobilized lipase activ-ities were obtained with the polypropylene based membrane. The retained lipase activity was obtained up to 76% in this study, and is comparable with the related literature[43–45].
3.5. Operational stability in lipid hydrolysis
The reusability of an immobilized enzyme is of key impor-tance for industrial applications, 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 inFig. 7shows that the same activity values to lipid hydrolysis were obtained within the first 5 cycles. After this, a steady decrease in the oil hydrolysis rate was observed, and this loss reached 19% after 12 cycles of batch operation, possibly result-ing from the inactivation of lipase upon use. This result showed that lipase immobilized on the polypropylene based membrane could be used successfully for industrial applications requiring long-term reaction stability. Thus, this immobilization method led to increased enzyme reusability.
3.6. Thermal stability
The effect of temperature on the stability of the free and immobi-lized lipase is shown inFig. 8. The pattern of heat stability indicated that a smaller rate of thermal inactivation was observed for the immobilized lipase on membrane than that of the free enzyme. At 55◦C, the free enzyme lost all its initial activity after a 120 min of heat treatment, while the immobilized enzyme showed significant resistance to thermal inactivation (retaining about 73% of its initial activity after the same period). At 65◦C, the free lipase lost all its ini-tial activity after 45 min heat treatment. Under the same conditions, the immobilized lipase retained about 55% of its initial activity. As in this figure, the free enzyme at 55 and 65◦C led to a steep loss of catalytic activity compared to immobilized counterpart, which is
0 10 20 30 40 50 60 70 80 90 100 0 50 100 150 Time (min) Residual activity (%) Free Lipase (55 °C) Free Lipase (65 °C) Immobilized-Lipase (55 °C) Immobilized-Lipase (65 °C)
Fig. 8. Thermal stability of the free and immobilized lipase at two different
tem-peratures. The results of thermal stability are presented in a normalized form with the highest value of each set being assigned the value of 100% activity. The free and immobilized lipase activity was assayed at 35◦C at pH 7.0.
typical of most enzymes[1,46], whereas a marked enhancement of thermal stability was achieved with enzyme immobilization since no activity decay was observed after one and half hour’s exposure at 55◦C. These results suggest that the thermostability of immobi-lized 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. 3.7. Storage stability
Enzymes are not stable during storage in solutions and their activities decrease gradually by the time. Free and immobilized lipase preparations were stored in phosphate buffer (1.0 M, pH 7.0) at 4◦C and activity measurements carried out for a period of 8 weeks (data not shown). The free enzyme lost its all-initial activity within 4 weeks. Immobilized preparations lost 36% activity dur-ing the same period. The immobilized lipase preserved its initial activity more than 67% at the end of 8 weeks storage period. The enhancement in stability provided by the present immobilization method is more than two-fold. Thus, the covalent immobilization definitely appears to hold the enzyme in a stable position in com-parison to the free counterpart.
3.8. Synthesis of green apple flavor by immobilized lipase
The effect of acid to alcohol molar ratio (i.e., valeric acid: ethyl alcohol molar ratio (i.e., 0.5:1.0, 1.0:1.0, 2.0:1.0 and 3.0:1.0) was investigated by using immobilized Candida rugosa lipase in a solvent-free system. The amount of synthesized ethyl valerate increased from 17.6% to 34.8% with increasing acid to alcohol molar ratio from 1/2 to 2, respectively. A maximum conversion of 34.8% was obtained with a molar ratio of 2. It has been reported that high alcohol concentrations may slow down the reaction rates[13,25]. On the other hand, as shown inFig. 9, when the molar ratio of acid: alcohol was further increased, the conversion (%) decreased, which could be due to the inhibition of the lipase activity by high valeric acid concentration. As previously reported, in the lipase-catalyzed esterification reaction, the first step consists of the preferential binding of the acid molecule to the enzyme molecule[47]. At high
0 5 10 15 20 25 30 35 40 0 1 2 3 4
Valeric acid / ethyl alchol molar ratio
Conversion yield (%)
Fig. 9. Effect of the molar ratio of acid:alcohol in the non-solvent system on the
ester synthesis efficiency. The enzymatic esterification reaction conditions: valeric acid: ethyl alcohol molar ratios (i.e., 0.5:1.0, 1.0:1.0, 2.0:1.0 and 3.0:1.0 containing of 20L distilled water) at 40◦C for 24 h.
alcohol molar ratio, a large increase in alcohol concentration may promote the binding of alcohol molecules to the lipase, during the first reaction step, competing with the acid. As a result, a decrease in the amount of bound acid occurs. Thus, this situation would lead to a decrease in the reaction rates, since the reaction will be limited by the amount of acid in the vicinity of the enzyme[13].
The synthesis of ethyl valerate by immobilized Candida rugosa lipase was also studied in hexane medium. The ethyl valerate syn-thesis experiments were carried out under 1:1 acid/alcohol molar ratio. The amount of synthesized ethyl valerate was 67.2% in the presence of n-hexane. The amount of the synthesized ethyl valerate was increased in the presence of n-hexane compared to a solvent-free system. As reported previously, the presence of an organic solvent can shift the equilibrium towards ester synthesis by a total transfer of ester into the organic phase[48]. Similar results related to the increment in the amount of ester synthesized in organic medium were reported in literature earlier. For example, Karra-Chaabouni et al.[49]produced ethyl valerate and hexyl acetate in hexane, heptane and solvent-free medium. They found remarkably higher ester yield in both hexane and heptane medium than those obtained in the absence of organic solvents.
Operational stability of the immobilized lipase was determined for 5 successive batch operations at 25◦C in solvent free system and in hexane. In solvent free system, the immobilized lipase remained almost the same as the original activity after 5 cycles. In hex-ane medium, a steady decrease in ester synthesis capability of the immobilized lipase was observed, and this loss reached to about 18% after 5 cycles of batch operation (data not shown). This activity loss could be a result of denaturation effect of hexane.
4. Conclusion
One of the most important aims of enzyme technology is to enhance the conformational stability of the enzyme. The extent of stabilization depends on the enzyme structure, the immobiliza-tion methods, and type of support. In the present study, a new polypropylene based hydrophobic support with functional amino groups was prepared by modification of PPC with hexamethylene diamine. The aminated membrane was used for immobilization of lipase via glutaraldehyde coupling. The properties of the membrane seem to provide an adequate approach for the immobilization of
lipase based on its hydrophobic properties. The immobilized lipase also exhibited a satisfactory performance for olive-oil hydrolysis in the activity tests performed in a batch mode. The immobilized lipase was more stable during operation and storage compared with free lipase. Finally, the ethyl valerate synthesis was conducted in a solvent-free system and in n-hexane medium by using the immobilized lipase. In the case of solvent free system, high conver-sion of 34.8% was achieved with an addition of 20L of water, using 696.9 U of immobilized lipase (10 cm2) at 40◦C and an acid to
alco-hol molar ratio of 2. The production of ethyl valerate in n-hexane medium was 67.2% with 1:1 acid/alcohol ratio. In this medium, the conversion was higher than that of the solvent free system which gives a moderate conversion up to 34.8%.
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