Article pubs.acs.org/IECR
Optimum Lipase Immobilized on Diamine-Grafted PVDF Membrane and Its Characterization Chia-Hung Kuo,† Guan-Jie Chen,‡ Yawo-Kuo Twu,§ Yung-Chuan Liu,*,⊥,‡ and Chwen-Jen Shieh*,† †
Biotechnology Center, ⊥Agricultural Biotechnology Center, and ‡Department of Chemical Engineering, National Chung Hsing University, 250 Kuo-kuang Road, Taichung, 402, Taiwan § Department of Bioindustry Technology, Da-Yeh University, 168 University Road, Chang-Hwa, 515, Taiwan S Supporting Information *
ABSTRACT: A facile and economic modification of polyvinylidene fluoride (PVDF) with an orientation of diamine is presented herein. The physical characterizations of native and diamine-grafted PVDF membranes are analyzed by three different techniques: the ninhydrin test, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The diamine-grafted PVDF is activated by glutaraldehyde for enzyme immobilization. The response surface methodology (RSM) is used to search the optimal immobilization conditions and to understand the significance of the factors affecting the responses of immobilized lipase. The optimal conditions for lipase immobilization are: a reaction time of 90 min, temperature of 35 °C, pH of 6, and an enzyme concentration of 7 mg/mL. An experiment performed under the optimum conditions obtains lipase activity of 60 U per g of membrane. A good agreement between the calculated and experimental values is thereby achieved. studied, including coating poly(DOPA),17 grafting acrylic acid18,19 and methacrylate polymers.20 Here, we have proposed a facile method to graft diamine onto the PVDF surface in order for conjugation with proteins or application in a coupling reaction. PVDF modified with an orientation of diamine introduces not only reactive groups of amine, but also a biofriendly interface on the surface. After the grafting of the diamine, the amino groups on the PVDF can be used as reactive sites for multipoint covalent attachment. The multipoint covalent attachment between the enzyme and the support maintains the conformation of the enzyme against distorting agents (heat, organic solvents, extreme pH values, etc).21 In general, glutaraldehyde activation of the aminated supports is one of the most popular techniques for immobilizing enzymes.21,22 This methodology is simple and efficient, and improves enzyme stability due to the formation of covalent linkages between the primary amine and aldehyde groups.23 However, to the best of our knowledge, the reaction conditions for lipase immobilization on diamine-grafted PVDF membranes have not previously been practiced and discussed. The present study determined that diamine could be simply and easily grafted onto a PVDF surface. The characterization property of the diamine-grafted PVDF was analyzed by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. The diamine-grafted PVDF membrane was activated by glutaraldehyde for lipase immobilization. The optimum conditions for immobilized lipase were computed using RSM and central composite rotatable design (CCRD).
1. INTRODUCTION Lipase (triacylglycerol ester hydrolase, EC 3.1.1.3) is an enzyme with many industrial applications, including hydrolysis, alcoholysis, acidolysis, amidolysis, esterification and transesterification.1 It is used in the food, detergent, beverage, cosmetics, biomedical and chemical industries.2 The lipase catalyzes the reverse synthesis reaction to produce esters in the organic solvent. Lipase is successfully utilized in organic chemical synthesis and has become increasingly important for synthesizing special compounds or biologically active enantiomers.3,4 In organic solvents, free lipase is generally insoluble, denatured, or the essential water layer is stripped away from the lipase molecule, resulting in decreased lipase activity or a very low reaction rate.5 Therefore, lipase immobilization is required to improve its stability and activity in organic solvents. Enzyme immobilization provides advantages such as: increased conformational stability, operational stability and easy recovery for reuse.6 The immobilization of lipase increases the esterification rate and yields have been reported.7,8 Polyvinylidene fluoride (PVDF) is a hydrophobic polymer which has been extensively applied in microfiltration (MF) and ultrafiltration (UF) membranes because of its high thermal stability, mechanical strength and resistance to acids, bases and solvents. Lipase is known to be a more hydrophobic enzyme compared to other proteins.9 The hydrophobic supports involve hydrophobic interfaces; these makes it possible for lipase to change the structure from a closed to an open conformation, which promotes hyperactivation after immobilization.10,11 The enhanced activity of lipase immobilized on hydrophobic materials has been previously demonstrated.12−15 Lipase immobilized on the membrane is able to simultaneously perform a catalytic reaction and product separation.16 However, PVDF itself is an inert chemical compound, as its surface requires relevant functional groups in order to couple with the enzyme. Several PVDF modification methods have been © 2012 American Chemical Society
Received: Revised: Accepted: Published: 5141
January 2, 2012 March 16, 2012 March 19, 2012 March 19, 2012 dx.doi.org/10.1021/ie300011q | Ind. Eng. Chem. Res. 2012, 51, 5141−5147
Industrial & Engineering Chemistry Research
Article
2. MATERIALS AND METHODS
lipase activity, a 5-level and 4-factor CCRD and RSM were applied to investigate the optimum levels of these variables and their relationships. The variables and their levels selected for the study were as follows: immobilization time of 0−120 min, immobilization temperature of 15−55 °C, immobilization pH of 4−8 and an enzyme concentration of 1−9 mg/mL. Table S. One shows the independent factors (xi), levels and experimental design in terms of coded and uncoded. To avoid bias, 27 runs were performed in a totally random order. 2.5. Statistical Analysis. The experimental data were analyzed by response surface regression (RSREG) procedures with SAS software to fit the following second-order polynomial eq 1
2.1. Materials. Lipase from C. rugosa (Amano AY-30, batch No. LAYC0552546, protein content 8.3% and 30,000 U/g) was purchased from Amano International Enzyme Co. (Nagoya, Japan). p-Nitrophenyl palmitate (p-NPP) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Ninhydrin was purchased from Alfa Aesar (Ward Hill, MA) and 1, 4-diaminobutane (1,4-DA) and glutaraldehyde (GA) were purchased from Acros Organics (Geel, Belgium). All of the other chemicals were of analytical reagent grade. The polyvinylidene fluoride (PVDF) membrane was purchased from Pall (Mexico) and had an average diameter of 47 mm and an average pore size of 0.45 μm. 2.2. Surface Modification of PVDF Membrane. The PVDF membrane (∼150 mg, d: 47 mm, pore size: 0.45 μm, thickness: 140 μm) was treated with 10 mL of 1 M 1,4diaminobutane solution (1 M carbonate buffer, pH 11.0) at 25 °C for 12 h to the grafting of a spacer arm onto the membrane surface. After that, the membrane was washed three times with distilled water, and the 1,4-diaminobutane-grafted PVDF membrane (PVDF-DA) was obtained. The PVDF-DA membrane was transferred into 10 mL of 0.1% (v/v) glutaraldehyde solution at 25 °C for 2 h to graft the glutaraldehyde onto the PVDF-DA membrane (PVDF-DA-GA). The PVDF-DA-GA membrane described above was thoroughly washed with distilled water and preserved at 4 °C until use. Scheme 1 shows the reaction scheme for the coupling reaction.
Y = βk 0 +
4
4
i=1
i=1
3
4
∑ βkixi + ∑ βkiixi 2 + ∑ ∑
βkijxixj
i=1 j=1+i (1)
where Y is the response (percent of molar conversion); βk0 is a constant; βki, βkii, and βkij are coefficients; and xi and xj are the uncoded independent variables. 2.6. Determination of Lipase Activity and Protein Content. Lipase activity was determined using p-nitrophenyl palmitate (p-NPP) as the substrate. The release of p-nitrophenol, resulting from the lipase-catalyzed hydrolysis of p-NPP, was measured by a reading of the absorbance at 410 nm. The lipase-immobilized PVDF membrane (∼150 mg) was added to a mixture of 2 mL of 0.5% (w/v) p-nitrophenyl palmitate (p-NPP) in ethanol and 2 mL of 0.05 M phosphate buffer solution (pH 7.0). The solution was incubated at 30 °C for 5 min, and 4 mL of 0.25 M Na2CO3 was added to terminate the reaction. After that, the 0.5 mL solution was diluted 10-fold with distilled water, and the absorbance at 410 nm was measured with a UV/vis spectrophotometer (Metertek SP-830, Metertech Inc., Taiwan). The molar extinction coefficient (ε410) for p-nitrophenol is 15000 M−1 cm−1. One unit (U) of enzyme activity is defined as the amount of enzyme which liberates 1 mmol p-nitrophenol per minute under assay conditions. Protein concentration was estimated by the Bradford method using protein dye reagent concentrate (BioRad, Hercules, California). Bovine serum albumin was used as the standard.
Scheme 1. Preparation of PVDF-DA-GA Membrane for Lipase Immobilization
2.3. Characterization of Diamine-Grafted PVDF Membrane. Scanning electron microscopy (SEM) of the native and diamine-grafted PVDF membranes utilized a JEOL Model JSM 5600 scanning electron microscope (JEOL Ltd., Japan). Fourier transformed infrared spectrometry (FTIR) was measured using a Perkin-Elmer PARAGON 500 spectrometer. The spectra (4000− 400 cm−1) were recorded with a resolution of 4 cm−1 and 64 scans per sample. The content of the amino groups on the PVDF-DA surface was determined by the ninhydrin method,24 with glycine used as the standard. Briefly, the PVDF-DA membrane was cut into small pieces (2 mg; diameter 5 mm) and added to a tube containing 0.2 mL of ninhydrin reagent. The tube was heated in boiling water for 3 min, and then diluted with 0.8 mL of EtOH. The amine concentration was determined by readings of UV absorbance at 570 nm. 2.4. Experimental Design for Lipase Immobilization onto a PVDF Membrane. The PVDF-DA-GA membrane was immersed in 20 mL of enzyme solution (50 mM phosphate buffer) and shaken (150 rpm) for lipase immobilization. In order to evaluate the effects of immobilization time (x1), temperature (x2), pH (x3) and enzyme concentration (x4) on
3. RESULTS AND DISCUSSION 3.1. Characterization of the Modified PVDF Membrane. In this study, PVDF membrane was grafted with 1,4DA. When the PVDF membrane was treated with a basic solution, fluorine and hydrogen were eliminated via nucleophilic substitution, and then 1,4-DA was grafted onto the surface. The color of the PVDF membrane changed to dark red after the grafting with 1,4-DA. Ninhydrin is usually used to quantify amino groups. The amino groups on the PVDF-DA membrane reacted with ninhydrin to form a purple-colored compound in solution, as shown in Figure 1. Based on the ninhydrin test, the presence of amino groups on the PVDF-DA membrane was confirmed. The amine content of the PVDF-DA membrane was determined to be 0.252 mmol/g. The FT-IR analyses of the native PVDF, PVDF-DA and PVDF-DA-GA were also studied. Infrared bands of CF2CH2 near 1735 cm−1 have been previously reported.25 In this study, as shown in Figure 2, the PVDF had a characteristic band at 1738 cm−1. After the graft with DA, the N−H bend of PVDF-DA was 5142
dx.doi.org/10.1021/ie300011q | Ind. Eng. Chem. Res. 2012, 51, 5141−5147
Industrial & Engineering Chemistry Research
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Figure 1. Ninhydrin test of native PVDF and PVDF-DA membranes.
Figure 2. FTIR spectra of native PVDF, PVDF-DA, and PVDF-DAGA membranes.
found at 1640 cm−1. Primary amine on the PVDF-DA membrane surface reacted with glutaraldehyde to form PVDF-DA-GA for enzyme immobilization. The CO stretch of PVDF-DA-GA was found at 1720 cm−1. Consequently, these results clearly demonstrated that PVDF was grafted with 1,4-DA and successfully activated by GA. The GA-grafted membrane, which easily formed the covalent bond with the amine group, is commonly used for enzyme immobilization.26,27 The scanning electron microscopy (SEM) micrographs of the native, amine- functionalized and lipase-bounded PVDF membranes are shown in Figure 3. The native PVDF membrane had a uniform porous surface structure (Figure 3A). The PVDF-DA membrane had a rougher surface structure after surface modification (Figure 3B). The PVDF-DA membrane was activated with glutaraldehyde, and then bounded with lipase. After immobilization, a rugged surface was evident because of the high concentration of immobilized enzymes on the membrane, as shown in Figure 3C. This change in the surface morphology of the support after enzyme immobilization was also reported for the PVC membrane.27 The size of the attached proteins was determined to be about 100 nm, based on the SEM image, as shown in Figure 3C; similar results have been reported in the literature.28 The porosity of the membranes, as illustrated in the above figures, did not change substantially. 3.2. Preliminary Study. The enzyme immobilization was carried out at room temperature in 20 mL of phosphate buffer solution (pH 7.5, 50 mM) containing 100 mg of lipase and
Figure 3. Scanning electron microscopy (SEM) micrographs of the (A) native PVDF, (B) PVDF-DA, and (C) lipase-bounded PVDF membranes.
150 mg of PVDF-DA-GA membrane. Figure 4 shows the time course of the lipase immobilization on the native PVDF and PVDF-DA-GA membranes. The results indicated that enzyme activity increased proportionally to the immobilization time due to the increase in enzyme binding capacity, but remained almost constant after 30 min. The enzyme activity increased to 16.50 and 35.32 U/g of membrane after 90 min for the native PVDF and PVDF-DA-GA membranes, respectively. The residual activity of the lipase immobilized on the native PVDF was much lower than that of the PVDF-DA-GA membrane. Therefore, the technique using PVDF-DA-GA membrane was chosen in this study for lipase immobilization. 3.3. Model Fitting. The RSREG procedure was employed to fit the second-order polynomial eq 1 to the experimental 5143
dx.doi.org/10.1021/ie300011q | Ind. Eng. Chem. Res. 2012, 51, 5141−5147
Industrial & Engineering Chemistry Research
Article
specific acvtivity (U/mg of protein) = 13.872901 − 0.112150x1 + 0.328680x2 + 15.161458x3 − 2.529255x4 − 0.001040x1x1 + 0.004475x2x1 − 0.011019x2x2 + 0.013306x3x1 − 0.019306x3x2 − 2.452771x3x3 − 0.018068x4x1 − 0.017328x4x2 + 1.890969x4x3 − 0.746161x4x4
(4)
The analysis of variance from Table 1 (ANOVA) indicated that the second-order polynomial model (eqs 2, 3 and 4) was statistically significant and adequate to represent the actual relationship between the responses (activity, protein attached and specific activity) and the variables, with a very small p-value (
