Fabrication of graphene oxide decorated with Fe3O4@SiO2 for immobilization of cellulase

  • Yue Li
  • Xiang-Yu Wang
  • Xiao-Ping Jiang
  • Jing-Jing Ye
  • Ye-Wang Zhang
  • Xiao-Yun Zhang
Research Paper


Fe3O4@SiO2–graphene oxide (GO) composites were successfully fabricated by chemical binding of functional Fe3O4@SiO2 and GO and applied to immobilization of cellulase via covalent attachment. The prepared composites were further characterized by transmission electron microscopy and Fourier transform infrared spectroscopy. Fe3O4 nanoparticles (NPs) were monodisperse spheres with a mean diameter of 17 ± 0.2 nm. The thickness of SiO2 layer was calculated as being 6.5 ± 0.2 nm. The size of Fe3O4@SiO2 NPs was 24 ± 0.3 nm, similar to that of Fe3O4@SiO2–NH2. Fe3O4@SiO2–GO composites were synthesized by linking of Fe3O4@SiO2–NH2 NPs to GO with the catalysis of EDC and NHS. The prepared composites were used for immobilization of cellulase. A high immobilization yield and efficiency of above 90 % were obtained after the optimization. The half-life of immobilized cellulase (722 min) was 3.34-fold higher than that of free enzyme (216 min) at 50 °C. Compared with the free cellulase, the optimal temperature of the immobilized enzyme was not changed; but the optimal pH was shifted from 5.0 to 4.0, and the thermal stability was enhanced. The immobilized cellulase could be easily separated and reused under magnetic field. These results strongly indicate that the cellulase immobilized onto the Fe3O4@SiO2–GO composite has potential applications in the production of bioethanol.


Graphene Magnetic nanoparticles Cellulase Immobilization Covalent attachment 



The financial support from National Science Foundation of China (No. 21376110) and Jiangsu University (08JDG004) was appreciated.


  1. Arica MY, Alaeddinoğlu NG, Patir S, Denizli A (2000) Invertase immobilized on spacer-arm attached poly (hydroxyethyl methacrylate) membrane: preparation and properties. J Appl Polym Sci 75:1685–1692. doi: 10.1002/(SICI)1097-4628(20000401)75:14<1685:AID-APP1>3.0.CO;2-6 CrossRefGoogle Scholar
  2. Ashtari K, Khajeh K, Fasihi J, Ashtari P, Ramazani A, Vali H (2012) Silica-encapsulated magnetic nanoparticles: enzyme immobilization and cytotoxic study. Int J Biol Macromol 50:1063–1069. doi: 10.1016/j.ijbiomac.2011.12.025 CrossRefGoogle Scholar
  3. Bao H, Zhang L, Chen G (2013) Immobilization of trypsin via graphene oxide-silica composite for efficient microchip proteolysis. J Chromatogr A 1310:74–81. doi: 10.1016/j.chroma.2013.08.040 CrossRefGoogle Scholar
  4. Basu S, Bhattacharyya P (2012) Recent developments on graphene and graphene oxide based solid state gas sensors. Sens Actuators B 173:1–21. doi: 10.1016/j.snb.2012.07.092 CrossRefGoogle Scholar
  5. Bayramoglu G, Senkal BF, Arica MY (2013) Preparation of clay–poly(glycidyl methacrylate) composite support for immobilization of cellulase. Appl Clay Sci 85:88–95. doi: 10.1016/j.clay.2013.09.010 CrossRefGoogle Scholar
  6. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. doi: 10.1016/0003-2697(76)90527-3 CrossRefGoogle Scholar
  7. Brinchi L, Cotana F, Fortunati E, Kenny JM (2013) Production of nanocrystalline cellulose from lignocellulosic biomass: technology and applications. Carbohydr Polym 94:154–169. doi: 10.1016/j.carbpol.2013.01.033 CrossRefGoogle Scholar
  8. Cao M, Li Z, Wang J, Ge W, Yue T, Li R, Colvin VL, Yu WW (2012) Food related applications of magnetic iron oxide nanoparticles: enzyme immobilization, protein purification, and food analysis. Trends Food Sci Technol 27:47–56. doi: 10.1016/j.tifs.2012.04.003 CrossRefGoogle Scholar
  9. Chakrabarti MH, Low CTJ, Brandon NP, Yufit V, Hashim MA, Irfan MF, Akhtar J, Ruiz-Trejo E, Hussain MA (2013) Progress in the electrochemical modification of graphene-based materials and their applications. Electrochim Acta 107:425–440. doi: 10.1016/j.electacta.2013.06.030 CrossRefGoogle Scholar
  10. Chen S (2003) Reversible immobilization of lysozyme via coupling to reversibly soluble polymer. Enzyme Microb Technol 33:643–649. doi: 10.1016/S0141-0229(03)00186-8 CrossRefGoogle Scholar
  11. Del Campo A, Sen T, Lellouche J-P, Bruce IJ (2005) Multifunctional magnetite and silica–magnetite nanoparticles: synthesis, surface activation and applications in life sciences. J Magn Magn Mater 293:33–40. doi: 10.1016/j.jmmm.2005.01.040 CrossRefGoogle Scholar
  12. Gülay S, Şanlı-Mohamed G (2012) Immobilization of thermoalkalophilic recombinant esterase enzyme by entrapment in silicate coated Ca-alginate beads and its hydrolytic properties. Int J Biol Macromol 50:545–551. doi: 10.1016/j.ijbiomac.2012.01.017 CrossRefGoogle Scholar
  13. Hanefeld U, Gardossi L, Magner E (2009) Understanding enzyme immobilisation. Chem Soc Rev 38:453–468. doi: 10.1039/B711564B CrossRefGoogle Scholar
  14. He F, Fan J, Ma D, Zhang L, Leung C, Chan HL (2010) The attachment of Fe3O4 nanoparticles to graphene oxide by covalent bonding. Carbon 48:3139–3144. doi: 10.1016/j.carbon.2010.04.052 CrossRefGoogle Scholar
  15. Hummers WS Jr, Offeman RE (1958) Preparation of graphitic oxide. J Am Chem Soc 80:1339. doi: 10.1021/ja01539a017 CrossRefGoogle Scholar
  16. Hung T-C, Fu C-C, Su C-H, Chen J-Y, Wu W-T, Lin Y-S (2011) Immobilization of cellulase onto electrospun polyacrylonitrile (PAN) nanofibrous membranes and its application to the reducing sugar production from microalgae. Enzyme Microb Technol 49:30–37. doi: 10.1016/j.enzmictec.2011.04.012 CrossRefGoogle Scholar
  17. Ince A, Bayramoglu G, Karagoz B, Altintas B, Bicak N, Arica MY (2012) A method for fabrication of polyaniline coated polymer microspheres and its application for cellulase immobilization. Chem Eng J 189–190:404–412. doi: 10.1016/j.cej.2012.02.048 CrossRefGoogle Scholar
  18. Kannoujia DK, Ali S, Nahar P (2009) Pressure-induced covalent immobilization of enzymes onto solid surface. Biochem Eng J 48:136–140. doi: 10.1016/j.bej.2009.09.005 CrossRefGoogle Scholar
  19. Kharrat N, Ali YB, Marzouk S, Gargouri Y-T, Karra-Châabouni M (2011) Immobilization of Rhizopus oryzae lipase on silica aerogels by adsorption: comparison with the free enzyme. Process Biochem 46:1083–1089. doi: 10.1016/j.procbio.2011.01.029 CrossRefGoogle Scholar
  20. Khoshnevisan K, Bordbar A-K, Zare D, Davoodi D, Noruzi M, Barkhi M, Tabatabaei M (2011) Immobilization of cellulase enzyme on superparamagnetic nanoparticles and determination of its activity and stability. Chem Eng J 171:669–673. doi: 10.1016/j.cej.2011.04.039 CrossRefGoogle Scholar
  21. Knezevic Z, Milosavic N, Bezbradica D, Jakovljevic Z, Prodanovic R (2006) Immobilization of lipase from Candida rugosa on Eupergit® C supports by covalent attachment. Biochem Eng J 30:269–278. doi: 10.1016/j.bej.2006.05.009 CrossRefGoogle Scholar
  22. Li C, Yoshimoto M, Fukunaga K, Nakao K (2007a) Characterization and immobilization of liposome-bound cellulase for hydrolysis of insoluble cellulose. Bioresour Technol 98:1366–1372. doi: 10.1016/j.biortech.2006.05.028 CrossRefGoogle Scholar
  23. Li S-F, Chen J-P, Wu W-T (2007b) Electrospun polyacrylonitrile nanofibrous membranes for lipase immobilization. J Mol Catal B 47:117–124. doi: 10.1016/j.molcatb.2007.04.010 CrossRefGoogle Scholar
  24. Li T, Li S, Wang N, Tain L (2008) Immobilization and stabilization of pectinase by multipoint attachment onto an activated agar-gel support. Food Chem 109:703–708. doi: 10.1016/j.foodchem.2008.01.012 CrossRefGoogle Scholar
  25. Lian P, Zhu X, Xiang H, Li Z, Yang W, Wang H (2010) Enhanced cycling performance of Fe3O4–graphene nanocomposite as an anode material for lithium-ion batteries. Electrochim Acta 56:834–840. doi: 10.1016/j.electacta.2010.09.086 CrossRefGoogle Scholar
  26. Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM, Fernandez-Lafuente R (2007) Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb Technol 40:1451–1463. doi: 10.1016/j.enzmictec.2007.01.018 CrossRefGoogle Scholar
  27. Mendes AA, de Castro HF, Andrade GSS, Tardioli PW, de Giordano RLC (2013) Preparation and application of epoxy–chitosan/alginate support in the immobilization of microbial lipases by covalent attachment. React Funct Polym 73:160–167. doi: 10.1016/j.reactfunctpolym.2012.08.023 CrossRefGoogle Scholar
  28. Olsson L, Hahn-Hägerdal B (1996) Fermentation of lignocellulosic hydrolysates for ethanol production. Enzyme Microb Technol 18:312–331. doi: 10.1016/0141-0229(95)00157-3 CrossRefGoogle Scholar
  29. Pan C, Hu B, Li W, Sun Y, Ye H, Zeng X (2009) Novel and efficient method for immobilization and stabilization of β-d-galactosidase by covalent attachment onto magnetic Fe3O4–chitosan nanoparticles. J Mol Catal B 61:208–215. doi: 10.1016/j.molcatb.2009.07.003 CrossRefGoogle Scholar
  30. Singh N, Srivastava G, Talat M, Raghubanshi H, Srivastava ON, Kayastha AM (2014) Cicer α-galactosidase immobilization onto functionalized graphene nanosheets using response surface method and its applications. Food Chem 142:430–438. doi: 10.1016/j.foodchem.2013.07.079 CrossRefGoogle Scholar
  31. Tang T, Fan H, Ai S, Han R, Qiu Y (2011) Hemoglobin (Hb) immobilized on amino-modified magnetic nanoparticles for the catalytic removal of bisphenol A. Chemosphere 83:255–264. doi: 10.1016/j.chemosphere.2010.12.075 CrossRefGoogle Scholar
  32. Teymourian H, Salimi A, Khezrian S (2013) Fe3O4 magnetic nanoparticles/reduced graphene oxide nanosheets as a novel electrochemical and bioelectrochemical sensing platform. Biosens Bioelectron 49:1–8. doi: 10.1016/j.bios.2013.04.034 CrossRefGoogle Scholar
  33. Wang J, Zheng S, Shao Y, Liu J, Xu Z, Zhu D (2010) Amino-functionalized Fe3O4@SiO2 core–shell magnetic nanomaterial as a novel adsorbent for aqueous heavy metals removal. J Colloid Interface Sci 349:293–299. doi: 10.1016/j.jcis.2010.05.010 CrossRefGoogle Scholar
  34. Wang S, Su P, Ding F, Yang Y (2013) Immobilization of cellulase on polyamidoamine dendrimer-grafted silica. J Mol Catal B 89:35–40. doi: 10.1016/j.molcatb.2012.12.011 CrossRefGoogle Scholar
  35. Wei H, Yang W, Xi Q, Chen X (2012) Preparation of Fe3O4@graphene oxide core–shell magnetic particles for use in protein adsorption. Mater Lett 82:224–226. doi: 10.1016/j.matlet.2012.05.086 CrossRefGoogle Scholar
  36. Wu JH, Ko SP, Liu HL, Jung M-H, Lee JH, Ju J-S, Kim YK (2008) Sub 5 nm Fe3O4 nanocrystals via coprecipitation method. Colloids Surf A 313–314:268–272. doi: 10.1016/j.colsurfa.2007.04.108 CrossRefGoogle Scholar
  37. Xu Y, Liu Z, Zhang X, Wang Y, Tian J, Huang Y, Ma Y, Zhang X, Chen Y (2009) A graphene hybrid material covalently functionalized with porphyrin: synthesis and optical limiting property. Adv Mater 21:1275–1279. doi: 10.1002/adma.200801617 CrossRefGoogle Scholar
  38. Yang K, Peng H, Wen Y, Li N (2010) Re-examination of characteristic FT-IR spectrum of secondary layer in bilayer oleic acid-coated Fe3O4 nanoparticles. Appl Surf Sci 256:3093–3097. doi: 10.1016/j.apsusc.2009.11.079 CrossRefGoogle Scholar
  39. Zhang Y, Wu H, Li L, Li J, Jiang Z, Jiang Y, Chen Y (2009) Enzymatic conversion of Baicalin into Baicalein by β-glucuronidase encapsulated in biomimetic core-shell structured hybrid capsules. J Mol Catal B 57:130–135. doi: 10.1016/j.molcatb.2008.08.005 CrossRefGoogle Scholar
  40. Zhu J, Xu M, Meng X, Shang K, Fan H, Ai S (2012a) Electro-enzymatic degradation of carbofuran with the graphene oxide–Fe3O4–hemoglobin composite in an electrochemical reactor. Process Biochem 47:2480–2486. doi: 10.1016/j.procbio.2012.10.006 CrossRefGoogle Scholar
  41. Zhu J, Xu M, Meng X, Shang K, Fan H, Ai S (2012b) Electro-enzymatic degradation of carbofuran with the graphene oxide–Fe3O4–hemoglobin composite in an electrochemical reactor. Process Biochem 47:2480–2486. doi: 10.1016/j.procbio.2012.10.006 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  1. 1.School of PharmacyJiangsu UniversityZhenjiangPeople’s Republic of China
  2. 2.School of Food and Biological EngineeringJiangsu UniversityZhenjiangPeople’s Republic of China

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