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Covalent immobilization of lipase from Candida rugosa onto poly(acrylonitrile-co-2-hydroxyethyl methacrylate) electrospun fibrous membranes for potential bioreactor application
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Bioresource Technology 99 (2008) 5459–5465
Covalent immobilization of lipase from Candida rugosa onto poly(acrylonitrile-co-2-hydroxyethyl methacrylate) electrospun fibrous membranes for potential bioreactor application Xiao-Jun Huang, An-Guo Yu, Zhi-Kang Xu * Institute of Polymer Science, Key Laboratory of Macromolecular Synthesis and Functionalization (Ministry of Education), Zhejiang University, Hangzhou 310027, PR China State Key Laboratory of Chemical Engineering, Zhejiang University, Hangzhou 310027, PR China Received 2 August 2007; received in revised form 2 November 2007; accepted 5 November 2007 Available online 14 January 2008
Abstract A simple way of fabricating enzymatic membrane reactor with high enzyme loading and activity retention from the conjugation between nanofibrous membrane and lipase was devised. Poly(acrylonitrile-co-2-hydroxyethyl methacrylate) (PANCHEMA) was electrospun into fibrous membrane and used as support for enzyme immobilization. The hydroxyl groups on the fibrous membrane surface were activated with epichlorohydrin, cyanuric chloride or p-benzoquinone, respectively. Lipase from Candida rugosa was covalently immobilized on these fibrous membranes. The resulted bioactive fibrous membranes were examined in catalytic efficiency and activity for hydrolysis. The observed enzyme loading on the fibrous membrane with fiber diameter of 80–150 nm was up to 1.6% (wt/wt), which was as thrice as that on the fibrous membrane with fiber diameter of 800–1000 nm. Activity retention for the immobilized lipase varied between 32.5% and 40.6% with the activation methods of hydroxyl groups. Stabilities of the immobilized lipase were obviously improved. In addition, continuous hydrolysis was carried out with an enzyme-immobilized fibrous membrane bioreactor and a steady hydrolysis conversion (3.6%) was obtained at a 0.23 mL/min flow rate under optimum condition. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Fibrous membrane; Electrospinning; Enzyme immobilization; Lipase; Membrane bioreactor
1. Introduction Lipases are the hydrolytic enzymes that can catalyze a wide range of reactions such as hydrolysis, alcoholysis, transesterifications, aminolysis and enantiomer resolution (Deng et al., 2005; Kose et al., 2002; Noureddini et al., 2005; Serra et al., 2003; Sharma and Chattopadhyay, 2000). These ubiquitous enzymes have found numerous commercial applications in the food, pharmaceutical, and detergent industries. For industrial applications, like most enzymes, lipases have often been immobilized onto insoluble or solid supports, which can offer better catalytic stabil-
*
Corresponding author. Fax: +86 571 8795 1773. E-mail address: [email protected] (Z.-K. Xu).
0960-8524/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.11.009
ity, feasible continuous operations, easy catalyst recycling, significant reduction in the operation costs and simple product purification (Amorim et al., 2003; Pahujani et al., 2007; Wang et al., 2007a,b; Yu et al., 2006). A promising property of lipases was the interfacial activation that could induce important conformational rearrangements yielding the ‘open state’ of lipases and improve their activity during immobilization (Fernandez-Lorente et al., 2006; Grazu et al., 2005; Mateo et al., 2007, 2003; Verger, 1997). However, in many cases the non-orientable immobilized lipases lost catalytic activity and efficiency to some extent. The properties and microstructure of supports have great impact on the performance of immobilized enzymes. It is, thus, important that the choice of support materials and immobilization methods for enzyme should be well justified.
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To achieve high enzyme loading and catalytic efficiency for large-scale operation and application, supports with high surface to volume ratio, an inherent characteristic of nanoscale materials, are often desirable. Therefore, in recent years, nanostructured supports such as mesoporous materials, nanoparticles, and nanofibers have been widely studied for enzyme immobilization (Drechsler et al., 2004; Dyal et al., 2003; Huang et al., 2006; Tang et al., 2007; Wang et al., 2006a,b; Ye et al., 2006a,b). Among these, it appears to us that electrospun nanofibrous membranes are the promising supports for enzyme immobilization due to the advantages including: (1) large specific surface providing relatively high quantity of enzyme loading per unit mass, (2) ultrafine fiber and high porosity entrusting the accessibility of active sites and low diffusion resistance necessary for high reaction rate and conversion, and (3) easy recoverability from reaction media or applicability for continuous operations in comparison with nanoparticles (Herricks et al., 2005; Huang et al., 2006; Jia et al., 2002; Lee et al., 2005; Nair et al., 2007; Wang et al., 2006a,b; Xie and Hsieh, 2003; Ye et al., 2006a,b). To maximize the enzyme activity, it is very important for the supports to provide a biocompatibility and/or inert environment for enzyme protein. A biofriendly interface on the support surface for enzyme immobilization may reduce some non-biospecific enzyme–support interaction and protein denaturation, create a specific microenvironment for the enzyme, and thus benefit the enzyme activity (Deng et al., 2004; Girard-Egrot et al., 2005; Gotoh et al., 1998; Huang et al., 2005; Ito et al., 2006; Mukhopadhyay et al., 2003). Poly(2-hydroxyethyl methacrylate) (pHEMA) and HEMA-based co-polymers are non-toxic and biocompatible synthetic materials with adequate mechanical strength for most biomedical applications (Belkas et al., 2005; Wang et al., 2006a,b; Yu and Ober, 2003). Methacrylate-based materials possessing moderate hydrophobic interface could benefit interfacial activation of lipase and thus promote a dramatic activation of lipases after their immobilization (Fernandez-Lafuente et al., 1998; Fernandez-Lorente et al., 2006; Mateo et al., 2007; Verger, 1997). In addition, it contains hydroxyl groups that act as attachment sites for bioactive species after activation. Therefore, in recent years HEMA-based polymers had been prepared in different forms (such as gels, beads and membranes) as the supports for enzyme immobilization (Arica and Bayramoglu, 2004; Gotoh et al., 1998; Liu and Chang, 2007; Shukla and Devi, 2005; Xu et al., 2006). In these cases, each had its own advantages and disadvantages. Some changes were observed in enzymatic activity, optimum pH and temperature, affinity to substrate, and stability. The extent of these changes depended on the nature of enzyme, type of support, and the immobilization condition. Although some properties of the immobilized enzyme were quite promising, the enzyme loading and catalytic efficiency were not very favorable. In this work, poly(acrylonitrile-co-2-methacryloyloxyethyl) (PANCHEMA) was synthesized and electrospun
into fibrous membranes. The prepared PANCHEMA fibrous membranes, which combine the advantages of HEMA and electrospinning technique, are expected to be attractive supports for enzyme immobilization. Lipase from Candida rugosa was covalently immobilized on these fibrous membranes using epichlorohydrin, cyanuric chloride or p-benzoquinone as covalent reagent, respectively. The performances such as enzyme loading, activity, reusability and storage stability of the immobilized lipase were described. In addition, the application of lipase-immobilized fibrous membrane bioreactor to the hydrolysis of p-nitrophenyl palmitate (p-NPP) was presented. 2. Experimental 2.1. Materials PANCHEMA was synthesized by water phase precipitation co-polymerization in our laboratory (Huang et al., 2005). The HEMA content in the co-polymer calculated from 1H NMR spectrum was 9.3 mol%. N,N0 -Dimethyl formamide (DMF) was purified by vacuum distillation before use. Lipase (from Candida rugosa), Bradford reagent, bovine serum albumin (BSA, molecular mass: 67,000 Da) and p-NPP were purchased from Sigma and used as received. All other reagents were analytical grade and used without further purification. 2.2. Preparation of PANCHEMA fibrous membranes by electrospinning To fabricate non-woven fibrous membranes using the electrospinning process, PANCHEMA was dissolved in DMF at room temperature with gentle stirring for 12 h to form homogeneous solution. After air bubbles were removed completely, each solution was placed in a syringe (50 mL) bearing an 1 mm inner diameter metal needle which was connected with a high voltage power supply (GDW-a, Tianjin Dongwen High Voltage Power Supply Plant, China). The grounded counter electrode was connected to an aluminum foil collector. Typically, electrospinning was performed at 10 kV voltage, 15 cm distance between the needle tip and the collector. The solution flow rate was controlled by a microinfusion pump (WZ-50C2, Zhejiang University Medical Instrument Co., LTD, China) to maintain at 2 mL/h from the needle outlet. It usually took one hour to obtain a sufficiently thick sample that could be detached from the aluminum foil collector. Fibrous membrane on the aluminum foil was dried under vacuum at 60 °C before it was detached. The fiber diameter was examined with field emission scanning electron microscope (FESEM). 2.3. Enzyme immobilization Different activation procedures were adopted for the immobilization of lipase on the PANCHEMA fibrous
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membranes (Arica, 2000; Horak et al., 1999; Rejikumar and Devi, 1998; Shukla and Devi, 2005; Xu et al., 2006). The activation of hydroxyl groups in PANCHEMA for covalent immobilization was achieved by reaction with epichlorohydrin, cyanuric chloride or p-benzoquinone, respectively. The activation and immobilization were carried out as the following processes. To identify the best method for the immobilization of lipase, three different protocols were tested as follow. 2.3.1. Method I In the process of activation through epichlorohydrin approx, 5 mg fibrous membrane was immersed in 5 mL epichlorohydrin and shaken at 25 °C for 3 h. The activated fibrous membrane was washed several times with acetone to remove excess epichlorohydrin, and then washed with phosphate buffer solution (PBS, 0.05 M, pH 7.0) before enzyme immobilization. 2.3.2. Method II For the activation of PANCHEMA fibrous membrane through cyanuric chloride, 5 mg fibrous membrane was treated with 0.25 g cyanuric chloride in 5 mL dioxane at 25 °C for 30 min. Then it was treated with 10 mL water and 10 mL 20% acetic acid for 30 min. The resulting activated fibrous membrane was washed with water, acetone and phosphate buffer solution (PBS, 0.05 M, pH 7.0) before enzyme immobilization. 2.3.3. Method III During the activation of PANCHEMA fibrous membrane through p-benzoquinone, 5 mg fibrous membrane was treated with 0.05 g p-benzoquinone dissolved in 5 mL 20% ethanol, and phosphate buffer solution (PBS, 0.05 M, pH 7.0) for 2 h at room temperature. The treated fibrous membrane was washed successively with 20% ethanol and water until the filtrate was free from p-benzoquinone and then with 1 M NaCl and phosphate buffer solution (PBS, 0.05 M, pH 7.0) before immobilization. Lipase solution (5.0 mg/mL) was prepared by solving appropriate amount of lipase powder in phosphate buffer (0.05 M, pH 7.0). The activated fibrous membrane was submerged in 5 mL lipase solution in a vertical orientation and shaken gently in a water bath at 30 °C for 3 h. Then, the fibrous membrane was taken out and rinsed with buffer until no soluble protein was detectable in washings. Protein concentration in solution was determined with Coomassie Brilliant Blue reagent following Bradford’s method (Bradford, 1976). BSA was used as standard to construct calibration curve. Amount of immobilized protein on the fibrous membrane was calculated from the protein mass balance among the initial and final lipase solutions and washings. Enzyme loading on the fibrous membrane was defined as the amount of protein (mg) per gram of the membrane. Each reported value was the mean of three experiments at least, and the standard deviation was within ca. ±5%.
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2.4. Activity assay of free and immobilized lipases Activity for the immobilized lipase in aqueous medium was determined according to the method reported previously (Huang et al., 2006; Chiou and Wu, 2004). In the standard condition, a reaction mixture was composed of 1.0 mL ethanol containing 14.4 mM p-NPP and 1.0 mL PBS in an Erlenmeyer flask. The reaction was started by immersing 5 mg immobilized lipase preparation in the flask. The mixture was incubated at a certain temperature under reciprocal agitation at 120 strokes per minute. After 5 min of reaction, the reaction was terminated by adding 2.0 mL 0.5 M Na2CO3 followed by centrifuging for 10 min (8400g). 0.50 mL supernatant was diluted 10 folds with de-ionized water, and measured at 410 nm in an UV–vis spectrophotometer (UV-1601, Shimadzu, Japan) against a blank without enzyme and treated in parallel. The reaction rate was calculated from the slope of the absorbance versus the time curve. Molar extinction coefficient of 14.5 103 M1 cm1 for p-nitrophenol (p-NP), which was determined from the absorbance of standard solutions of p-NP in the reaction medium, was used. One enzyme unit is the amount of biocatalyst liberating 1.0 lmol p-NP/min in the condition. Specific activity was defined as the number of lipase unit per milligram of protein and it was expressed as lmol p-NP min1 mg protein1. Activity retention value was the ratio of specific activity of immobilized lipase with that of free one. Each data was the average of three parallel experiments at least. The standard error for the result was below 5% of the average value. 2.5. Stability measurements For the reusability measurement, 5 mg immobilized lipase and 2.0 mL of substrate were added in an Erlenmeyer flask. After 5 min reaction, the released p-NP was measured as in activity assay. The immobilized lipase was taken out from the flask and washed with PBS to remove any residual substrate on the nanofibrous membrane. It was then reintroduced into fresh substrate and the same measurement was repeated 10 times. In order to prevent the influence of storage time on the enzyme activity, there was just 30 s between two following cycles and all measurements were carried out in one day. The storage stability was determined as follows. Free and immobilized lipases were respectively stored in phosphate buffer (PBS, 0.05 M, pH 7.0) at 4 °C for a month. Parts of them were periodically withdrawn for activity assay. The residual activities were then determined as described above. 2.6. Enzyme-immobilized fibrous membrane bioreactor A simple enzyme-immobilized membrane bioreactor was set up in our lab. PANCHEMA fibrous membrane (fiber diameter = 80–150 nm, dry membrane weight = 10 mg)
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was directly collected on a round wire mesh with area of 9 10 cm2. The round-shaped PANCHEMA fibrous membrane with the wire mesh was installed into a dead-end reactor cell. A peristaltic pump was used between the substrate reservoir and the reactor cell for better control of feed flow rate. Epichlorohydrin, washing solutions and lipase solution were successively circulated in the reactor cell by the peristaltic pump. The condition of activation and enzyme immobilization was the same as described above. The temperature of substrate reservoir or reactor cell was controlled to ±0.5 °C by circulation water through the reservoir or reactor jacket. p-NPP (7.2 mM) substrate solution was continuously flowed through the membrane bioreactor using the peristaltic pump. The continuous hydrolysis process was conducted at 42 °C for 10 min. The hydrolytic products, p-NP, leaving the reactor cell was collected and measured at 410 nm in an UV–vis spectrophotometer against a blank without enzyme and treated in parallel. It was measured periodically and used for evaluating the activity and the efficiency of reactor for continuous operation. The hydrolysis conversion was defined as the fraction of the amount of hydrolytic p-NP compared with the total amount of p-NPP. 3. Results and discussion 3.1. Preparation of fibrous membranes from PANCHEMA Electrospinning has been recognized as an effective technique for the preparation of polymeric fibers with diameter from several micrometers down to tens of nanometers (Dzenis, 2004). Among various polymers, acrylonitrilebased homo- and co-polymers were most recently fabricated into nanofibers with reinforcing, superhydrophobic and/or catalytic properties (Ge et al., 2004; Zhang and Hsieh, 2006; Zussman et al., 2006). Several factors such as polymer molecular weight, concentration, electric field strength, and solvent properties can significantly influence the size and morphology of the electrospun fibers. In this work, PANCHEMA with a molecular weight of 100,000–110,000 g/mol was synthesized by a water phase precipitation co-polymerization process and then electrospun into non-woven fibrous membranes. It was found that the fiber diameter of the resulted fibrous membranes could be controlled by varying the concentration of PANCHMEA solution. Almost homogenous networks (fibrous membranes) with fiber diameter ranging from 80 nm to 1000 nm were obtained.
3.2. Lipase immobilization on the electrospun fibrous membranes For large-scale application, high enzyme loading is always desired to enhance the efficiency of biotransformation and to reduce the size of bioreactor. Enzyme loading on the electrospun fibrous membranes is expected to be high considering the large surface area/mass ratio. In the present work, enzyme immobilization through covalent coupling was achieved using activating agent epichlorohydrin, cyanuric chloride or p-benzoquinone, respectively, for the activation of hydroxyl groups on the surfaces of PANCHEMA fibrous membranes. To enhance the enzyme loading, the activation of hydroxyl groups followed by enzyme coupling was carried out at the optimized conditions. It was found that the enzyme loading increased as the fiber diameter decreased in spite of the different activated methods. An enzyme loading of 1.55–1.65 wt.% was observed on fibers of 80–150 nm, while it was about 0.55–0.60 wt% on fibers of 800–1000 nm. The increase of a factor of 3 in the enzyme loading was attributed to the decrease of the fiber diameter into nano-scale, which can increase the outer surface area (when the density of the PANCHEMA fiber is taken as 1.18 g/cm3, the fiber diameter of 80–150 nm have a surface area of 11.3–21.2 m2 per 1 gram, while that of 800–1000 nm with a surface area of 1.69–2.12 m2 per 1 gram) and the exposure of the hydroxyl groups on nanofibers available for enzyme coupling. In this regard, it could clearly demonstrate that increasing the outer surface area of the supports is the efficient way to enhance the enzyme loading. 3.3. Activity of immobilized lipase Activities for lipases immobilized on the PANCHEMA fibrous membranes through different activation methods are compared in Table 1. It can be seen that the total enzyme loading was nearly equal for the immobilized lipase prepared by the three methods, while the activity retention of the immobilized lipase prepared by method I (40.6 %) was slightly higher than that prepared by method II (36.4 %) and by method III (32.5%). Compared with cyanuric chloride and p-benzoquinone, epichlorohydrin can covalently link with available amino, hydroxyl, or sulfhydryl groups of enzyme. The random linkage with these groups of enzyme could reduce the chance of the directly covalent bonding between the active site of enzyme and the support surface. In addition, it can form a flexible and little longer spacer
Table 1 Immobilization of lipase on the electrospun poly(acrylonitrile-co-2-hydroxyethyl methacrylate) fibrous membranes Method of activation
Fiber diameter (nm)
Loading enzyme (mg/g)
Specific activity (U/mg)a
Activity retention (%)
Epichlorohydrin Cyanuric chloride Benzoquinone
80–150 80–150 80–150
16.2 ± 1.1 16.5 ± 1.3 15.5 ± 1.2
17.0 15.3 12.1
40.6 ± 0.6 36.4 ± 0.4 32.5 ± 0.8
a
Specific activity for free lipase is 42 U/mg.
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3.4. Enzyme stability studies Reusability and storage stability are of considerable importance for various applications of biocatalysts in a commercial point of view. An increased stability can make the immobilized enzyme more advantageous than its free one. To investigate the reusability, the enzyme-immobilized fibrous membrane was washed with PBS (0.05 M, pH 7.0) after one catalysis run and reintroduced into a fresh p-NPP solution for another hydrolysis at 42 °C. Fig. 1 shows the effect of repeated use on the activity of the immobilized lipases. It can be seen that the activities of the immobilized lipases decayed obviously with recycled uses. After 10 reuses, the lipase immobilized by the three activation methods retained over 30 % residual activity. The activity loss could be related to the inactivation of the enzyme caused by the denaturation of the protein. The free and immobilized lipases were stored in phosphate buffer (0.05 M, pH 7.0) at 4 °C and measured for a period of 30 days. The storage stability of the immobilized lipases compared with that of the free one is shown in Fig. 2. It can be seen that the free lipase lost all its initial activity within 30 days. While the residual activity of lipase immobilized on the PANCHEMA fibrous membranes retained over 60% of its initial activity during the same period. These results indicated that the immobilization procedures had considerably improved the storage stability. 3.5. Enzyme-immobilized fibrous membrane bioreactor PANCHEMA (10 mg) fibrous membrane (fiber diameter = 80–150 nm, membrane area = 9.10 cm2) was installed
Residual activity (%)
100
80
60
40
100
Residual activity (%)
arm between enzyme and support comparing with cyanuric chloride and p-benzoquinone. This resulted both in a reduction in denaturation of the enzyme protein and in enhanced substrate accessibility to the enzyme’s active site.
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80
60
40
20
0 0
5
10
15
20
25
30
Storage time (Days) Fig. 2. Storage stability at 4 °C: free lipase (j), the immobilized lipase prepared by method I (d), method II (N), and method III (.) (fiber diameter = 80–150 nm).
into a reactor cell and method I was employed for the preparation of the enzyme-immobilized fibrous membrane bioreactor. Because the condition of activation and immobilization was the same as described above, the amount of enzyme attached to the fibrous membrane after continuous immobilization process should be about 162 mg. Hydrolysis was carried out in the bioreactor under different flow rate varying from 0.2 to 2 mL/min at 42 °C. It was found that when the flow rate was as low as 0.23 mL/min, 3.6% of the hydrolytic conversion could be obtained in the bioreactor under the optimum conditions. The hydrolysis conversion decreased to 1.3% at a higher flow rate of 1.86 mL/min. The decrease of hydrolysis conversion with increasing the flow rate was mainly due to the decrease of contact time between enzyme and substrate. It can image that a relatively higher conversion can be obtained by increasing the weight of the fibrous membrane in the bioreactor. In addition, it is well known that the nanofibrous membrane possess much higher porosity together with high surface area and can produce relatively high flow rate under very low operating pressure. Thus, the small pressure drop and the high flow rate of the nanofibrous membrane are strongly desire properties by an ideal enzyme-immobilized membrane bioreactor because it is exactly the most important advantage of enzyme-immobilized nanofibrous membrane bioreactor over the traditional enzyme-immobilized membranes and fixed bed bioreactors. 4. Conclusion
20 0
2
4
6
8
10
Recycle number Fig. 1. Reusability of the immobilized lipases: the immobilized lipase prepared by method I (j), method II (d), and method III (N) (fiber diameter = 80–150 nm).
In this work, we thoroughly studied the comprehensive properties of immobilized lipases on PANCHEMA fibrous membranes. The observed lipase loading was dramatically depended on the fiber diameter of fibrous membrane and increased with the decreasing of the fiber diameter. Epoxy-activated PANCHEMA fibrous membrane seemed to be almost-ideal system for the enzyme immobilization
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and could preserve relatively high activity for the immobilized enzyme. The stabilities of the immobilized lipase were obviously improved compared with the free one. We think that, the lipase-immobilized nanofibrous membrane bioreactor possessing high enzyme loading and catalytic efficiency may have great potentials as biocatalysts for the applications in a wide range of reactions such as hydrolysis, alcoholysis, aminolysis, and transesterification. Acknowledgements Financial support from the National Natural Science Foundation of China for Distinguished Young Scholars (to Prof. Dr. Z.-K. Xu, grant no. 50625309) is gratefully acknowledged. Dr. X.-J. Huang thanks the financial supports from the National Postdoctoral Science Foundation of China (Grant 20060400337) and Postdoctoral Science & Research Foundation of Zhejiang Province (Grant 2006-bsh-17). References Amorim, R.V.S., Melo, E.S., Carneiro-da-Cunha, M.G., Ledingham, W.M., Campos-Takaki, G.M., 2003. Chitosan from Syncephalastrum racemosum used as a film support for lipase immobilization. Bioresour. Technol. 89, 35–39. Arica, M.Y., 2000. Epoxy-derived pHEMA membrane for use bioactive macromolecules immobilization: covalently bound urease in a continuous model system. J. Appl. Polym. Sci. 77, 2000–2008. Arica, M.Y., Bayramoglu, G., 2004. Polyethyleneimine-grafted poly (hydroxyethyl methacrylate-co-glycidyl methacrylate) membranes for reversible glucose oxidase immobilization. Biochem. Eng. J. 20, 73–77. Belkas, J.S., Munro, C.A., Shoichet, M.S., Johnston, M., Midha, R., 2005. Long-term in vivo biomechanical properties and biocompatibility of poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) nerve conduits. Biomaterials 26, 1741–1749. Bradford, M., 1976. A rapid and sensitive method for the quantition of microgram quantities of protein utilizing the principle of dyebinding. Anal. Biochem. 72, 248–254. Chiou, S.H., Wu, W.T., 2004. Immobilization of Candida rugosa lipase on chitosan with activation of the hydroxyl groups. Biomaterials 25, 197–204. Deng, H.T., Xu, Z.K., Huang, X.J., Wu, J., Seta, P., 2004. Adsorption and activity of Candida rugosa lipase on polypropylene hollow fiber membrane modified with phospholipid analogous polymers. Langmuir 20, 10168–10173. Deng, H.T., Xu, Z.K., Dai, Z.W., Wu, J., Seta, P., 2005. Immobilization of Candida rugosa lipase on polypropylene microfiltration membrane modified by glycopolymer: hydrolysis of olive oil in biphasic bioreactor. Enzyme Microb. Technol. 36, 996–1002. Drechsler, U., Fischer, N.O., Frankamp, B.L., Rotello, V.M., 2004. Highly efficient biocatalysts via covalent immobilization of Candida rugosa lipase on ethylene glycol-modified gold–silica nanocomposites. Adv. Mater. 16, 271–274. Dyal, A., Loos, K., Noto, M., Chang, S.W., Spagnoli, C., Shafi, K., Ulman, A., Cowman, M., Gross, R.A., 2003. Activity of Candida rugosa lipase immobilized on gamma-Fe2O3 magnetic nanoparticles. J. Am. Chem. Soc. 125, 1684–1685. Dzenis, Y., 2004. Spinning continuous fibers for nanotechnology. Science 304, 1917–1919. Fernandez-Lafuente, R., Armisen, P., Sabuquillo, P., Fernandez-Lorente, G., Guisan, J.M., 1998. Immobilization of lipases by selective adsorption on hydrophobic supports. Chem. Phys. Lipids 93, 185–197.
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X.-J. Huang et al. / Bioresource Technology 99 (2008) 5459–5465 Pahujani, S., Kanwar, S.S., Chauhan, G., Gupta, R., 2007. Glutaraldehyde activation of polymer Nylon-6 for lipase immobilization: enzyme characteristics and stability. Bioresour. Technol. Available online. Rejikumar, S., Devi, S., 1998. Preparation and characterization of urease bound on crosslinked poly(vinyl alcohol). J. Mol. Catal. B-Enzym. 4, 61–66. Serra, S., Brenna, E., Fuganti, C., Maggioni, F., 2003. Lipase-catalyzed resolution of p-menthan-3-ols monoterpenes: preparation of the enantiomer-enriched forms of menthol, isopulegol, trans- and cispiperitol, and cis-isopiperitenol. Tetrahedron-Asymmetr. 14, 3313–3319. Sharma, A., Chattopadhyay, S., 2000. Enantio-reversal in Candida rugosa lipase-catalyzed esterification of 3-hydroxybutyric acid. J. Mol. Catal. B-Enzyme 10, 531–534. Shukla, S.P., Devi, S., 2005. Covalent coupling of peroxidase to a copolymer of acrylamide (AAm)-2-hydroxyethyl methaacrylate (HEMA) and its use in phenol oxidation. Process Biochem. 40, 147–154. Tang, Z.X., Qian, J.Q., Shi, L.E., 2007. Characterizations of immobilized neutral lipase on chitosan nano-particles. Mater. Lett. 61, 37–40. Verger, R., 1997. Interfacial activation of lipases: facts and artifacts. Trends Biotechnol. 15, 32–38. Wang, L., Li, C.M., Ryan, A.J., Armes, S.P., 2006a. Synthesis and peptide-induced degradation of biocompatible fibers based on highly branched poly(2-hydroxyethyl methacrylate). Adv. Mater. 18, 1566–1570. Wang, Z.G., Xu, Z.K., Wan, L.S., Wu, J., Innocent, C., Seta, P., 2006b. Nanofibrous membranes containing carbon nanotubes: electrospun for redox enzyme immobilization. Macromol. Rapid Commun. 27, 516–521. Wang, Y.J., Hu, Y., Xu, H., Luo, G.S., Dai, Y.Y., 2007a. Immobilization of lipase with a special microstructure in composite hydrophilic CA/
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Covalent immobilization of lipase from Candida rugosa onto poly(acrylonitrile-co-2-hydroxyethyl methacrylate) electrospun fibrous membranes for potential bioreactor application Xiao-Jun Huang, An-Guo Yu, Zhi-Kang Xu * Institute of Polymer Science, Key Laboratory of Macromolecular Synthesis and Functionalization (Ministry of Education), Zhejiang University, Hangzhou 310027, PR China State Key Laboratory of Chemical Engineering, Zhejiang University, Hangzhou 310027, PR China Received 2 August 2007; received in revised form 2 November 2007; accepted 5 November 2007 Available online 14 January 2008
Abstract A simple way of fabricating enzymatic membrane reactor with high enzyme loading and activity retention from the conjugation between nanofibrous membrane and lipase was devised. Poly(acrylonitrile-co-2-hydroxyethyl methacrylate) (PANCHEMA) was electrospun into fibrous membrane and used as support for enzyme immobilization. The hydroxyl groups on the fibrous membrane surface were activated with epichlorohydrin, cyanuric chloride or p-benzoquinone, respectively. Lipase from Candida rugosa was covalently immobilized on these fibrous membranes. The resulted bioactive fibrous membranes were examined in catalytic efficiency and activity for hydrolysis. The observed enzyme loading on the fibrous membrane with fiber diameter of 80–150 nm was up to 1.6% (wt/wt), which was as thrice as that on the fibrous membrane with fiber diameter of 800–1000 nm. Activity retention for the immobilized lipase varied between 32.5% and 40.6% with the activation methods of hydroxyl groups. Stabilities of the immobilized lipase were obviously improved. In addition, continuous hydrolysis was carried out with an enzyme-immobilized fibrous membrane bioreactor and a steady hydrolysis conversion (3.6%) was obtained at a 0.23 mL/min flow rate under optimum condition. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Fibrous membrane; Electrospinning; Enzyme immobilization; Lipase; Membrane bioreactor
1. Introduction Lipases are the hydrolytic enzymes that can catalyze a wide range of reactions such as hydrolysis, alcoholysis, transesterifications, aminolysis and enantiomer resolution (Deng et al., 2005; Kose et al., 2002; Noureddini et al., 2005; Serra et al., 2003; Sharma and Chattopadhyay, 2000). These ubiquitous enzymes have found numerous commercial applications in the food, pharmaceutical, and detergent industries. For industrial applications, like most enzymes, lipases have often been immobilized onto insoluble or solid supports, which can offer better catalytic stabil-
*
Corresponding author. Fax: +86 571 8795 1773. E-mail address: [email protected] (Z.-K. Xu).
0960-8524/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.11.009
ity, feasible continuous operations, easy catalyst recycling, significant reduction in the operation costs and simple product purification (Amorim et al., 2003; Pahujani et al., 2007; Wang et al., 2007a,b; Yu et al., 2006). A promising property of lipases was the interfacial activation that could induce important conformational rearrangements yielding the ‘open state’ of lipases and improve their activity during immobilization (Fernandez-Lorente et al., 2006; Grazu et al., 2005; Mateo et al., 2007, 2003; Verger, 1997). However, in many cases the non-orientable immobilized lipases lost catalytic activity and efficiency to some extent. The properties and microstructure of supports have great impact on the performance of immobilized enzymes. It is, thus, important that the choice of support materials and immobilization methods for enzyme should be well justified.
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To achieve high enzyme loading and catalytic efficiency for large-scale operation and application, supports with high surface to volume ratio, an inherent characteristic of nanoscale materials, are often desirable. Therefore, in recent years, nanostructured supports such as mesoporous materials, nanoparticles, and nanofibers have been widely studied for enzyme immobilization (Drechsler et al., 2004; Dyal et al., 2003; Huang et al., 2006; Tang et al., 2007; Wang et al., 2006a,b; Ye et al., 2006a,b). Among these, it appears to us that electrospun nanofibrous membranes are the promising supports for enzyme immobilization due to the advantages including: (1) large specific surface providing relatively high quantity of enzyme loading per unit mass, (2) ultrafine fiber and high porosity entrusting the accessibility of active sites and low diffusion resistance necessary for high reaction rate and conversion, and (3) easy recoverability from reaction media or applicability for continuous operations in comparison with nanoparticles (Herricks et al., 2005; Huang et al., 2006; Jia et al., 2002; Lee et al., 2005; Nair et al., 2007; Wang et al., 2006a,b; Xie and Hsieh, 2003; Ye et al., 2006a,b). To maximize the enzyme activity, it is very important for the supports to provide a biocompatibility and/or inert environment for enzyme protein. A biofriendly interface on the support surface for enzyme immobilization may reduce some non-biospecific enzyme–support interaction and protein denaturation, create a specific microenvironment for the enzyme, and thus benefit the enzyme activity (Deng et al., 2004; Girard-Egrot et al., 2005; Gotoh et al., 1998; Huang et al., 2005; Ito et al., 2006; Mukhopadhyay et al., 2003). Poly(2-hydroxyethyl methacrylate) (pHEMA) and HEMA-based co-polymers are non-toxic and biocompatible synthetic materials with adequate mechanical strength for most biomedical applications (Belkas et al., 2005; Wang et al., 2006a,b; Yu and Ober, 2003). Methacrylate-based materials possessing moderate hydrophobic interface could benefit interfacial activation of lipase and thus promote a dramatic activation of lipases after their immobilization (Fernandez-Lafuente et al., 1998; Fernandez-Lorente et al., 2006; Mateo et al., 2007; Verger, 1997). In addition, it contains hydroxyl groups that act as attachment sites for bioactive species after activation. Therefore, in recent years HEMA-based polymers had been prepared in different forms (such as gels, beads and membranes) as the supports for enzyme immobilization (Arica and Bayramoglu, 2004; Gotoh et al., 1998; Liu and Chang, 2007; Shukla and Devi, 2005; Xu et al., 2006). In these cases, each had its own advantages and disadvantages. Some changes were observed in enzymatic activity, optimum pH and temperature, affinity to substrate, and stability. The extent of these changes depended on the nature of enzyme, type of support, and the immobilization condition. Although some properties of the immobilized enzyme were quite promising, the enzyme loading and catalytic efficiency were not very favorable. In this work, poly(acrylonitrile-co-2-methacryloyloxyethyl) (PANCHEMA) was synthesized and electrospun
into fibrous membranes. The prepared PANCHEMA fibrous membranes, which combine the advantages of HEMA and electrospinning technique, are expected to be attractive supports for enzyme immobilization. Lipase from Candida rugosa was covalently immobilized on these fibrous membranes using epichlorohydrin, cyanuric chloride or p-benzoquinone as covalent reagent, respectively. The performances such as enzyme loading, activity, reusability and storage stability of the immobilized lipase were described. In addition, the application of lipase-immobilized fibrous membrane bioreactor to the hydrolysis of p-nitrophenyl palmitate (p-NPP) was presented. 2. Experimental 2.1. Materials PANCHEMA was synthesized by water phase precipitation co-polymerization in our laboratory (Huang et al., 2005). The HEMA content in the co-polymer calculated from 1H NMR spectrum was 9.3 mol%. N,N0 -Dimethyl formamide (DMF) was purified by vacuum distillation before use. Lipase (from Candida rugosa), Bradford reagent, bovine serum albumin (BSA, molecular mass: 67,000 Da) and p-NPP were purchased from Sigma and used as received. All other reagents were analytical grade and used without further purification. 2.2. Preparation of PANCHEMA fibrous membranes by electrospinning To fabricate non-woven fibrous membranes using the electrospinning process, PANCHEMA was dissolved in DMF at room temperature with gentle stirring for 12 h to form homogeneous solution. After air bubbles were removed completely, each solution was placed in a syringe (50 mL) bearing an 1 mm inner diameter metal needle which was connected with a high voltage power supply (GDW-a, Tianjin Dongwen High Voltage Power Supply Plant, China). The grounded counter electrode was connected to an aluminum foil collector. Typically, electrospinning was performed at 10 kV voltage, 15 cm distance between the needle tip and the collector. The solution flow rate was controlled by a microinfusion pump (WZ-50C2, Zhejiang University Medical Instrument Co., LTD, China) to maintain at 2 mL/h from the needle outlet. It usually took one hour to obtain a sufficiently thick sample that could be detached from the aluminum foil collector. Fibrous membrane on the aluminum foil was dried under vacuum at 60 °C before it was detached. The fiber diameter was examined with field emission scanning electron microscope (FESEM). 2.3. Enzyme immobilization Different activation procedures were adopted for the immobilization of lipase on the PANCHEMA fibrous
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membranes (Arica, 2000; Horak et al., 1999; Rejikumar and Devi, 1998; Shukla and Devi, 2005; Xu et al., 2006). The activation of hydroxyl groups in PANCHEMA for covalent immobilization was achieved by reaction with epichlorohydrin, cyanuric chloride or p-benzoquinone, respectively. The activation and immobilization were carried out as the following processes. To identify the best method for the immobilization of lipase, three different protocols were tested as follow. 2.3.1. Method I In the process of activation through epichlorohydrin approx, 5 mg fibrous membrane was immersed in 5 mL epichlorohydrin and shaken at 25 °C for 3 h. The activated fibrous membrane was washed several times with acetone to remove excess epichlorohydrin, and then washed with phosphate buffer solution (PBS, 0.05 M, pH 7.0) before enzyme immobilization. 2.3.2. Method II For the activation of PANCHEMA fibrous membrane through cyanuric chloride, 5 mg fibrous membrane was treated with 0.25 g cyanuric chloride in 5 mL dioxane at 25 °C for 30 min. Then it was treated with 10 mL water and 10 mL 20% acetic acid for 30 min. The resulting activated fibrous membrane was washed with water, acetone and phosphate buffer solution (PBS, 0.05 M, pH 7.0) before enzyme immobilization. 2.3.3. Method III During the activation of PANCHEMA fibrous membrane through p-benzoquinone, 5 mg fibrous membrane was treated with 0.05 g p-benzoquinone dissolved in 5 mL 20% ethanol, and phosphate buffer solution (PBS, 0.05 M, pH 7.0) for 2 h at room temperature. The treated fibrous membrane was washed successively with 20% ethanol and water until the filtrate was free from p-benzoquinone and then with 1 M NaCl and phosphate buffer solution (PBS, 0.05 M, pH 7.0) before immobilization. Lipase solution (5.0 mg/mL) was prepared by solving appropriate amount of lipase powder in phosphate buffer (0.05 M, pH 7.0). The activated fibrous membrane was submerged in 5 mL lipase solution in a vertical orientation and shaken gently in a water bath at 30 °C for 3 h. Then, the fibrous membrane was taken out and rinsed with buffer until no soluble protein was detectable in washings. Protein concentration in solution was determined with Coomassie Brilliant Blue reagent following Bradford’s method (Bradford, 1976). BSA was used as standard to construct calibration curve. Amount of immobilized protein on the fibrous membrane was calculated from the protein mass balance among the initial and final lipase solutions and washings. Enzyme loading on the fibrous membrane was defined as the amount of protein (mg) per gram of the membrane. Each reported value was the mean of three experiments at least, and the standard deviation was within ca. ±5%.
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2.4. Activity assay of free and immobilized lipases Activity for the immobilized lipase in aqueous medium was determined according to the method reported previously (Huang et al., 2006; Chiou and Wu, 2004). In the standard condition, a reaction mixture was composed of 1.0 mL ethanol containing 14.4 mM p-NPP and 1.0 mL PBS in an Erlenmeyer flask. The reaction was started by immersing 5 mg immobilized lipase preparation in the flask. The mixture was incubated at a certain temperature under reciprocal agitation at 120 strokes per minute. After 5 min of reaction, the reaction was terminated by adding 2.0 mL 0.5 M Na2CO3 followed by centrifuging for 10 min (8400g). 0.50 mL supernatant was diluted 10 folds with de-ionized water, and measured at 410 nm in an UV–vis spectrophotometer (UV-1601, Shimadzu, Japan) against a blank without enzyme and treated in parallel. The reaction rate was calculated from the slope of the absorbance versus the time curve. Molar extinction coefficient of 14.5 103 M1 cm1 for p-nitrophenol (p-NP), which was determined from the absorbance of standard solutions of p-NP in the reaction medium, was used. One enzyme unit is the amount of biocatalyst liberating 1.0 lmol p-NP/min in the condition. Specific activity was defined as the number of lipase unit per milligram of protein and it was expressed as lmol p-NP min1 mg protein1. Activity retention value was the ratio of specific activity of immobilized lipase with that of free one. Each data was the average of three parallel experiments at least. The standard error for the result was below 5% of the average value. 2.5. Stability measurements For the reusability measurement, 5 mg immobilized lipase and 2.0 mL of substrate were added in an Erlenmeyer flask. After 5 min reaction, the released p-NP was measured as in activity assay. The immobilized lipase was taken out from the flask and washed with PBS to remove any residual substrate on the nanofibrous membrane. It was then reintroduced into fresh substrate and the same measurement was repeated 10 times. In order to prevent the influence of storage time on the enzyme activity, there was just 30 s between two following cycles and all measurements were carried out in one day. The storage stability was determined as follows. Free and immobilized lipases were respectively stored in phosphate buffer (PBS, 0.05 M, pH 7.0) at 4 °C for a month. Parts of them were periodically withdrawn for activity assay. The residual activities were then determined as described above. 2.6. Enzyme-immobilized fibrous membrane bioreactor A simple enzyme-immobilized membrane bioreactor was set up in our lab. PANCHEMA fibrous membrane (fiber diameter = 80–150 nm, dry membrane weight = 10 mg)
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was directly collected on a round wire mesh with area of 9 10 cm2. The round-shaped PANCHEMA fibrous membrane with the wire mesh was installed into a dead-end reactor cell. A peristaltic pump was used between the substrate reservoir and the reactor cell for better control of feed flow rate. Epichlorohydrin, washing solutions and lipase solution were successively circulated in the reactor cell by the peristaltic pump. The condition of activation and enzyme immobilization was the same as described above. The temperature of substrate reservoir or reactor cell was controlled to ±0.5 °C by circulation water through the reservoir or reactor jacket. p-NPP (7.2 mM) substrate solution was continuously flowed through the membrane bioreactor using the peristaltic pump. The continuous hydrolysis process was conducted at 42 °C for 10 min. The hydrolytic products, p-NP, leaving the reactor cell was collected and measured at 410 nm in an UV–vis spectrophotometer against a blank without enzyme and treated in parallel. It was measured periodically and used for evaluating the activity and the efficiency of reactor for continuous operation. The hydrolysis conversion was defined as the fraction of the amount of hydrolytic p-NP compared with the total amount of p-NPP. 3. Results and discussion 3.1. Preparation of fibrous membranes from PANCHEMA Electrospinning has been recognized as an effective technique for the preparation of polymeric fibers with diameter from several micrometers down to tens of nanometers (Dzenis, 2004). Among various polymers, acrylonitrilebased homo- and co-polymers were most recently fabricated into nanofibers with reinforcing, superhydrophobic and/or catalytic properties (Ge et al., 2004; Zhang and Hsieh, 2006; Zussman et al., 2006). Several factors such as polymer molecular weight, concentration, electric field strength, and solvent properties can significantly influence the size and morphology of the electrospun fibers. In this work, PANCHEMA with a molecular weight of 100,000–110,000 g/mol was synthesized by a water phase precipitation co-polymerization process and then electrospun into non-woven fibrous membranes. It was found that the fiber diameter of the resulted fibrous membranes could be controlled by varying the concentration of PANCHMEA solution. Almost homogenous networks (fibrous membranes) with fiber diameter ranging from 80 nm to 1000 nm were obtained.
3.2. Lipase immobilization on the electrospun fibrous membranes For large-scale application, high enzyme loading is always desired to enhance the efficiency of biotransformation and to reduce the size of bioreactor. Enzyme loading on the electrospun fibrous membranes is expected to be high considering the large surface area/mass ratio. In the present work, enzyme immobilization through covalent coupling was achieved using activating agent epichlorohydrin, cyanuric chloride or p-benzoquinone, respectively, for the activation of hydroxyl groups on the surfaces of PANCHEMA fibrous membranes. To enhance the enzyme loading, the activation of hydroxyl groups followed by enzyme coupling was carried out at the optimized conditions. It was found that the enzyme loading increased as the fiber diameter decreased in spite of the different activated methods. An enzyme loading of 1.55–1.65 wt.% was observed on fibers of 80–150 nm, while it was about 0.55–0.60 wt% on fibers of 800–1000 nm. The increase of a factor of 3 in the enzyme loading was attributed to the decrease of the fiber diameter into nano-scale, which can increase the outer surface area (when the density of the PANCHEMA fiber is taken as 1.18 g/cm3, the fiber diameter of 80–150 nm have a surface area of 11.3–21.2 m2 per 1 gram, while that of 800–1000 nm with a surface area of 1.69–2.12 m2 per 1 gram) and the exposure of the hydroxyl groups on nanofibers available for enzyme coupling. In this regard, it could clearly demonstrate that increasing the outer surface area of the supports is the efficient way to enhance the enzyme loading. 3.3. Activity of immobilized lipase Activities for lipases immobilized on the PANCHEMA fibrous membranes through different activation methods are compared in Table 1. It can be seen that the total enzyme loading was nearly equal for the immobilized lipase prepared by the three methods, while the activity retention of the immobilized lipase prepared by method I (40.6 %) was slightly higher than that prepared by method II (36.4 %) and by method III (32.5%). Compared with cyanuric chloride and p-benzoquinone, epichlorohydrin can covalently link with available amino, hydroxyl, or sulfhydryl groups of enzyme. The random linkage with these groups of enzyme could reduce the chance of the directly covalent bonding between the active site of enzyme and the support surface. In addition, it can form a flexible and little longer spacer
Table 1 Immobilization of lipase on the electrospun poly(acrylonitrile-co-2-hydroxyethyl methacrylate) fibrous membranes Method of activation
Fiber diameter (nm)
Loading enzyme (mg/g)
Specific activity (U/mg)a
Activity retention (%)
Epichlorohydrin Cyanuric chloride Benzoquinone
80–150 80–150 80–150
16.2 ± 1.1 16.5 ± 1.3 15.5 ± 1.2
17.0 15.3 12.1
40.6 ± 0.6 36.4 ± 0.4 32.5 ± 0.8
a
Specific activity for free lipase is 42 U/mg.
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3.4. Enzyme stability studies Reusability and storage stability are of considerable importance for various applications of biocatalysts in a commercial point of view. An increased stability can make the immobilized enzyme more advantageous than its free one. To investigate the reusability, the enzyme-immobilized fibrous membrane was washed with PBS (0.05 M, pH 7.0) after one catalysis run and reintroduced into a fresh p-NPP solution for another hydrolysis at 42 °C. Fig. 1 shows the effect of repeated use on the activity of the immobilized lipases. It can be seen that the activities of the immobilized lipases decayed obviously with recycled uses. After 10 reuses, the lipase immobilized by the three activation methods retained over 30 % residual activity. The activity loss could be related to the inactivation of the enzyme caused by the denaturation of the protein. The free and immobilized lipases were stored in phosphate buffer (0.05 M, pH 7.0) at 4 °C and measured for a period of 30 days. The storage stability of the immobilized lipases compared with that of the free one is shown in Fig. 2. It can be seen that the free lipase lost all its initial activity within 30 days. While the residual activity of lipase immobilized on the PANCHEMA fibrous membranes retained over 60% of its initial activity during the same period. These results indicated that the immobilization procedures had considerably improved the storage stability. 3.5. Enzyme-immobilized fibrous membrane bioreactor PANCHEMA (10 mg) fibrous membrane (fiber diameter = 80–150 nm, membrane area = 9.10 cm2) was installed
Residual activity (%)
100
80
60
40
100
Residual activity (%)
arm between enzyme and support comparing with cyanuric chloride and p-benzoquinone. This resulted both in a reduction in denaturation of the enzyme protein and in enhanced substrate accessibility to the enzyme’s active site.
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80
60
40
20
0 0
5
10
15
20
25
30
Storage time (Days) Fig. 2. Storage stability at 4 °C: free lipase (j), the immobilized lipase prepared by method I (d), method II (N), and method III (.) (fiber diameter = 80–150 nm).
into a reactor cell and method I was employed for the preparation of the enzyme-immobilized fibrous membrane bioreactor. Because the condition of activation and immobilization was the same as described above, the amount of enzyme attached to the fibrous membrane after continuous immobilization process should be about 162 mg. Hydrolysis was carried out in the bioreactor under different flow rate varying from 0.2 to 2 mL/min at 42 °C. It was found that when the flow rate was as low as 0.23 mL/min, 3.6% of the hydrolytic conversion could be obtained in the bioreactor under the optimum conditions. The hydrolysis conversion decreased to 1.3% at a higher flow rate of 1.86 mL/min. The decrease of hydrolysis conversion with increasing the flow rate was mainly due to the decrease of contact time between enzyme and substrate. It can image that a relatively higher conversion can be obtained by increasing the weight of the fibrous membrane in the bioreactor. In addition, it is well known that the nanofibrous membrane possess much higher porosity together with high surface area and can produce relatively high flow rate under very low operating pressure. Thus, the small pressure drop and the high flow rate of the nanofibrous membrane are strongly desire properties by an ideal enzyme-immobilized membrane bioreactor because it is exactly the most important advantage of enzyme-immobilized nanofibrous membrane bioreactor over the traditional enzyme-immobilized membranes and fixed bed bioreactors. 4. Conclusion
20 0
2
4
6
8
10
Recycle number Fig. 1. Reusability of the immobilized lipases: the immobilized lipase prepared by method I (j), method II (d), and method III (N) (fiber diameter = 80–150 nm).
In this work, we thoroughly studied the comprehensive properties of immobilized lipases on PANCHEMA fibrous membranes. The observed lipase loading was dramatically depended on the fiber diameter of fibrous membrane and increased with the decreasing of the fiber diameter. Epoxy-activated PANCHEMA fibrous membrane seemed to be almost-ideal system for the enzyme immobilization
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and could preserve relatively high activity for the immobilized enzyme. The stabilities of the immobilized lipase were obviously improved compared with the free one. We think that, the lipase-immobilized nanofibrous membrane bioreactor possessing high enzyme loading and catalytic efficiency may have great potentials as biocatalysts for the applications in a wide range of reactions such as hydrolysis, alcoholysis, aminolysis, and transesterification. Acknowledgements Financial support from the National Natural Science Foundation of China for Distinguished Young Scholars (to Prof. Dr. Z.-K. Xu, grant no. 50625309) is gratefully acknowledged. Dr. X.-J. Huang thanks the financial supports from the National Postdoctoral Science Foundation of China (Grant 20060400337) and Postdoctoral Science & Research Foundation of Zhejiang Province (Grant 2006-bsh-17). References Amorim, R.V.S., Melo, E.S., Carneiro-da-Cunha, M.G., Ledingham, W.M., Campos-Takaki, G.M., 2003. Chitosan from Syncephalastrum racemosum used as a film support for lipase immobilization. Bioresour. Technol. 89, 35–39. Arica, M.Y., 2000. Epoxy-derived pHEMA membrane for use bioactive macromolecules immobilization: covalently bound urease in a continuous model system. J. Appl. Polym. Sci. 77, 2000–2008. Arica, M.Y., Bayramoglu, G., 2004. Polyethyleneimine-grafted poly (hydroxyethyl methacrylate-co-glycidyl methacrylate) membranes for reversible glucose oxidase immobilization. Biochem. Eng. J. 20, 73–77. Belkas, J.S., Munro, C.A., Shoichet, M.S., Johnston, M., Midha, R., 2005. Long-term in vivo biomechanical properties and biocompatibility of poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) nerve conduits. Biomaterials 26, 1741–1749. Bradford, M., 1976. A rapid and sensitive method for the quantition of microgram quantities of protein utilizing the principle of dyebinding. Anal. Biochem. 72, 248–254. Chiou, S.H., Wu, W.T., 2004. Immobilization of Candida rugosa lipase on chitosan with activation of the hydroxyl groups. Biomaterials 25, 197–204. Deng, H.T., Xu, Z.K., Huang, X.J., Wu, J., Seta, P., 2004. Adsorption and activity of Candida rugosa lipase on polypropylene hollow fiber membrane modified with phospholipid analogous polymers. Langmuir 20, 10168–10173. Deng, H.T., Xu, Z.K., Dai, Z.W., Wu, J., Seta, P., 2005. Immobilization of Candida rugosa lipase on polypropylene microfiltration membrane modified by glycopolymer: hydrolysis of olive oil in biphasic bioreactor. Enzyme Microb. Technol. 36, 996–1002. Drechsler, U., Fischer, N.O., Frankamp, B.L., Rotello, V.M., 2004. Highly efficient biocatalysts via covalent immobilization of Candida rugosa lipase on ethylene glycol-modified gold–silica nanocomposites. Adv. Mater. 16, 271–274. Dyal, A., Loos, K., Noto, M., Chang, S.W., Spagnoli, C., Shafi, K., Ulman, A., Cowman, M., Gross, R.A., 2003. Activity of Candida rugosa lipase immobilized on gamma-Fe2O3 magnetic nanoparticles. J. Am. Chem. Soc. 125, 1684–1685. Dzenis, Y., 2004. Spinning continuous fibers for nanotechnology. Science 304, 1917–1919. Fernandez-Lafuente, R., Armisen, P., Sabuquillo, P., Fernandez-Lorente, G., Guisan, J.M., 1998. Immobilization of lipases by selective adsorption on hydrophobic supports. Chem. Phys. Lipids 93, 185–197.
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