Bioprocess and Biosystems Engineering

, Volume 41, Issue 7, pp 1051–1060 | Cite as

Immobilization of cellulase on thermo-sensitive magnetic microspheres: improved stability and reproducibility

  • Juan Han
  • Junhui Rong
  • Yun Wang
  • Qian Liu
  • Xu Tang
  • Cheng Li
  • Liang Ni
Research Paper


Magnetic double-shell hybrid microspheres (Fe3O4@SiO2@p(NIPAM-co-GMA)) have been developed as a promising supported substrate for the immobilization of cellulase. Since the surface of the magnetic microspheres not only contains an epoxy group from GMA (glycidyl methacrylate) that can covalently bind to the enzyme, but also has an intelligent temperature response property from NIPAM (N-isopropylacrylamide), the cellulase can be covalently bonded to the magnetic microspheres and have a temperature-sensitive capability. The immobilized cellulase has the recovery ability of cellulase activity after a high-temperature inactivation. The average amount and activity of immobilized enzymes, respectively, was 233 mg g−1, 57.4 U mg−1 under the optimized conditions. The experimental results show that the immobilized cellulase has a wider catalytic temperature range, better temperature and storage stability. The residual activity still remained about 65.6% of the initial activity after the sixth catalysis run, which indicated that the immobilized enzyme had high reproducibility.

Graphical abstract


Immobilized enzyme Cellulase Magnetic microspheres Thermo-sensitive 



This work was supported by the National Natural Science Foundation of China (nos. 21676124, 31470434 and 21576124), China Postdoctoral Science Foundation funded project (no. 2017M610308), and Jiangsu Postdoctoral Science Foundation funded project (no. 1701107B).


  1. 1.
    Klemm D, Heublein B, Fink HP, Bohn A (2005) Cellulose: fascinating biopolymer and sustainable raw material. Angew Chem Int Ed Engl 44:3358–3393CrossRefPubMedGoogle Scholar
  2. 2.
    Huber GW, Iborra S, Corma A (2006) Synthesis of transportation fuels from biomass: chemistry, catalysts, and engineering. Chem Rev 106:4044–4098CrossRefGoogle Scholar
  3. 3.
    Abraham RE, Verma ML, Barrow CJ, Puri M (2014) Suitability of magnetic nanoparticle immobilised cellulases in enhancing enzymatic saccharification of pretreated hemp biomass. Biotechnol Biofuels 7Google Scholar
  4. 4.
    Khoshnevisan K, Vakhshiteh F, Barkhi M, Baharifar H, Poor-Akbar E, Zari N, Stamatis H, Bordbar AK, Khoshnevisan K, Vakhshiteh F (2017) Immobilization of cellulase enzyme onto magnetic nanoparticles: applications and recent advances. 442, pp 66–73Google Scholar
  5. 5.
    Heidarizadeh M, Doustkhah E, Rostamnia S, Rezaei PF, Harzevili FD, Zeynizadeh B (2017) Dithiocarbamate to modify magnetic graphene oxide nanocomposite (Fe3O4-GO): a new strategy for covalent enzyme (lipase) immobilization to fabrication a new nanobiocatalyst for enzymatic hydrolysis of PNPD. Int J Biol Macromol 101:696–702CrossRefPubMedGoogle Scholar
  6. 6.
    Khoshnevisan K, Barkhi M, Ghasemzadeh A, Tahami HV, Pourmand S (2016) Fabrication of coated/uncoated magnetic nanoparticles to determine their surface properties. Mater Manuf Processes 31:1206–1215CrossRefGoogle Scholar
  7. 7.
    Lee YC, Dutta S, Wu KCW (2014) Integrated, cascading enzyme-/chemocatalytic cellulose conversion using catalysts based on mesoporous silica nanoparticles. Chemsuschem 7:3181–3181CrossRefGoogle Scholar
  8. 8.
    Das R, Mishra H, Srivastava A, Kayastha A (2017) Covalent immobilization of β-amylase onto functionalized molybdenum sulfide nanosheets, its kinetics and stability studies: A gateway to boost enzyme application. Chem Eng J 328:215–227CrossRefGoogle Scholar
  9. 9.
    Cho EJ, Jung S, Kim HJ, Lee YG, Nam KC, Lee HJ, Bae HJ (2012) Co-immobilization of three cellulases on Au-doped magnetic silica nanoparticles for the degradation of cellulose. Chem Commun 48:886–888CrossRefGoogle Scholar
  10. 10.
    Kamat RK, Ma WF, Yang YK, Zhang YT, Wang CC, Kumar CV, Lin Y (2013) Adsorption and hydrolytic activity of the polycatalytic cellulase nanocomplex on cellulose. ACS Appl Mater Interfaces 5:8486–8494CrossRefPubMedGoogle Scholar
  11. 11.
    Pan MR, Sun YF, Zheng J, Yang WL (2013) Boronic acid-functionalized core–shell–shell magnetic composite microspheres for the selective enrichment of glycoprotein. ACS Appl Mater Interfaces 5:8351–8358CrossRefPubMedGoogle Scholar
  12. 12.
    Yi DK, Selvan ST, Lee SS, Papaefthymiou GC, Kundaliya D, Ying JY (2005) Silica-coated nanocomposites of magnetic nanoparticles and quantum dots. J Am Chem Soc 127:4990–4991CrossRefPubMedGoogle Scholar
  13. 13.
    Khoshnevisan K, Barkhi M, Zare D, Davoodi D, Tabatabaei M (2012) Preparation and characterization of CTAB-coated Fe3O4 nanoparticles. Synth React Inorg M 42:644–648CrossRefGoogle Scholar
  14. 14.
    Bayramoglu G, Doz T, Ozalp VC, Arica MY (2017) Improvement stability and performance of invertase via immobilization on to silanized and polymer brush grafted magnetic nanoparticles. Food Chem 221:1442–1450CrossRefPubMedGoogle Scholar
  15. 15.
    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 189:404–412CrossRefGoogle Scholar
  16. 16.
    Huang F, Wang JZ, Qu AT, Shen LL, Liu JJ, Liu JF, Zhang ZK, An YL, Shi LQ (2014) Maintenance of amyloid beta peptide homeostasis by artificial chaperones based on mixed-shell polymeric micelles. Angew Chem Int Ed Engl 53:8985–8990CrossRefPubMedGoogle Scholar
  17. 17.
    Ganguli S, Yoshimoto K, Tomita S, Sakuma H, Matsuoka T, Shiraki K, Nagasaki Y (2009) Regulation of lysozyme activity based on thermotolerant protein/smart polymer complex formation. J Am Chem Soc 131:6549–6553CrossRefPubMedGoogle Scholar
  18. 18.
    Liu X, Liu Y, Zhang ZK, Huang F, Tao Q, Ma RJ, An YL, Shi LQ (2013) Temperature-responsive mixed-shell polymeric micelles for the refolding of thermally denatured proteins. Chem Eur J 19:7437–7442CrossRefPubMedGoogle Scholar
  19. 19.
    Ma WF, Xu SA, Li JM, Guo J, Lin Y, Wang CC (2011) Hydrophilic dual-responsive magnetite/PMAA core/shell microspheres with high magnetic susceptibility and pH sensitivity via distillation–precipitation polymerization. J Polym Sci Part A: Polym Chem 49:2725–2733CrossRefGoogle Scholar
  20. 20.
    Bourgeatlami E, Lang J (1999) Encapsulation of inorganic particles by dispersion polymerization in polar media. J Colloid Interface Sci 210:281CrossRefGoogle Scholar
  21. 21.
    Stöber W, Fink A, Bohn E (1968) Controlled growth of monodisperse silica spheres in the micron size range. J Colloid Interface Sci 26:62–69CrossRefGoogle Scholar
  22. 22.
    Ma W, Zhang Y, Li L, Zhang Y, Yu M, Guo J, Lu H, Wang C (2013) Ti4+-immobilized magnetic composite microspheres for highly selective enrichment of phosphopeptides. Adv Funct Mater 23:107–115CrossRefGoogle Scholar
  23. 23.
    Bai F, Xinlin Yang A, Huang W (2004) Synthesis of narrow or monodisperse poly(divinylbenzene) microspheres by distillation–precipitation polymerization. Macromolecules 37:3641–3649CrossRefGoogle Scholar
  24. 24.
    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 72:248–254CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Khoshnevisan K, Bordbar AK, 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 171:669–673CrossRefGoogle Scholar
  26. 26.
    IUPAC (2009) Measurement of cellulase activities. Pure Appl Chem 59:257–268Google Scholar
  27. 27.
    Pardo AG, Forchiassin F (1999) Influence of temperature and pH on cellulase activity and stability in Nectria catalinensis. Rev Argent Microbiol 31:31–35PubMedGoogle Scholar
  28. 28.
    Gokhale AA, Lu J, Lee I (2013) Immobilization of cellulase on magnetoresponsive graphene nano-supports. J Mol Catal B-Enzym 90:76–86CrossRefGoogle Scholar
  29. 29.
    Bayramoglu G, Senkal BF, Arica MY (2013) Preparation of clay-poly(glycidyl methacrylate) composite support for immobilization of cellulase. Appl Clay Sci 85:88–95CrossRefGoogle Scholar
  30. 30.
    Ungurean M, Paul C, Peter F (2013) Cellulase immobilized by sol–gel entrapment for efficient hydrolysis of cellulose. Bioproc Biosyst Eng 36:1327–1338CrossRefGoogle Scholar
  31. 31.
    Liu X, Liu Y, Zhang Z, Huang F, Tao Q, Ma R, An Y, Shi L (2013) Temperature-responsive mixed-shell polymeric micelles for the refolding of thermally denatured proteins. Chemistry 19:7437–7442CrossRefPubMedGoogle Scholar
  32. 32.
    Zang LM, Qiu JH, Wu XL, Zhang WJ, Sakai E, Wei Y (2014) Preparation of magnetic chitosan nanoparticles as support for cellulase immobilization. Ind Eng Chem Res 53:3448–3454CrossRefGoogle Scholar
  33. 33.
    Yan Q, Yuan JY, Yuan WZ, Zhou M, Yin YW, Pan CY (2008) Copolymer logical switches adjusted through core-shell micelles: from temperature response to fluorescence response. Chem Commun 6188–6190Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Juan Han
    • 1
  • Junhui Rong
    • 2
  • Yun Wang
    • 2
  • Qian Liu
    • 2
  • Xu Tang
    • 2
  • Cheng Li
    • 2
  • Liang Ni
    • 2
  1. 1.School of Food and Biological EngineeringJiangsu UniversityZhenjiangChina
  2. 2.School of Chemistry and Chemical EngineeringJiangsu UniversityZhenjiangChina

Personalised recommendations