Nanofibrous poly(acrylonitrile-co-maleic acid) membranes functionalized with gelatin and chitosan for lipase immobilization

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Biomaterials 27 (2006) 4169–4176 www.elsevier.com/locate/biomaterials

Nanofibrous poly(acrylonitrile-co-maleic acid) membranes functionalized with gelatin and chitosan for lipase immobilization Peng Yea,b, Zhi-Kang Xua,b,, Jian Wuc, Christophe Innocentd, Patrick Setad a

Institute of Polymer Science, Zhejiang University, Hangzhou 310027, PR China Key Laboratory of Macromolecule Synthesis and Functionalization, Ministry of Education; Hangzhou 310027, PR China c Department of Chemistry, Zhejiang University, Hangzhou 310027, PR China d Institute of Europe´e des Membranes, UMR CNRS no.5635, 34293 Montpellier Cedex 05, France

b

Received 29 December 2005; accepted 15 March 2006 Available online 3 April 2006

Abstract Nanofibrous membranes with an average diameter of 100 and 180 nm were fabricated from poly(acrylonitrile-co-maleic acid) (PANCMA) by the electrospinning process. These nanofibrous membranes contain reactive groups which can be used to covalently immobilize biomacromolecules. Two natural macromolecules, chitosan and gelatin, were tethered on these nanofibrous membranes to fabricate dual-layer biomimetic supports for enzyme immobilization in the presence of 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxyl succinimide (NHS). Lipase from Candida rugosa was then immobilized on these dual-layer biomimetic supports using glutaraldehyde (GA), and on the nascent PANCMA fibrous membrane using EDC/NHS as coupling agent, respectively. The properties of the immobilized lipases were assayed. It was found that there is an increase of the activity retention of the immobilized lipase on the chitosan-modified nanofibrous membrane (45.671.8%) and on the gelatin-modified one (49.771.8%), compared to that on the nascent one (37.671.8%). The kinetic parameters of the free and immobilized lipases, Km and Vmax, were also assayed. In comparison with the immobilized lipase on the nascent nanofibrous membrane, there is an increase of the Vmax value for the immobilized lipases on the chitosan- and gelatin-modified nanofibrous membranes. Results also indicate that the pH and thermal stabilities of lipases increase upon immobilization. The residual activities of the immobilized lipases are 55% on the chitosan-modified nanofibrous membrane and 60% on the gelatin-modified one, after 10 uses. r 2006 Elsevier Ltd. All rights reserved. Keywords: Electrospinning; Nanofibrous membranes; Biomacromolecules; Enzyme immobilization; Lipase

1. Introduction As biocatalysts, enzymes exhibit a number of advantages such as high level of catalytic efficiency and high degree of selectivities, including chemical-, regio- and stero-selectivity [1–3]. However, there exist various practical problems in the enzyme applications, for example, high cost and instability. To overcome these problems, immobilizing enzyme onto insoluble or solid supports has been regarded as a useful strategy to improve their thermal and operational stability and recoverability. Corresponding author. Institute of Polymer Science, Zhejiang University, Hangzhou 310027, PR China. Fax: +86 571 8795 1773. E-mail address: [email protected] (Z.-K. Xu).

0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.03.027

In recent years, there is a trend to use nanostructured materials as supports for enzyme immobilization, since the large surface area to volume ratio of nanosize materials can effectively improve the enzyme loading per unit volume of support and the catalytic efficiency of the immobilized enzyme. Both nanoparticles and nanofibers were explored for this purpose [4–7]. In the cases of nanoparticles, nevertheless, their dispersion in reaction solution and the subsequent recovery for reuse are often difficult. On the contrary, nanofibers can be easily recovered from reaction media and be applied for continuous operations. Jia et al. [6] immobilized a-chymotrypsin on the surface of polystyrene nanofibers (120 nm) produced by electrospinning and showed that the enzyme loading was 1.4% (wt/wt). Jiang et al. [7] studied the feasibility of lysozyme immobilization

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by electrospinning the enzyme-containing dextran solution into nanofibrous membranes (200–400 nm). On the other hand, for enzyme immobilization, the biocompatibility of support is also one important requirement [8–11], as the biocompatible surface can reduce some non-biospecific enzyme-support interactions, create a specific microenvironment for the enzymes and thus benefit the enzyme activity [12]. To increase the biocompatibility of the support, various surface modification protocols have often been used to introduce a biofriendly interface on the support surface, such as coating, adsorption, self-assembly and graft polymerization. Among these methods, it is relatively easy and effective to directly tether natural macromolecules on the support surface to form a biomimetic layer for enzyme immobilization. In fact, this protocol has been used in tissue engineering recently [13,14]. In our previous work [15], a dual-layer biomimetic membrane support had been prepared for enzyme immobilization, by tethering chitosan on the poly(acrylonitrile-comaleic acid) (PANCMA) hollow fiber ultrafiltration membrane surface with reactive carboxyl groups. In this work, to further raise enzyme loading on the support and to reduce the diffusion resistance for the immobilized enzyme, PANCMA was fabricated into nanofibrous membranes by the electrospinning process. Two natural macromolecules, chitosan and gelatin, were then tethered on the PANCMA nanofibrous membrane surface to produce novel dual-layer biomimetic membrane supports with nanostructure, respectively. Chitosan (D-glucosamine), which is present in variety of sources and commercially obtained from wastes generated by fishing industry, is considered to be a suitable support for enzyme immobilization since it is nontoxic and user-friendly, and has protein affinity [16,17]. Gelatin (a protein) with characteristics of easily available and low in cost is derived from collagen [18]. It shows biological properties that is almost identical with those of collagen. These features make gelatin widely used in a variety of biomedical applications. Since these two biomaterials containing reactive groups (amino groups) have been successfully used as enzyme immobilization supports, it is reasonable to choose them for the formation of biomimetic layers on the PANCMA nanofibrous membrane surface for enzyme immobilization. Lipase, one kind of the most utility enzyme [19–22], was immobilized on these nanofibrous membranes with GA and 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxyl succinimide (NHS) as coupling agent, respectively. The effect of immobilization process on the activity, pH and thermal stabilities, kinetic parameters and reusability of the enzyme was investigated. 2. Experimental 2.1. Materials Lipase (from Candida rugosa), Bradford reagent, bovine serum albumin (BSA, molecular mass: 67,000 Da), EDC, NHS, p-nitrophenyl palmitate (p-NPP) and 2-morpholino- ethane sulfonic acid (MES) were purchased

from Sigma and used as received. Gelatin was purchased from Gouyao Chemical Reagent Co., LTD., China. All other chemicals are of analytical grade and used without further purification. PANCMA hollow fiber ultrafiltration membrane was fabricated in our lab according to the reported process [23–25]. The outer and the inner diameters of the hollow fiber ultrafiltration membrane are 850 and 545 mm, respectively, with water flux of 146 L/m2 h atm, BSA rejection of 96%, and breaking strength of 135 N/cm2. The molar fraction of maleic acid in the copolymer is 7.5%.

2.2. Preparation of the PANCMA nanofibrous membranes by electrospinning PANCMA was dissolved in dimethylformamide (DMF) at room temperature with gentle stirring for 12 h to form a homogeneous solution (4 wt%). After air bubbles were removed completely, the solution was placed in a plastic syringe (50 mL) bearing a 1 mm inner diameter metal needle, which was connected with a high-voltage power supply (DW-P3031AC, Tianjin Dongwen High-voltage Power Supply Plant, China). The grounded counter electrode was connected to the tinfoil collector. Typically, electrospinning was performed at 8, 10, 12 and 14 kV. The distance between the needle tip and the collector was 180 mm. The flow rate of the solution was controlled by a syringe pump (WZ-50C2, Zhejiang University Medical Instrument Co., LTD, China) to maintain at 0.3 mL/h from the needle outlet. It usually took 5 h to obtain a sufficiently thick membrane that can be detached from the tinfoil collector. The membrane on tinfoil was dried under vacuum at 60 1C before it was detached. The morphology of the electrospun nanofibrous membrane was examined under a field emission scanning electron microscope (FESEM, FEI, SIRION-100, USA).

2.3. Preparation of low molecular weight gelatin A sample of 7.8 g gelatin with high molecular weight (HMW gelatin with molecular mass of 160,000 Da) was stirred for 1 h and dissolved absolutely in 100 mL de-ionized water to form a solution (78 mg/mL) at 80 1C. Then, 0.6 g citric acid monohydrate was added to the solution to modulate the pH value of the solution to 3.0 and stirred for 4 h at 80 1C. After this, 0.1 M NaOH solution was added to modulate the pH value of the solution to 7.0 to terminate the hydrolysis. The product was collected with rotatory evaporator at 60 1C, washed several times with de-ionized water and dried in vacuum oven at room temperature. Following this process, the low molecular weight gelatin (LMW gelatin with and molecular mass of 7000 Da) was obtained. The Mw was characterized with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). The low molecular weight chitosan was prepared according to the reported process [15].

2.4. Preparation of the dual-layer biomimetic membranes An appropriate amount of the nascent nanofibrous membrane was thoroughly washed with de-ionized water, and then rinsed with acetic acid buffer solution (50 mM, pH 5.0). After this the membrane was submerged into the low molecular weight chitosan or gelatin solution (15 mg/mL in acetic acid buffer solution, 50 mM, pH 5.0) in the presence of EDC/NHS (10 mg/mL, the molar ratio of EDC to NHS ¼ 1:1) and shook gently for 24 h at room temperature. Finally, the modified membrane was taken out, washed several times with de-ionized water to remove free chitosan or gelatin and then dried in atmosphere. Following this process, the chitosanmodified or gelatin-modified nanofibrous membrane was obtained.

2.5. Immobilization of lipase on the dual-layer biomimetic membranes Schematic representatives for the preparation of supports and the enzyme immobilization are shown in Fig. 1. Lipase was immobilized onto

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Fig. 1. Schematic representative for the preparation of the support and enzyme immobilization. the chitosan- or gelatin-modified membranes by a typical glutaraldehyde (GA) activation procedure. An appropriate amount of the membrane was submerged into a GA solution composed of 25% GA water solution and de-ionized water (5%, v/v), and shook for 12 h at room temperature. The activated support was taken out, washed several times with de-ionized water to remove excess GA, then washed with phosphate buffer solution (PBS, 50 mM, pH 6.0) and submerged into an enzyme solution (2 mg/mL in PBS, 50 mM, pH 6.0). The immobilization process was carried out at 4 1C in a shaking water bath for 3 h. After this, the membrane was taken out, thoroughly rinsed with PBS (50 mM, pH 6) and then immersed into a sodium borohydride solution (5 mg/mL) dissolved in a pH 8.0 borate buffer at 5 1C for 10 min. Finally, the membrane was taken out and thoroughly rinsed with de-ionized water. The amount of immobilized protein on the support was determined by measuring the initial and final concentrations of protein within the enzyme solutions and washings using Coomassie Brilliant Blue reagent following Bradford’s method [26]. BSA was used as standard to construct a calibration curve. The immobilization capacity of the protein on the support was defined as the amount of protein (mg) per gram of the support.

PBS (50 mM, pH 7.5) in an Erlenmeyer flask. The reaction was started by addition of 0.10 mL free lipase preparation (or 25 mg immobilized lipase preparation). The mixture was incubated at 37 1C under reciprocal agitation at 120 strokes per minute. After 5 min of reaction, agitation was stopped, and then the reaction was terminated by adding 2.0 mL of 0.5 N Na2CO3 followed by centrifuging for 10 min (10,000 rpm). The supernatant of 0.50 mL 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 pnitrophenol (p-NP), which was determined from the absorbance of standard solutions of p-NP in the reaction medium, was used. One enzyme unit was the amount of biocatalyst liberating 1.0 mmol of p-NP per minute in these conditions. Activity was defined as the number of lipase unit per gram of support. Specific activity was defined as the number of enzyme unit per milligram of protein. Activity retention was defined as the ratio of the activity of the amount of the enzyme coupled on the hollow fiber membrane to the activity of the same amount of the free one.

2.6. Immobilization of lipase on the nascent PANCMA membrane

2.8. pH and thermal stability measurements

Lipase was immobilized onto the nascent PANCMA nanofibrous membrane by an EDC/NHS activation procedure. An appropriate amount of the membrane was thoroughly washed with de-ionized water, and then rinsed with MES buffer (50 mM, pH 6.0). After this, the pretreated membrane was submerged into the EDC/NHS solution (20 mg/mL in MES buffer, 50 mM, pH 6.0, the molar ratio of EDC to NHS ¼ 1:1) and shaken gently for 6 h at room temperature. The activated membrane was taken out, washed several times with PBS (50 mM, pH 5.5) and submerged into an enzyme solution (2 mg/mL in PBS, pH 5.5). The immobilization process was carried out at 4 1C in a shaking water bath for 1 h. Finally, the membranes were taken out, thoroughly rinsed with PBS (50 mM, pH 5.5) and then rinsed with de-ionized water. The amount of immobilized protein on the membrane was determined as described above.

The pH stabilities of the free and immobilized lipases were assayed by immersing them in PBS (50 mM) in the pH range of 3–10 for 1 h at 25 1C and then determining their activities. The thermal stabilities of the free and immobilized lipases were assayed by immersing them in PBS (50 mM, pH 7.0) for 2 h at 50 1C and periodically determining their activities.

2.7. Activity assay of free and immobilized lipases The reaction rate of the free and immobilized lipase preparations was determined according to the method reported by Chiou et al. [27] with only minor modification. In the standard conditions, the reaction mixture was composed of 1.0 mL ethanol containing 14.4 mM p-NPP and 1.0 mL

3. Results and discussion 3.1. Fabrication of the PANCMA Nanofibrous Membranes Electrospinning has been proven effective in preparing polymeric fibers with diameter between tens of nanometers and several micrometers [28–32]. Several parameters, including viscosity, surface tension, net charge density and voltage, can significantly impact the size and morphology of the fibrous material. In fact, one of the most significant parameters influencing the fiber morphology is

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Fig. 2. SEM photographs of the electrospun PANCMA fibrous membranes. Voltages are (a) 8 kV, (b) 10 kV, (c) 12 kV and (d) 14 kV.

the applied voltage. To compare the effect of the voltage on the formation of electrospun nanofiber membrane, the PANCMA concentration was fixed at 4 wt%, the solution flow rate was set at 3 mL/h and the distance between the electrode and the collecting plate was maintained 180 mm for all of the samples, when the voltage was changed from 8 to 14 kV.Fig. 2 shows the SEM photographs of the fibrous membranes prepared in our cases. When the voltage is 8 and 10 kV, as presented in Fig. 2(a) and (b), relative homogenous fibers can be spun with an average diameter of 100 and 180 nm, respectively. As the voltage is increased to 12 kV, beads are deposited on the collector and thin fibers coexist among the beads (Fig. 2(c)). For the electrospinning process, when the voltage is low, the jets of polymer solution would not break up rather than travel and split into filaments to the collecting plate and form fibers. At high voltage, on the other hand, the jets are unstable and likely to break up and form droplets, which are the precursors of the beads observed in the electrospun fibrous membranes. Furthermore, several electrospun fibers broke up and more beads formed at the voltage of 14 kV (Fig. 2(d)). Thus, the nanofibrous membrane fabricated at 10 kV with diameter of 100 nm was chosen and applied in next experiments. 3.2. Immobilization of lipase Table 1 shows the activity of the free and immobilized lipases under the optimum reaction conditions. It can be seen that the amounts of bound protein are 2.3670.06 mg/g

on the hollow fiber membrane [15], 21.270.7 mg/g on the nascent nanofiber membrane, 22.570.75 mg/g on the chitosan-modified nanofiber membrane and 20.770.75 mg/ g on the gelatin-modified nanofiber membrane. In comparison with the free enzyme, the immobilized lipase under its optimum reaction condition retains 33.971.6% of the activity on the hollow fiber membrane [15], 37.671.8% on the nascent nanofiber membrane, 45.671.8% on the chitosan-modified nanofiber membrane, and 49.771.8% on the gelatin-modified nanofiber membrane. Interestingly, compared to that on the hollow fiber membrane, there is a significant increase of the amount of bound protein on these nanofiber membranes. This result can be explained by the fact that the remarkable high specific area of the nanofiber membrane with the reduction of the fiber diameter can provide more potential reaction sites for the covalent coupling of enzyme. On the other hand, the activity retention of the immobilized enzyme on the nascent nanofiber membrane is also higher than that on the hollow fiber membrane. The main reason for this could be the remarkable reduction of the diffusion resistance for the immobilized lipase on this nanofiber membrane. As mentioned in the introduction, to increase the biocompatibility of the nascent nanofiber membrane, two natural macromolecules, chitosan and gelatin, were tethered on the membrane surface to form biomimetic layers. The loading amounts are 142 mg/g for chitosan and 126 mg/g for gelatin. After this, lipase was immobilized on the modified membranes using GA as coupling agent. It can be observed that there is an increase of the activity

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Table 1 Activity of the free and immobilized lipases under optimum reaction conditions Samples

Free lipase Lipase immobilized on membrane Lipase immobilized on membrane Lipase immobilized on nanofiber membrane Lipase immobilized on nanofiber membrane

Temperature (1C)

pH

Bound protein (mg/g)

Specific activity (U/mg protein)

Activity retention (%)

the hollow fiber

37 45

7.5 7.5

— 2.3670.06

42.1 14.3

100 33.971.6

the nascent nanofiber

45

7.5

21.270.71

15.8

37.671.8

the chitosan-modified

45

7.0

22.570.75

19.2

45.671.8

the gelatin-modified

45

7.5

20.770.75

20.9

49.771.8

retention of the immobilized enzyme from 37.671.8% on the nascent nanofiber membrane to 45.671.8% on the chitosan-modified nanofiber membrane and 49.771.8% on the gelatin-modified nanofiber membrane. Several speculates could be used to explain this phenomenon. Firstly, the biomimetic layer on the support surface could create a biocompatible microenvironment for the immobilized lipase, which usually leads to high activity retention of the immobilized enzyme. Secondly, the immobilization of lipase on the hydrophilic biomimetic layer might benefit the exposition of its active site [27] and the decrease of random coupling between lipase and the support, due to the special structure of lipase, hydrophobic region surrounding its active site [33]. Finally, the use of GA, which introduced a penta-carbon chain, might be in favor of the contact of lipase with substrate. Furthermore, the activity retention of the immobilized enzyme on the gelatin-modified membrane is higher than that on the chitosan-modified membrane. Two possible factors could explain this result. Firstly, compared to gelatin, there are a large number of amino groups in chitosan, which are potential reaction sites for covalent coupling with enzyme. Thus, it becomes easier to immobilize one enzyme molecule through multiple point chemical bonds on the chitosan-modified membrane, which substantially reduces its enzymatic activity [34,35]. Secondly, the gelatin layer might create a more favorable microenvironment for the immobilized lipase to retain its activity than the chitosan layer. 3.3. pH and thermal stabilities The thermal stabilities of the free and immobilized lipases are given in Fig. 3(a). It can be seen that the free lipase losses its initial activity within around 100 min, while the immobilized lipases retain their initial activity of about 66% for the chitosan-modified nanofiber membrane and 62% for the gelatin-modified one after a 120 min of heat treatment, respectively. These results indicate that the thermal stability of immobilized lipases is much better than that of the free one owing to the formation of covalent bond between the enzyme and the supports, which prevents

the conformation transition of the enzyme at high temperature. Fig. 3(b) shows the pH stabilities of the free and immobilized lipases on these supports. There is no activity loss for the free lipase in the pH range from 4 to 6, while it is stable up to a pH value of 7 for the immobilized lipases. These data demonstrate that the pH stability of lipase could be enhanced by the immobilization process. 3.4. Reuse stability of the immobilized lipase For immobilized enzyme, one of the most important advantages is reuse stability, which can effectively reduce the cost in industry applications. To evaluate the reuse stability, the lipase-immobilized membranes were washed with PBS (50 mM, pH 7.5) after any run and reintroduced into a fresh solution. This process was repeated up to 10 cycles. Fig. 4 shows the effect of repeated use on the activities of these immobilized enzymes. After 10 reuses, the residual activity of the immobilized enzymes is 55% for the chitosan-modified nanofiber membrane and 60% for the gelatin-modified nanofiber membrane. These results are due to the inactivation of the enzyme caused by the denaturation of protein and the leakage of protein from the supports upon use. 3.5. Kinetic parameters The kinetic parameters Km and Vmax from double reciprocal plot are listed in Table 2. It was found that the Km value is 0.45, 1.36, 0.98, 1.22 and 1.05 mM for the free, the hollow fiber membrane bound, the nascent nanofiber membrane-bound the chitosan-modified nanofiber membrane bound, and the gelatin-modified nanofiber membrane bound enzymes, respectively. Obviously, there is an increase in the Km value for these immobilized enzymes. The increase in the Km values is either due to the conformational changes of the enzyme resulting in a lower possibility to form substrate–enzyme complex or due to the lower accessibility of the substrate to the active site of the immobilized enzyme caused by the increased diffusion limitation.

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Fig. 4. Reuse stability of the immobilized lipases: on the chitosanmodified nanofibrous membrane (n), on the gelatin-modified nanofibrous membrane (J).

Fig. 3. pH and thermal stabilities of the free and immobilized lipases: the free (’), on the chitosan-modified nanofibrous membrane (n), on the gelatin-modified nanofibrous membrane (J).

From Table 2, it can be seen that the Km values of the immobilized lipase on the nanofibrous membranes are lower than that on the hollow fiber membrane. This result is largely attributed to the presence of the large specific surface area of the nanofiber membrane, which can create a more favorable interface for the mass transfer of substrate or product to or from the active site of the enzyme. Furthermore, the Km value of the immobilized lipase on the gelatin-modified nanofiber membranes is lower than that on the chitosan-modified nanofiber membrane. There are two reasons to explain this result. On the one hand, compared to the chitosan-modified membrane bound enzyme, the immobilized enzyme on the gelatin-modified membrane which is coupled by less multiple chemical bonds could easily convert its conformation to form substrate-enzyme complex. On the other hand, some properties of the gelatin layer might lead to the lower Km

value. Gelatin is a protein which contains hydrophilic and hydrophobic groups, unlike chitosan belongs to polysaccharide. The presence of the hydrophobic groups in the gelatin layer could benefit the adsorption of hydrophobic substrate molecules on the membrane surface to close the active site of enzyme through hydrophobic interaction, and hence influences the kinetic parameter Km. As shown in Table 2, the Vmax values of enzymes demonstrate a decrease upon immobilization from 46.4 U/mg for the free lipase to 16.1, 16.5, 22.1, and 23.3 U/mg for the immobilized lipases on the hollow fiber membrane, the nascent nanofiber membrane, the chitosanmodified nanofiber membrane and the gelatin-modified nanofiber membrane, respectively. It was found that the Vmax value of the immobilized lipase on the nascent membrane is lower than that on the dual-layer biomimetic supports. This is attributed to the biocompatible microenvironment for the immobilized lipase created by the biomimetic-layer on the support surface, which is corresponding to the activity retention of the immobilized lipase on these supports and could be explained by the same reasons. 4. Conclusion PANCMA was fabricated into nanofibrous membrane and two natural biomacromolecules, chitosan and gelatin, were tethered on the membrane surface to prepare duallayer biomimetic support for lipase immobilization. This protocol established such environments in which protein and polysaccharide could contact with enzyme at similar conditions. It was found that there is an increase of the activity retention of the immobilized lipase on the gelatinmodified nanofiber membrane (49.771.8%) and on the

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Table 2 Activity and kinetic parameters of the free and immobilized lipases Samples Free lipase Lipase immobilized Lipase immobilized Lipase immobilized Lipase immobilized

on on on on

the the the the

hollow fiber membrane nascent nanofibrous membrane chitosan-modified nanofibrous membrane gelatin-modified nanofibrous membrane

chitosan-modified nanofiber membrane (45.671.8%), compared to that on the nascent nanofiber membrane (37.671.8%). The kinetic parameter Km values of the immobilized lipase on the nanofiber membranes are lower than that on the hollow fiber membrane. In comparison with the chitosan-modified membrane, there is a decrease of the Km value for the immobilized enzyme on the gelatinmodified membrane. After immobilization, the pH, thermal and reuse stabilities of the immobilized enzyme can be enhanced. Thus, the method described in this work indicates that the nanofiber membrane modified with chitosan and gelatin could be potential supports in the enzyme immobilization technology for industrial applications. Acknowledgement The authors are grateful to the financial support from the National Natural Science Foundation of China (Grant no. 50273032) and Programme Sino-Franc- ais de Recherches Avance´es (Grant no. PRA E03-04). References [1] Schmid A, Dordick JS, Hauer B, Kiener A, Wubbolts M, Witholt B. Industrial biocatalysis today and tomorrow. Nature 2001;409:258–68. [2] Reetz MT, Wilensek S, Zha D, Jaeger JE. Directed evolution of an enantioselective enzyme through combinatorial multiplecassette mutagenesis. Angew Chem Int Ed Engl 2001;40:3589–91. [3] Palomo JM, Segura RL, Mateo C, Fernandez-Lafuente R, Guisan JM. Improving the activity of lipases from thermophilic organisms at mesophilic temperatures for biotechnology applications. Biomacromolecules 2004;5:249–54. [4] Dyal A, Loos K, Noto M, Chang SW, Spagnoli C, Shafi KVPM, et al. Activity of Candida rugosa lipase immobilized on g-Fe2O3 magnetic nanoparticles. J Am Chem Soc 2003;125:1684–5. [5] Drechsler U, Fischer NO, Frankamp BL, Rotello VM. Highly efficient biocatalysts via covalent immobilization of Candida rugosa lipase on ethylene glycol-modified gold-silica nanocomposites. Adv Mater 2004;16:271–4. [6] Jia H, Zhu G, Vugrinovich B, Kataphinan W, Reneker DH, Wang P. Enzyme-carrying polymeric nanofibers prepared via electrospinning for use as unique biocatalysts. Biotechnol Prog 2002;18:1027–32. [7] Jiang HL, Fang DF, Hsiao BS, Chu B, Chen W. Optimization and characterization of dextran membranes prepared by electrospinning. Biomacromolecules 2004;5:326–33. [8] Deng H- T, Xu Z- K, Huang X- J, Wu J, Seta P. Adsorption and activity of Candida rugosa lipase on polypropylene hollow fiber membrane modified with phospholipid analogous polymers. Langmuir 2004;20:10168–73.

Vmax (U/mg)

Km (mM)

46.4 16.1 16.5 22.1 23.3

0.45 1.36 0.98 1.22 1.05

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