Preparation of Magnetic Chitosan Nanoparticles As Support for

Article pubs.acs.org/IECR

Preparation of Magnetic Chitosan Nanoparticles As Support for Cellulase Immobilization Limin Zang,† Jianhui Qiu,*,† Xueli Wu,† Wenjuan Zhang,† Eiichi Sakai,† and Yi Wei‡ †

Department of Machine Intelligence and Systems Engineering, Faculty of System Science and Technology, Akita Prefectural University, Yurihonjo, Akita 015-0055, Japan ‡ College of Chemistry and Chemical Engineering, Lanzhou University, Gansu 730000, China ABSTRACT: A simple preparation process was developed for magnetic nanoparticles, consisting of chitosan coated on Fe3O4 nanoparticles, to be used as support for enzyme immobilization. Cellulase was covalently immobilized on this magnetic support using glutaraldehyde as a coupling agent. The structure, morphology, and magnetic property of the support were studied by X-ray diffraction, vibrating-sample magnetometer, thermogravimetric analysis, transmission electron microscopy, and Fourier transform infrared (FT-IR) spectroscopy. The properties of the immobilized cellulase were investigated by regarding activity, optimum operational pH and temperature, thermal stability, and reusability. The amount of cellulase on the nanoparticles reached 112.3 mg/g. The characterization and determination results showed that the immobilized cellulase had higher operational stability than the free enzyme over wider temperature and pH ranges and good reusability after recovery by magnetic separation. Therefore, these magnetic Fe3O4−chitosan nanoparticles are expected to be a useful support for enzyme. supports are combined by covalent bonds, are widely used.21−23 The physical adsorption method is simple and rarely changes the advanced structure of enzymes, thus decreasing the loss of activity. However, the enzymes will fall off if the connection is not firm, which hinders practical application of the method when reusability is necessary.24 Despite some studies reported that covalent binding may result in a loss of activity, taking into account the high cost of cellulase, covalent binding is still suitable for industrial application because of the advantages, such as prevention of enzyme loss from the support and improved reusability and stability.25 Moreover, there are some methods for covalent binding via glutaraldehyde (GDA) activation that are simple and effective.26−28 Various researchers have used chitosan as a nanoparticleforming material to modify magnetic particles since it has outstanding advantages, especially with numerous functional groups such as amino, hydroxymethyl, and hydroxyl groups which can be adapted to interact with enzyme.29,30 The pKa value of the amino groups of chitosan is about 6.5. This means that chitosan can be dissolved and has a coil-like structure in an acid solution.31,32 This is favorable to promote the loading capacity of cellulase on chitosan due to the fact that the optimum pH of cellulase is found to be pH 5. However, the mechanical properties of chitosan without any modification are poor, which cause structure destruction during long-time use. Besides, recovery of chitosan via filtration or centrifugation is unsuitable for large-scale applications because it would take a long time. In recent years, magnetic chitosan supports are prepared via various techniques, including in situ formation approaches,33 photochemical polymerization,34 suspension

1. INTRODUCTION Ethanol produced from agricultural and municipal lignocellulosic wastes which have large amounts of cellulose and hemicellulose is of great interest since it can be produced economically without using food resources, has a high octane number, and reduces green house gas emissions.1,2 Therefore, it is thought to be a “green” alternative to fossil fuels. For bioethanol production, cellulose and hemicellulose are hydrolyzed to fermentable reducing sugars, and then, the sugars are fermented to produce ethanol. Enzymatic hydrolysis is usually done under mild conditions and corrosion of equipment is not a problem as in acid or alkaline hydrolysis.3 Therefore, enzymatic hydrolysis of lignocellulosic materials is a critical step in producing fermentable reducing sugars.4−6 Cellulase enzyme complex, composing of three components of enzyme, is necessary for the enzymatic hydrolysis of lignocellulosic materials to produce glucose. The practical applications of cellulase are limited because of its hydrophilic nature.7 For diverse practical applications, cellulase should be stable and reusable over wide pH and temperature ranges.8 Therefore, improvement of the stability and reusability of cellulase has received great attention. Immobilization of bioactive materials onto solid supports provides a way to improve stability and reusability, modify the catalytic properties, and in certain cases get higher activity or selectivity.9−14 Among supports, magnetic supports for biological and biomedical materials are of particular interest.15−20 They have several merits, including nontoxicity, large surface area, and the ability to produce desired magnetic properties so that they can be separated using magnets and reused. Recovery of enzyme by magnets is more feasible in large-scale applications compared with other recovery methods such as filtration and centrifugation. Among various immobilization techniques, physical adsorption, where interactions including van der Waals force, ionic bonds, and hydrogen bonds between enzymes and supports occur, and covalent binding, where enzymes and © 2014 American Chemical Society

Received: Revised: Accepted: Published: 3448

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polymerization,35 phase-inversion techniques,36 and microwave irradiation.37 However, these methods are time-consuming or require specialized equipment. The relevance between the solubility of chitosan and pH of the solution was utilized to prepare Fe3O4−chitosan nanoparticles in this work. Then, the as-prepared nanoparticles were activated by GDA so as to immobilize cellulase by chemical binding. Meanwhile, some chitosan performed condensation reaction via GDA which is helpful to improve the mechanical properties of chitosan. The optimum pH and temperature values, thermal stability, and reusability of our immobilized cellulase were investigated. Compared with the previous reports, the main findings of this work are the following: (1) The Fe3O4−chitosan nanoparticles with high magnetic sensitivity can be easily separated from the reaction mixture. (2) The process for immobilization is mild enough that it does not denature the enzyme during preparation. (3) The mechanical properties of chitosan are improved after cross-linking by GDA which is favorable for wide practical application.

thermal analysis (DTA) were performed with a Shimadzu DTG60 (Japan) at a heating rate of 10 °C/min in N2 atmosphere. The chemical structures of the nanoparticles were confirmed by Fourier transform infrared (FT-IR, IRT-7000, Jasco, Japan) spectroscopy. The transmission electron microscopy (TEM) images were recorded by a Hitachi H-8100 (Japan) microscope. 2.5. Determination of Cellulase Loading Efficiency and Activity. The cellulase concentration was determined according to Bradford protein assay method,40 and the cellulase loading efficiency was calculated from the following equation: loading efficiency (%) =

C0V0 − CiVi × 100% C0V0

Where C0 is the protein concentration and V0 is the volume of the cellulase solution before immobilization, respectively. Ci is the protein concentration and Vi is the volume of the filtrate after immobilization, respectively. The activity of cellulase was determined according to the standard procedure of IUPAC with some modifications.41,42 Here, 0.5 mL of cellulase solution at the appropriate dilution was mixed with 0.5 mL of 1% CMC solution (dissolved in 0.1 M acetate buffer, pH 5.0) to proceed the hydrolysis. The reaction was carried out at 50 °C for 0.5 h, and the amount of glucose produced during the reaction was used to calculate the activity by measuring absorbance at 540 nm using DNS as reagent. One International Unit (IU) of cellulase activity is defined as the amount of cellulase that hydrolyzes CMC to produce 1 μmol glucose per minute. 2.6. Reusability Assay. From the viewpoint of practical applications, 33.6 mg cellulase immobilized on 0.3 g support was utilized to hydrolyze 100 mL 1% CMC for at 50 °C 24 h. After separation by a magnet, the immobilized cellulase was added to a fresh substrate solution. The reusability assay was repeated 10 cycles. The activity after each cycle and the glucose productivity during 24 h were used to evaluate the reusability of the immobilized cellulase.

2. MATERIALS AND METHODS 2.1. Materials. Cellulase was purchased from Meiji Seika Pharma Co., Ltd. (Tokyo, Japan). Chitosan, carboxy methyl cellulose sodium salt (CMC), iron(II) chloride tetrahydrate (FeCl2·4H2O), iron(III) chloride hexahydrate (FeCl3·6H2O), acetic acid, glutaraldehyde (GDA, 25%, v/v), sodium hydroxide (NaOH), and ammonium hydroxide (NH4OH, 28 wt %) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). 2.2. Synthesis of Fe3O4−Chitosan Supports. The coprecipitating method was used to synthesize Fe3O4 nanoparticles.38 With vigorous stirring, 5.4 g FeCl3·6H2O and 1.99 g FeCl2·4H2O were dissolved in 50 mL of deionized water. Then 28 wt % NH4OH was added to the mixture until the pH reached 10. The reaction was performed at 80 °C for 2 h under N2 protection. Subsequently, the Fe3O4 nanoparticles were washed with deionized water until the pH of the wash was neutral. After 0.25 g chitosan was completely dissolved in 50 mL of acetic acid solution (1%, v/v), 2.0 g dry Fe3O4 nanoparticles prepared above were added to it. With vigorous stirring for 30 min, the Fe3O4 nanoparticles were homogeneously dispersed in the chitosan solution. Then, 50 mL of 1 M NaOH solution was added to mixture and the chitosan coated Fe3O4 nanoparticles were obtained. The obtained particles were washed with deionized water until the pH of the wash was 7 and stored at 4 °C for immobilization. 2.3. Immobilization of Cellulase. Cellulase was immobilized via GDA activation method.39 The prepared Fe3O4− chitosan nanoparticles were used as support to immobilize cellulase. Typically, 1.0 g support was reacted with 30 mL of 2.5% (v/v) GDA solution with stirring at 25 °C for 2 h so as to activate the support by offering aldehyde groups. The GDAactivated support was washed with deionized water three times to remove unreacted GDA and then mixed with 50 mL of 6 mg/mL cellulase solution (dissolved in 0.1 M acetate buffer, pH 5.0) with gentle stirring at 25 °C for 2 h for immobilization by chemical binding. Afterward, the products were magnetically separated and washed with acetate buffer to remove unimmobilized cellulase 2.4. Characterization. The X-ray diffraction (XRD) measurements were carried out with a PANalytical X’Pert Pro (UK) instrument. A vibrating-sample (VSM, Riken Denshi Co. Ltd., Japan) magnetometer was used to obtain magnetization curves. Thermogravimetric analysis (TGA) and differential

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of Fe3O4−Chitosan Supports. The schematic representation for the preparation of the Fe3O4−chitosan supports and cellulase immobilization was illustrated in Scheme 1a. The solubility of chitosan depends on the pH of the solution. Chitosan was first dissolved in an acid solution, and the protonated chitosan is regarded as polycations whereas the Fe3O4 nanoparticles are negative. There is a strong electrostatic interaction43 between chitosan and Fe3O4 nanoparticles. When the pH of the solution was adjusted to alkaline, chitosan was precipitated onto the surface of Fe3O4 to form Fe3O4−chitosan nanoparticles as support.38 The amino group on the Fe3O4−chitosan was converted to aldehyde group via GDA, and then, cellulase was covalently bound to the support by Schiff base linkage. During GDA activation, the condensation reaction of chitosan occurred, which was helpful for improving the mechanical properties of chitosan (Scheme 1b). The XRD patterns of Fe 3 O 4 , Fe 3 O 4 −chitosan, and immobilized cellulase are shown in Figure 1. For Fe3O4 nanoparticles, the peaks at 2θ = 30.1°, 35.4°, 43.1°, 53.2°, 56.9°, and 62.5° correspond to the (220), (311), (400), (422), (511), and (440), respectively. After coating with chitosan and immobilization of cellulase, the same characteristic peaks exhibited, which revealed that the nanoparticles maintained the crystalline structure of Fe3O4. 3449

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Scheme 1. Schematic Diagram of Preparation of Immobilizd Cellulase (a); Condensation Reaction of Chitosan Adducts with GDA44 (b)

Figure 1. XRD patterns of Fe3O4 (a), Fe3O4−chitosan (b), and immobilized cellulase (c).

Figure 2. Magnetization curves for Fe3O4 (a) and immobilized cellulase (b) at room temperature.

The magnetization curves of the Fe3O4 and immobilized cellulase at room temperature are shown in Figure 2. Immobilized cellulase possessed high saturation magnetization (σs) about 46.6 emu/g, while for the pure Fe3O4 was 58.9 emu/g. The results showed that coating with chitosan and cellulase immobilization had an effect on the magnetism of resulting nanoparticles owing to the existence of diamagnetic chitosan and cellulase. Even so, such magnetism allows the supports to be recovered rapidly from the enzyme reaction system by applying an external magnetic field, which is a significant advantage for enzyme immobilization. The thermal properties of the nanoparticles were studied by TGA and DTA as shown in Figures 3 and 4. Fe3O4 (Figure 3a) was thermally stable, and there was no obvious weight loss over

Figure 3. TGA curves of Fe3O4 (a), Fe3O4−chitosan (b), Fe3O4− chitosan−GDA (c), immobilized cellulase (d), and chitosan (e).

the entire testing temperature range. For other samples (Figure 3b−e), the weight loss below 200 °C was attributed to the release of water molecules.32 The polysaccharide units of chitosan (Figure 3e) decomposed from 200 °C,45 and the overall weight loss was 60.95% at 800 °C. The content of chitosan in Fe3O4−chitosan nanoparticles estimated from the TGA curves of Fe3O4 (Figure 3a) and Fe3O4−chitosan (Figure 3b) was 10.36% (0.116 g chitosan/g Fe3O4), which was consistent with the mass ratio of feeding material (m(chitosan):m(Fe3O4) = 0.125:1). The weight percentage of cellulase in immobilized cellulase 3450

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(Figure 5e), the CO stretching vibration at 1381 cm−1 and NH bending vibration at 1595 cm−1 appeared.39 And, the characteristic peaks at 1030 and 1149 cm−1 for saccharide structure also arose. In Figure 5b−d, both of the characteristic peaks of chitosan and FeO appeard. Upon GDA activation (Figure 5c), chitosan condensated with GDA (Scheme 1), and a new band at 1630 cm−1 representing the CN vibration appeared. The characteristic bands of proteins at 1651 and 1541 cm−1 in the spectrum of free cellulase (Figure 5f) also appeared in the spectrum of immobilized cellulase (Figure 5d), which indicated that cellulase was immobilized onto the support. 3.3. Particle Morphology Analysis. The particle micrographs and sizes of Fe3O4, Fe3O4−chitosan, and immobilized cellulase were obtained, and images are given in Figure 6. All of the particles had nanometer size, and the diameters were found to be 10−18 nm. For support materials, such small dimensions are conducive to immobilizing more enzyme since they have larger specific surface area.39 In Figure 6a, many magnetic Fe3O4 nanoparticles were aggregated resulting from the magnetic feature of Fe3O4 nanoparticles.39 After coating with chitosan (Figure 6b), the particles dispersed more uniformly than pure Fe3O4 because the chitosan shell would decrease the magnetism of the nanoparticles and increase the repulsive force between the nanoparticles.38 As shown in Figure 6c, some nanoparticles were aggregated because some chitosan underwent a condensation reaction via GDA. But the agglomeration was slighter in comparison with Figure 6a. 3.4. Properties of the Immobilized Cellulase. Cellulase was immobilized on Fe3O4−chitosan nanoparticles by chemical binding via GDA. The cellulase loading efficiency was 72.4%, and the cellulase immobilized on the supports was 112.3 mg/g support, with an activity of 5.23 IU/mg cellulase. In addition, the main performance parameters of the immobilized cellulase were compared with those in other reports, and this is summarized in Table 1. 3.4.1. Optimum pH and Temperature for Free and Immobilized Cellulase. The effect of pH on the activity was investigated by carrying out the enzyme assay in the pH range of 3−8 at 50 °C, and the results are shown in Figure 7a. The maximum activity was at pH 5 for immobilized cellulase, which was the same as free cellulase. It is worth noting that the activity of immobilized cellulase was generally higher over most of the pH values as compared to the free cellulase, which indicated that immobilized cellulase showed preferable pH adaptability. The influence of the temperature on the activity was studied from 30 to 70 °C at pH 5. Figure 7b indicated that the optimum temperature of the immobilized and free cellulase was 50 °C. The free callulase had higher activity than the immobilized cellulase at 50 °C. However, at higher temperature the immobilized cellulase showed greater activity, which indicated

Figure 4. DTA curves of Fe3O4 (a), Fe3O4−chitosan (b), Fe3O4− chitosan−GDA (c), immobilized cellulase (d), and chitosan (e).

estimated from the TGA curves of Fe3O4−chitosan−GDA (Figure 3c) and immobilized cellulase (Figure 3d) was 10.26% (114.8 mg cellulase/g support), which was consistent with the results of the Bradford assay. For temperatures of 400−500 °C, the heat flow curves of Fe3O4 (Figure 4a) and chitosan (Figure 4e) were stable; whereas the heat flow curves of Fe3O4−chitosan− GDA (Figure 4c) and immobilized cellulase (Figure 4d) had a significant endothermic peak at 432.5 and 408.3 °C, respectively. This indicated that some chitosan performed condensation reaction via GDA (Scheme 1b), which improved the thermal stability of the coated chitosan. The results agreed with a previous report.45 3.2. FT-IR Studies. The FT-IR spectra of Fe3O4, Fe3O4− chitosan, Fe 3 O 4 −chitosan−GDA, immobilized cellulase, chitosan, and free cellulase are given in Figure 5. For chitosan

Figure 5. FT-IR spectra of Fe3O4 (a), Fe3O4−chitosan (b), Fe3O4− chitosan−GDA (c), immobilized cellulase (d), chitosan (e), and free cellulase (f).

Figure 6. TEM images of Fe3O4 (a), Fe3O4−chitosan (b), and immobilized cellulase (c). 3451

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Table 1. Supports, Immobilizing Techniques, and Reusability of the Current Work and Other Reports reusability support clay/PGMA sol−gel matrix magnetoresponsive graphene commercial activated carbon modified PVA coated chitosan magnetic chitosan

immobilizing technique

cellulase loading amount

times

time for each cycle

residual activity

adsorption covalent entrapment covalent adsorption adsorption covalent

43.4 mg/g 32.7 mg/g 43.9 mg protein/g 2.5 mg protein/g 1565 mg/g 0.144 mg protein/10 beads 112.3 mg/g

12 12 6 4 5 8 10

1h 1h 24 h 1h 1h 5 min 24 h

37% 79% 20% 55% 70% 52% 50%

refs 46 47 48 49 50 current work

Figure 8. Thermal stability of immobilized and free cellulase.

Figure 7. Effect of pH (a) and temperature (b) on activity of the immobilized and free cellulase. Figure 9. Influence of repeats on the activities of the immobilized cellulase.

that the immobilized cellulase had better heat resistance than the free cellulase. 3.4.2. Thermal Stability. Figure 8 plots thermal stability trends for the immobilized and free cellulase during 5 h test at 60 °C. In order to compare the thermal stability in a short time, 60 °C was chosen as test temperature which is 10 °C higher than the optimum temperature. The activity of both immobilized and free cellulase decreased; however, the former decreased slowly and less than the latter. The residual activity of the immobilized cellulase was 58.7% after 5 h test; whereas, the free cellulase had kept only 52.9%. These results might result from the screen function of the Fe3O4−chitosan supports as has been reported by others.32,51 The promising stability of the immobilized cellulase is very important for practical applications. 3.4.3. Reusability. From the viewpoint of practical applications, the immobilized cellulase was applied to hydrolyze 100 mL 1% CMC for 24 h, which is longer than previous reports.48−50 The immobilized cellulase was recycled 10 times in the present experiment. The activity assay was made after

Figure 10. Influence of repeats on the glucose productivity of the immobilized cellulase at 24 h, pH 5, and 50 °C. 3452

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(6) Sakamoto, T.; Hasunuma, T.; Hori, Y.; Yamada, R.; Kondo, A. Direct ethanol production from hemicellulosic materials of rice straw by use of an engineered yeast strain codisplaying three types of hemicellulolytic enzymes on the surface of xylose-utilizing Saccharomyces cerevisiae cells. J. Biotechnol. 2012, 158, 203−210. (7) Ho, K. M.; Mao, X.; Gu, L.; Li, P. Facile route to enzyme immobilization: core-shell nanoenzyme particles consisting of welldefined poly(methyl methacrylate) cores and cellulase shells. Langmuir 2008, 24, 11036−11042. (8) George, S. P.; Ahmad, A.; Rao, M. B. Studies on carboxymethyl cellulase produced by an alkalothermophilic actinomycete. Bioresour. Technol. 2001, 77, 171−175. (9) Jordan, J.; Kumar, C. S.S.R.; Theegala, C. Preparation and characterization of cellulase-bound magnetite nanoparticles. J. Mol. Catal. B−Enzym. 2011, 68, 139−146. (10) Bornscheuer, U. T. Immobilizing enzymes: how to create more suitable biocatalysts. Angew. Chem., Int. Ed. 2003, 42, 3336−3337. (11) Polizzi, K. M.; Bommarius, A. S.; Broering, J. M.; ChaparroRiggers, J. F. Stability of biocatalysts. Curr. Opin. Chem. Biol. 2007, 11, 220−225. (12) Mateo, C.; Palomo, J. M.; Fernandez-Lorente, G.; Guisan, J. M.; Fernandez-Lafuente, R. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb. Technol. 2007, 40, 1451−1463. (13) Fernandez-Lafuente, R. Stabilization of multimeric enzymes: strategies to prevent subunit dissociation. Enzyme Microb. Technol. 2009, 45, 405−418. (14) Iyer, P. V.; Ananthanarayan, L. Enzyme stability and stabilization-aqueous and non-aqueous environment. Process Biochem. 2008, 43, 1019−1032. (15) Liao, M. H.; Chen, D. H. Immobilization of yeast alcohol dehydrogenase on magnetic nanoparticles. Biotechnol. Lett. 2001, 23, 1723−1727. (16) Huang, S. H.; Liao, M. H.; Chen, D. H. Direct binding and characterization of lipase onto magnetic nanoparticles. Biotechnol. Prog. 2003, 19, 1095−1100. (17) Koneracká, M.; Kopčanský, P.; Antalík, M.; Timko, M.; Ramchand, C. N.; Lobo, D.; Mehta, R. V.; Upadhyay, R. V. Immobilization of proteins and enzymes to fine magnetic particles. J. Magn. Magn. Mater. 1999, 201, 427−430. (18) Kondo, A.; Fukuda, H. Preparation of thermo-sensitive magnetic hydrogen microspheres and application to enzyme immobilization. J. Ferment. Bioeng. 1997, 84, 337−341. (19) Niemeyer, C. M. Nanoparticles, proteins, and nucleic acids, biotechnology meets materials science. Angew. Chem., Int. Ed. 2001, 4, 4128−4148. (20) Wilheim, C.; Gazeau, F.; Roger, J.; Pons, N.; Salis, M. F.; Perzynski, R. Binding of biological effectors on magnetic nanoparticles measured by a magnetically induced transient birefringence experiment. Phys. Rev. E. 2002, 65, 031404. (21) Lee, Y. C.; Chen, C. T.; Chiu, Y. T.; Wu, K. C.-W. An Effective Cellulose-to-Glucose-to-Fructose Conversion Sequence by Using Enzyme Immobilized Fe3O4-Loaded Mesoporous Silica Nanoparticles as Recyclable Biocatalysts. ChemCatChem 2013, 5, 2153−2157. (22) Brady, D.; Jordaan, J. Advances in enzyme immobilization. Biotechnol. Lett. 2009, 31, 1639−1650. (23) Hirsh, S. L.; Bilek, M. M. M.; Nosworthy, N. J.; Kondyurin, A.; dos Remedios, C. G.; McKenzie, D. R. A Comparison of Covalent Immobilization and Physical Adsorption of a Cellulase Enzyme Mixture. Langmuir 2010, 26, 14380−14388. (24) Chiou, S. H.; Wu, W. T. Immobilization of Candida rugosa lipase on chitosan with activation of the hydroxyl groups. Biomaterials 2004, 25, 197−204. (25) Chang, R. H.-Y.; Jang, J.; Wu, K. C.-W. Cellulase immobilized mesoporous silica nanocatalysts for efficient cellulose-to-glucose conversion. Green Chem. 2011, 13, 2844−2850. (26) Migneault, I.; Dartiguenave, C.; Bertrand, M. J.; Waldron, K. C. Glutaraldehyde: behavior in aqueous solution, reaction with proteins,

each recycle (24 h), and the change in residual activity was apparent from Figure 9. The activity of the immobilized cellulase decreased with number of times of reuse compared to its initial value and the immobilized cellulase kept over 50% after 10 times reuse. This resultant activity loss might be caused by several factors, including loss of constituents of the cellulase complex, end-product inhibition, and protein denaturation.9 Another reason for the loss in activity could be that some physically adsorbed cellulase might have been present initially, but it fell off during the reusability assay. However, the glucose productivity in the 24 h period remained higher than 80% of the original after 10 cycles (Figure 10), which is very important and attractive for practical applications.

4. CONCLUSIONS In this paper, magnetic chitosan supports were prepared by coating chitosan onto the surface of Fe3O4 nanoparticles. Subsequently, cellulase was successfully immobilized on Fe3O4−chitosan nanoparticles via GDA activation. The high magnetic sensitivity of the immobilized cellulase (46.6 emu/g) makes the separation of cellulase from reaction mixture easy. The optimum pH was 5 and the optimum temperature was 50 °C for immobilized cellulase. The amount of cellulase on Fe3O4−chitosan nanoparticles was found to be as high as 112.3 mg/g, with an activity of 5.23 IU/mg cellulase. The thermal stability of the immobilized cellulase was preferable to the free cellulase. The immobilized cellulase retained 50% of its initial activity after 10 cycles. Meanwhile, the glucose productivity during 24 h remained higher than 80% of the original after 10 cycles. All the present results suggested that the Fe3O4−chitosan support was suitable for covalent immobilization of cellulase, and the resulting immobilized enzyme has good potential for application to large-scale glucose production.

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AUTHOR INFORMATION

Corresponding Author

*Tel. and fax: +81 184 27 2134. E-mail address: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Environment Research and Technology Development Fund (3K113018) of the Ministry of the Environment, Japan.

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REFERENCES

(1) Lynd, L. R. Overview and evaluation of fuel ethanol from cellulosic biomass: technology, economics, the environment, and policy. Annu. Rev. Energy. Environ. 1996, 21, 403−465. (2) Saxena, R. C.; Adhikari, D. K.; Goyal, H. B. Biomass-based energy fuel through biochemical routes: A review. Renew. Sust. Energ. Rev. 2008, 13, 156−167. (3) Sun, Y.; Cheng, J. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour. Technol. 2002, 83, 1−11. (4) Zhou, H.; Lou, H.; Yang, D.; Zhu, J. Y.; Qiu, X. Lignosulfonate to enhance enzymatic saccharification of lignocelluloses: role of molecular weight and substrate lignin. Ind. Eng. Chem. Res. 2013, 52, 8464−8470. (5) Govumoni, S. P.; Koti, S.; Kothagouni, S. Y.; Venkateshwar, S.; Linga, V. R. Evaluation of pretreatment methods for enzymatic saccharification of wheat straw for bioethanol production. Carbohydr. Polym. 2013, 91, 646−650. 3453

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and application to enzyme crosslinking. Biotechniques 2004, 37, 790− 802. (27) López-Gallego, F.; Betancor, L.; Mateo, C.; Hidalgo, A.; AlonsoMorales, N.; Dellamora-Ortiz, G.; Guisán, J. M.; Fernández-Lafuente, R. Enzyme stabilization by glutaraldehyde crosslinking of adsorbed proteins on aminated supports. J. Biotechnol. 2005, 119, 70−75. (28) Betancor, L.; López-Gallego, F.; Hidalgo, A.; Alonso-Morales, N.; Dellamora-Ortiz, G.; Mateo, C.; Fernández-Lafuente, R.; Guisán, J. M. Different mechanisms of protein immobilization on glutaraldehyde activated supports: effect of support activation and immobilization conditions. Enzyme Microb. Technol. 2006, 39, 877−882. (29) Jiang, D. S.; Long, S. Y.; Huang, J.; Xiao, H. Y.; Zhou, J. Y. Immobilization of pycnoporus sanguineus laccase on magnetic chitosan microspheres. Biochem. Eng. J. 2005, 25, 15−23. (30) Xu, D.; Hein, S.; Loo, S. L.; Wang, K. The fixed-bed study of dye removal on chitosan beads at high pH. Ind. Eng. Chem. Res. 2008, 47, 8796−8800. (31) MacLaughlin, F. C.; Mumper, R. J.; Wang, J. U.; Tagliaferri, J. M.; Gill, I.; Hinchcliffe, M.; Rolland, A. P. Chitosan and depolymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery. J. Controlled Release 1998, 56, 259−272. (32) Wang, J. Z.; Zhao, G. H.; Li, Y. F.; Liu, X.; Hou, P. P. Reversible immobilization of glucoamylase onto magnetic chitosan nanocarriers. Appl. Microbiol. Biotechnol. 2013, 97, 681−692. (33) Tsai, Z. T.; Wang, J. F.; Kuo, H. Y.; Shen, C. R.; Wang, J. J.; Yen, T. C. In situ preparation of high relaxivity iron oxide nanoparticles by coating with chitosan: a potential MRI contrast agent useful for cell tracking. J. Magn. Magn. Mater. 2009, 322, 208−213. (34) Zhang, L. Y.; Zhu, X. J.; Zheng, S. Y.; Sun, H. W. Photochemical preparation of magnetic chitosan beads for immobilization of pullulanase. Biochem. Eng. J. 2009, 46, 83−87. (35) Zhao, W.; Yang, R. J.; Qian, T. T.; Hua, X.; Zhang, W. B.; Katiyo, W. Preparation of novel poly(hydroxyethyl methacrylate-coglycidyl methacrylate)-grafted core-shell magnetic chitosan microspheres and immobilization of lactase. Int. J. Mol. Sci. 2013, 14, 12073−12089. (36) Fernández-Lucas, J.; Harris, R.; Mata-Casar, I.; Heras, A.; De La Mata, I.; Arroyo, M. Magnetic chitosan beads for covalent immobilization of nucleoside 2′-deoxyribosyltransferase: Application in nucleoside analogues synthesis. J. Ind. Microbio.l Biotechnol. 2013, 40, 955−966. (37) Pospiskova, K.; Safarik, I. Low-cost, easy-to-prepare magnetic chitosan microparticles for enzymes immobilization. Carbohydr. Polym. 2013, 96, 545−548. (38) Kuo, C. H.; Liu, Y. C.; Chang, C. M.; Chen, J. H.; Chang, C.; Shieh, C. J. Optimum conditions for lipase immobilization on chitosan-coated Fe3O4 nanoparticles. Carbohydr. Polym. 2012, 87, 2538−2545. (39) Xie, W. L.; Wang, J. L. Immobilized lipase on magnetic chitosan microspheres for transesterification of soybean oil. Biomass Bioenerg. 2012, 36, 373−380. (40) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248−254. (41) Ghose, T. K. Measurement of cellulase activities. Pure Appl. Chem. 1987, 59, 257−268. (42) Khoshnevisan, K.; Bordbar, A. K.; Zare, D.; Davoodi, D.; Noruzi, M.; Barkhi, M.; Tabatabaei, M. Immobilization of cellulase enzyme on superparamagnetic nanoparticles and determination of its activity and stability. Chem. Eng. J. 2011, 171, 669−673. (43) Zhao, D. L.; Wang, X. X.; Zeng, X. W.; Xia, Q. S.; Tang, J. T. Preparation and inductive heating property of Fe3O4−chitosan composite nanoparticles in an AC magnetic field for localized hyperthermia. J. Alloy. Compd. 2009, 477, 739−743. (44) Abd El-Ghaffar, M. A.; Hashem, M. S. Chitosan and its amino acids condensation adducts as reactive natural polymer supports for cellulase immobilization. Carbohydr. Polym. 2010, 81, 507−516.

(45) Chen, J. P.; Yang, P. C.; Ma, Y. H.; Wu, T. Characterization of chitosan magnetic nanoparticles for in situ delivery of tissue plasminogen activator. Carbohydr. Polym. 2011, 84, 364−372. (46) Bayramoglu, G.; Senkal, B. F.; M. Arica, Y. Preparation of clay− poly(glycidyl methacrylate) composite support for immobilization of cellulase. Appl. Clay Sci. 2013, 85, 88−95. (47) Ungurean, M.; Paul, C.; Peter, F. Cellulase immobilized by sol− gel entrapment for efficient hydrolysis of cellulose. Bioprocess. Biosyst. Eng. 2013, 36, 1327−1338. (48) Gokhale, A. A.; Lu, J.; Lee, I. Immobilization of cellulase on magnetoresponsive graphene nano-supports. J. Mol. Catal. B−Enzym. 2013, 90, 76−86. (49) Daoud, F. B.; Kaddour, S.; Sadoun, T. Adsorption of cellulase Aspergillus niger on a commercial activated carbon: Kinetics and equilibrium studies. Colloid Surf. B−Biointerfaces 2010, 75, 93−99. (50) Dinçer, A.; Telefoncu, A. Improving the stability of cellulase by immobilization on modified polyvinyl alcohol coated chitosan beads. J. Mol. Catal. B−Enzym. 2007, 45, 10−14. (51) Bai, Y. X.; Li, Y. F.; Lin, L. Synthesis of a mesoporous functional copolymer bead carrier and its properties for glucoamylase immobilization. Appl. Microbiol. Biot. 2009, 83, 457−464.

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dx.doi.org/10.1021/ie404072s | Ind. Eng. Chem. Res. 2014, 53, 3448−3454