Applied Microbiology and Biotechnology

, Volume 97, Issue 2, pp 681–692 | Cite as

Reversible immobilization of glucoamylase onto magnetic chitosan nanocarriers

  • Jianzhi Wang
  • Guanghui Zhao
  • Yanfeng LiEmail author
  • Xiao Liu
  • Pingping Hou
Biotechnologically relevant enzymes and proteins


A simple preparation process for the monodispersed pH-sensitive core-shell magnetic microspheres was carried out consisting of chitosan self-assembled on magnetic iron oxide nanoparticles. Meanwhile, glucoamylase was immobilized as a model enzyme on this carrier of Fe3O4/CS microspheres by ionic adsorption. The morphology, inner structure, and high magnetic sensitivity of the resulting magnetic chitosan microspheres were studied, respectively, with a field emission scanning electron microscope (SEM), transmission electron microscope (TEM), FT-IR spectroscopy, thermogravimetric analysis (TGA), and a vibrating sample magnetometer (VSM). Subsequently, the properties of glucoamylase immobilized on the regenerated supports were also investigated by determining storage stability, pH stability, reusability, magnetic response, and regeneration of supports. The results from characterization and determination remarkably indicated that the immobilized glucoamylase obtained presents excellent storage stability, pH stability, reusability, magnetic response, and regeneration of supports. Therefore, this kind of magnetic Fe3O4/CS microspheres with perfect monodispersity should be an ideal support for enzyme immobilization.


Magnetic chitosan Ionic adsorption Immobilization Reversible Glucoamylase 



The authors would like to acknowledge the financial supports from the National Natural Science Foundation of China (No. 21074049), the National Natural Science Foundation for the scientific research ability training of undergraduate students majoring in chemistry by the two patters based on the tutorial system and top students (J1103307), and the Opening Foundation of State Key Laboratory of Applied Organic Chemistry (SKLAOC-2009-35).


  1. Arica MY, Yavuz H, Patir S, Adil D (2000) Immobilization of glucoamylase onto spacer-arm attached magnetic poly methylmethacrylate microspheres: characterization and application to a continuous flow reactor. J Mol Catal B: Enzym 11:127–138CrossRefGoogle Scholar
  2. Bahar T, Celebi SS (1999) Immobilization of glucoamylase on magnetic poly(styrene) particles. J Appl Polym Sci 72:69–73CrossRefGoogle Scholar
  3. Bahar T, Celebi SS (2000) Preparation of low-cost magnetic nitrocellulose microbeads. React Funct Polym 45:235–242CrossRefGoogle Scholar
  4. Bai YX, Li YF, Lin L (2009) Synthesis of a mesoporous functional copolymer bead carrier and its properties for glucoamylase immobilization. Appl Microbiol Biot 83:457–464CrossRefGoogle Scholar
  5. Belessi V, Zboril R, Tucek J, Mashlan M, Tzitzios V, Petridis D (2008) Ferrofluids from magnetic chitosan hybrids. Chem Mater 20:3298–3305CrossRefGoogle Scholar
  6. Bomatí-Miguel O, Tartaj P, Morales MP, Bonville P, Golla-Schindler U, Zhao XQ, Veintemillas-Verdaguer S (2006) Core-shell iron–iron oxide nanoparticles synthesized by laser-induced pyrolysis. Small 2:1476–1483CrossRefGoogle Scholar
  7. Bornscheuer UT (2003) Immobilizing enzymes: how to create more suitable biocatalysts. Angew Chem Int Ed 42:3336–3337CrossRefGoogle Scholar
  8. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. J Mang Magn Mater 72:248–254Google Scholar
  9. Crabb WD, Mitchinson C (1997) Enzymes involved in the processing of starches to sugars. Trends Biotechnol 15:349–352CrossRefGoogle Scholar
  10. Cui M, Wang FJ, Shao ZQ, Lu FS, Wang WJ (2011) Influence of DS of CMC on morphology and performance of magnetic microcapsules. Cellulose 18:1265–1271CrossRefGoogle Scholar
  11. Domínguez de María P, Sinisterra JV, Tsai SW, Alcántara AR (2006) Carica papaya lipase (CPL): an emerging and versatile biocatalyst. Biotechnol Adv 24:493–499CrossRefGoogle Scholar
  12. Dong HC, Venkat M, Krzysztof M (2009) Thermally responsive PM(EO)2MA magnetic microgels via activators generated by electron transfer atom transfer radical polymerization in miniemulsion. Chem Mater 21:3965–3972CrossRefGoogle Scholar
  13. Francis Suh JK, Howard WT (2000) Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review. Biomaterials 21:2589–2598CrossRefGoogle Scholar
  14. Fuentes M, Pessela BCC, Maquiese JV, Ortiz C, Segura RL, Palomo JM, Abian O, Torres R, Mateo C, Fernández-Lafuente R, Guisán JM (2004a) Reversible and strong immobilization of proteins by ionic exchange on supports coated with sulfate-dextran. Biotechnol Prog 20:1134–1139CrossRefGoogle Scholar
  15. Fuentes M, Maquiese JV, Pessela BCC, Abian O, Fernández-Lafuente R, Mateo C, Guisán JM (2004b) New cationic exchanger support for reversible immobilization of proteins. Biotechnol Prog 20:284–288CrossRefGoogle Scholar
  16. Gan Q, Wang T (2007) Chitosan nanoparticle as protein delivery carrier—systematic examination of fabrication conditions for efficient loading and release. Colloid Surface B 59:24–34CrossRefGoogle Scholar
  17. Ganesh Kumar A, Perinbam K, Kamatchi P, Nagesh N, Sekaran G (2010) In situ immobilization of acid protease on mesoporous activated carbon packed column for the production of protein hydrolysates. Bioresource Technol 101:1377–1379CrossRefGoogle Scholar
  18. Gao WW, Chan JM, Farokhzad OC (2010) pH-responsive nanoparticles for drug delivery. Mol Pharmaceut 6:1913–1920CrossRefGoogle Scholar
  19. Garcia-Galan C, Berenguer-Murcia A, Fernandez-Lafuente R, Rodrigues RC (2011) Potential of different enzyme immobilization strategies to improve enzyme performance. Adv Synth Catal 353:2885–2904CrossRefGoogle Scholar
  20. Ge JP, Hu YX, Biasini M, Beyermann WP, Yin YD (2007) Superparamagnetic magnetite colloidal nanocrystal clusters. Angew Chem Int Ed 46:4342–4345CrossRefGoogle Scholar
  21. Jiang DS, Long SY, Huang J, Xiao HY (2005) Immobilization of Pycnoporus sanguineus laccase on magnetic chitosan microspheres. Biochem Eng J 25:15–23CrossRefGoogle Scholar
  22. Lei ZL, Pang XL, Na L, Lin L, Li YL (2009) A novel two-step modifying process for preparation of chitosan-coated Fe3O4/SiO2 microspheres. J Mater Process Tech 209:3218–3225CrossRefGoogle Scholar
  23. Li GY, JiangYR HKL, Ding P, Chen J (2008) Preparation and properties of magnetic Fe3O4–chitosan nanoparticles. J Alloy Compd 466:451–456CrossRefGoogle Scholar
  24. Liu XW, Hu QY, Fang Z, Zhang XJ, Zhang BB (2009) Magnetic chitosan nanocomposites: a useful recyclable tool for heavy metal ion removal. Langmuir 25:3–8CrossRefGoogle Scholar
  25. Lu AH, Salabas EL, Schiith F (2007) Magnetic nanoparticles: synthesis, protection, functionalization, and application. Angew Chem Int Ed 46:1222–1224CrossRefGoogle Scholar
  26. MacLaughlin FC, Mumper RJ, Wang JU, Tagliaferri JM, Gill I, Hinchcliffe M, Rolland AP (1998) Chitosan and depolymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery. J Control Rel 56:259–272CrossRefGoogle Scholar
  27. Mateo C, Abian O, Fernandez-Lafuente R, Guisán JM (2000a) Reversible enzyme immobilization via a very strong and nondistorting ionic adsorption on support-polyethylenimine composites. Biotechnol Bioeng 68:98–105CrossRefGoogle Scholar
  28. Mateo C, Abian O, Fernandez-Lafuente R, Guisán JM (2000b) Increase in conformational stability of enzymes immobilized on epoxy-activated supports by favoring additional multipoint covalent attachment. Enzyme Microb Tech 26:509–515CrossRefGoogle Scholar
  29. Mateo C, Palomo JM, Fernandez-Lorente G, Guisán JM, Fernandez-Lafuente R (2007) Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb Tech 40:1451–1463CrossRefGoogle Scholar
  30. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 81:426–428CrossRefGoogle Scholar
  31. Mohapatra S, Pal D, Ghosh SK, Pramanik P, Nanosci J (2007) Design of superparamagnetic iron oxide nanoparticle for purification of recombinant proteins. Nanotechnol 7:3193–319Google Scholar
  32. Montes T, Grazu V, López-Gallego F, Hermoso JA, Guisán JM, Fernández-Lafuente R (2006) Chemical modification of protein surfaces to improve their reversible enzyme immobilization on ionic exchangers. Biomacromolecules 7:3052–3058CrossRefGoogle Scholar
  33. Nakamura T, Ogata Y, Akichika S, Shitara A, Nakamura A, Ohta K (1995) Continuous production of fructose syrups from inulin by immobilized inulinase from Aspergillus niger mutant 817. J Ferment and Bioeng 80:164–165CrossRefGoogle Scholar
  34. Oh JT, Kim JH (2000) Preparation and properties of immobilized amyloglucosidase on nonporous PS/PNaSS microspheres. Enzyme Microb Tech 27:356–361CrossRefGoogle Scholar
  35. Osman B, Kara A, Beşirli N (2011) Immobilization of glucoamylase onto lewis metal ion chelated magnetic affinity sorbent: kinetic, isotherm and thermodynamic studies. J Macromol Sci A 48:387–399Google Scholar
  36. Pan CL, Hu B (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: Enzym 61:208–215CrossRefGoogle Scholar
  37. Park J, Yosun HW, Park JG, Noh HJ, Kim JY, Park JH (2004) Ultra-large-scale syntheses of monodisperse nanocrystals. Nat Mater 3:891–895CrossRefGoogle Scholar
  38. Peng ZG, Hidajat K, Uddin MS (2004) Adsorption of bovine serum albumin on nanosized magnetic particles. J Colloid Interf Sci 271:277–283CrossRefGoogle Scholar
  39. Recillas M, Silva LL, Peniche C, Goycoolea FM, Rinaudo M, Argüelles-Monal WM (2009) Thermoresponsive behavior of chitosan-g-N-isopropylacrylamide copolymer solutions. Biomacromolecules 10:1633–1641CrossRefGoogle Scholar
  40. Salgın S, Takaç S, Özdamar TH (2006) Adsorption of bovine serum albumin on polyether sulfone ultrafiltration membranes: determination of interfacial interaction energy and effective diffusion coefficient. J Membrane Sci 278:251–260CrossRefGoogle Scholar
  41. Shamim N, Hong L, Hidajat K, Uddin MS (2006) Thermosensitive-polymer-coated magnetic nanoparticles: adsorption and desorption of bovine serum albumin. J Colloid Interf Sci 304:1–8CrossRefGoogle Scholar
  42. Tanriseven A, Uludağ YB, Doğan Ş (2002) A novel method for the immobilization of glucoamylase to produce glucose from maltodextrin. Enz Microbiol Tech 30:406–409CrossRefGoogle Scholar
  43. Torres R, Pessela BCC, Mateo C, Ortiz C, Fuentes M, Guisán JM, Fernandez-Lafuente R (2004) Reversible immobilization of glucoamylase by ionic adsorption on sepabeads coated with polyethyleneimine. Biotechnol Prog 20:1297–1300CrossRefGoogle Scholar
  44. Tsai ZT, Wang JF, Kuo HY, Shen CR, Wang JJ, Yen TC (2009) 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 322:208–213CrossRefGoogle Scholar
  45. Wang X, Zhuang J, Peng Q, Li YD (2005) A general strategy for nanocrystal synthesis. Nature 437:121–124CrossRefGoogle Scholar
  46. Wang F, Guo C, Liu HZ, Liu CZ (2007) Reversible immobilization of glucoamylase by metal affinity adsorption on magnetic chelator particles. J Mol Catal B: Enzym 48:1–7CrossRefGoogle Scholar
  47. Wang YJ, Wang XH, Luo GS, Dai YY (2008) Adsorption of bovin serum albumin (BSA) onto the magnetic chitosan nanoparticles prepared by a microemulsion system. Bioresource Technol 99:3881–3884CrossRefGoogle Scholar
  48. Woo K, Lee HJ, Ahn JP, Park YS (2003) Sol-Gel mediated synthesis of Fe2O3 nanorods. Adv Mater 15:1761–1764CrossRefGoogle Scholar
  49. Wu Y, Wang TJ, Luo GS, Dai YY (2009) In situ preparation of magnetic Fe3O4–chitosan nanoparticles for lipase immobilization by cross-linking and oxidation in aqueous solution. Bioresource Technol 100:3459–3464CrossRefGoogle Scholar
  50. Xu XY, Yu ZM, Zhu YW, Wang BC (2005) Effect of sodium oleate adsorption on the colloidal stability and zeta potential of detonation synthesized diamond particles in aqueous solutions. Diam Relat Mater 14:206–212CrossRefGoogle Scholar
  51. Yamamoto H, Amaike M (1997) Biodegradation of cross-linked chitosan gels by a microorganism. Macromolecules 30:3936–3937CrossRefGoogle Scholar
  52. Yang Y, Bai YX, Li YF, Xia CG (2008) Preparation and application of polymer-grafted magnetic nanoparticles for lipase immobilization. J Magn Magn Mater 320:2350–2355CrossRefGoogle Scholar
  53. Yu SY, Hu JH, Pan XY, Pi Y, Jiang M (2006) Stable and pH-sensitive nanogels prepared by self-assembly of chitosan and ovalbumin. Langmuir 22:2754–2759CrossRefGoogle Scholar
  54. Yuan Q, Venkatasubramanian R, Hein S, Misra RDK (2008) A stimulus-responsive magnetic nanoparticle drug carrier: magnetite encapsulated by chitosan-grafted-copolymer. Acta Biomater 4:1024–1037CrossRefGoogle Scholar
  55. Zhao ZP, Wang Z, Wang SC (2003) Formation, charged characteristic and BSA adsorption behavior of carboxymethyl chitosan/PES composite MF membrane. J Membrane Sci 217:151–158CrossRefGoogle Scholar
  56. Zhao GH, Wang JZ, Li YF, Chen X, Liu YP (2011a) Enzymes immobilized on superparamagnetic Fe3O4@Clays nanocomposites: preparation, characterization, and a new strategy for the regeneration of supports. J Phys Chem C 115:6350–6359CrossRefGoogle Scholar
  57. Zhao GH, Li YF, Wang JZ, Zhu H (2011b) Reversible immobilization of glucoamylase onto magnetic carbon nanotubes functionalized with dendrimer. Appl Microbiol Biotechnol 91:591–601CrossRefGoogle Scholar
  58. Zhi J, Wang YJ, Lu YC, Ma JY, Luo GS (2006) In situ preparation of magnetic chitosan/Fe3O4 composite nanoparticles in tiny pools of water-in-oil microemulsion. React Funct Polym 66:1552–1558CrossRefGoogle Scholar
  59. Zhu LZ, Ma JW, Jia NQ, Zhao Y, Shen HB (2009) Chitosan-coated magnetic nanoparticles as carriers of 5-fluorouracil: preparation, characterization and cytotoxicity studies. Colloid Surface B 68:1–6CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Jianzhi Wang
    • 1
  • Guanghui Zhao
    • 1
  • Yanfeng Li
    • 1
    Email author
  • Xiao Liu
    • 1
  • Pingping Hou
    • 1
  1. 1.State Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Institute of Biochemical Engineering & Environmental TechnologyLanzhou UniversityLanzhouChina

Personalised recommendations