Skip to main content
SpringerLink
Log in

Cellulase immobilized by sol–gel entrapment for efficient hydrolysis of cellulose

  • Original Paper
  • Published:
Bioprocess and Biosystems Engineering Aims and scope Submit manuscript

Abstract

Cellulase from Trichoderma reesei (Celluclast 1.5 L, Novozyme) was immobilized by sol–gel encapsulation, using binary or ternary mixtures of tetramethoxysilane (TMOS) with alkyl- or aryl-substituted trimethoxysilanes as precursors. Optimization of immobilization conditions resulted in 92 % recovery of total enzymatic activity in the best immobilized preparate. The immobilized cellulase exhibiting the highest activity, obtained from tetramethoxysilane and methyltrimethoxysilane precursors at 3:1 molar ratio, was investigated in the hydrolysis reaction of microcrystalline cellulose (Avicel PH101). Although the optimal values did not change significantly, both temperature and pH stabilities of the sol–gel entrapped cellulase improved compared to the native enzyme. Immobilization also conferred superior resistance against the inactivation effect of glucose. Reuse of the sol–gel entrapped cellulase showed 40 % retention of the initial activity after five batch hydrolysis cycles, demonstrating the potential of this biocatalyst for large-scale application.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  1. Nigan PS, Singh A (2011) Production of liquid biofuels from renewable resources. Prog Energy Combust 37:52–68

    Article  Google Scholar 

  2. Mabee WE, Saddler JN (2010) Bioethanol from lignocellulosics: status and perspectives in Canada. Bioresour Technol 101:4806–4813

    Article  CAS  Google Scholar 

  3. Scott EL, Kootstra AMJ, Sanders JPM (2010) In: Singh OM, Harvey SP (eds) Sustainable biotechnology. Sources of renewable energy. Springer, Dordrecht

    Google Scholar 

  4. Naik SN, Goud VV, Rout PK, Dalai AK (2010) Production of first and second generation biofuels: a comprehensive review. Renew Sustain Energy Rev 14:578–597

    Article  CAS  Google Scholar 

  5. Jordan J, Kumar CSSR, Theegala C (2011) Preparation and characterization of cellulase-bound magnetite nanoparticles. J Mol Cat B: Enzymatic 68:139–146

    Article  CAS  Google Scholar 

  6. Dincer A, Telefoncu A (2007) Improving the stability of cellulase by immobilization on modified polyvinyl alcohol coated chitosan beads. J Mol Cat B: Enzymatic 45:10–14

    Article  CAS  Google Scholar 

  7. Jones PO, Vasudevan PT (2010) Cellulose hydrolysis by immobilized Trichoderma reesei cellulose. Biotechnol Lett 32:103–106

    Article  CAS  Google Scholar 

  8. Kharrat N, BenAli Y, Marzouk S, Gargouri Y, Karra-Châabouni M (2011) Immobilization of Rhizopus oryzae lipase on silica aerogels by adsorption: comparison with the free enzyme. Process Biochem 46:1083–1089

    Google Scholar 

  9. Pazarlioğlu NK, Sariişik M, Telefoncu A (2005) Treating denim fabrics with immobilized commercial cellulases. Process Biochem 40:767–771

    Article  Google Scholar 

  10. Sinegani AAS, Emtiazi G, Shariatmadari H (2005) Sorption and immobilization of cellulase on silicate clay minerals. J Coll Interface Sci 290:39–44

    Article  CAS  Google Scholar 

  11. Lozano P, Bernal B, Bernal JM, Pucheault M, Vaultier M (2011) Stabilizing immobilized cellulase by ionic liquids for saccharification of cellulose solutions in 1-butyl-3-methylimidazolium chloride. Green Chem 13:1406–1410

    Article  CAS  Google Scholar 

  12. Hartono SB, Qiao SZ, Liu J, Jack K, Ladewig BP, Hao Z, Lu GQM (2010) Functionalized mesoporous silica with very large pores for cellulase immobilization. J Phys Chem 114:8353–8362

    CAS  Google Scholar 

  13. Lupoi JS, Smith EA (2011) Evaluation of nanoparticle-stabilized cellulase for improved ethanol yield in simultaneous saccharification and fermentation reactions. Biotechnol Bioeng 108:2835–2843

    Article  CAS  Google Scholar 

  14. Takimoto A, Shiomi T, Ino K, Tsunoda T, Kawai A, Mizukami F, Sakaguchi K (2008) Encapsulation of cellulase with mesoporous silica (SBA-15). Micropor Mesopor Mat 116:601–606

    Article  CAS  Google Scholar 

  15. Darias R, Villalonga R (2001) Functional stabilization of cellulase by covalent modification with chitosan. J Chem Technol Biotechnol 76:489–493

    Article  CAS  Google Scholar 

  16. Xu J, Huo S, Yuan Z, Zhang Y, Xu H (2011) Characterization of direct cellulase immobilization with superparamagnetic nanoparticles. Biocat Biotransform 29:71–76

    Article  CAS  Google Scholar 

  17. Chang RH-Y, Jang J, Wu KC-W (2011) Cellulase immobilized mesoporous silica nanocrystals for efficient cellulose-to-glucose conversion. Green Chem 13:2844–2850

    Article  CAS  Google Scholar 

  18. Abd El-Ghaffar MA, Hashem MS (2010) Chitosan and its amino acids condensation adducts as reactive natural polymer supports for cellulase immobilization. Carbohydr Polym 81:507–516

    Article  CAS  Google Scholar 

  19. Mao X, Guo G, Huang J, Du Z, Huang Z, Ma L, Li P, Gu L (2006) A novel method to prepare chitosan powder and its application in cellulase immobilization. J Chem Technol Biotechnol 81:89–195

    Article  Google Scholar 

  20. Feng T, Du Y, Yang J, Li J, Shi X (2006) Immobilization of a nonspecific chitosan hydrolytic enzyme for application in preparation of water-soluble low-molecular-weight chitosan. J Appl Polym Sci 101:1334–1339

    Article  CAS  Google Scholar 

  21. Lee S-M, Jin LH, Kim JH, Han SO, Na HB, Hyeon T, Koo Y-M, Kim J, Lee J-H (2010) β-Glucosidase coating on polymer nanofibers for improved cellulosic ethanol production. Bioprocess Biosyst Eng 33:141–147

    Article  CAS  Google Scholar 

  22. Kourkoutas Y, Bekatorou A, Banat IM, Marchant R, Koutinas AA (2004) Immobilization technologies and support materials suitable in alcohol beverages production: a review. Food Microbiol 21:377–397

    Article  CAS  Google Scholar 

  23. Pierre AC (2004) The sol-gel encapsulation of enzymes. Biocatal Biotransform 22:145–170

    Article  CAS  Google Scholar 

  24. Vidinha P, Augusto V, Almeida M, Fonseca I, Fidalgo A, Ilharco L, Cabral JMS, Barreiros S (2006) Sol–gel encapsulation: an efficient and versatile immobilization technique for cutinase in non-aqueous media. J Biotechnol 121:23–33

    Article  CAS  Google Scholar 

  25. Zarcula C, Kiss C, Corîci L, Croitoru R, Csunderlik C, Peter F (2009) Combined sol-gel entrapment and adsorption method to obtain solid-phase lipase biocatalyst. Rev Chim (Bucharest) 60(9):922–927

    CAS  Google Scholar 

  26. Tomin A, Weiser D, Hellner G, Bata Z, Corici L, Peter F, Koczka B, Poppe L (2011) Fine-tuning the second generation sol–gel lipase immobilization with ternary alkoxysilane precursor systems. Process Biochem 46:52–58

    Article  CAS  Google Scholar 

  27. Das S, Berke-Schlessel D, Ji H-F, McDonough J, Wei Y (2011) Enzymatic hydrolysis of biomass with recyclable use of cellobiase enzyme immobilized in sol-gel routed mesoporous silica. J Mol Cat B: Enzymatic 70:49–54

    Article  CAS  Google Scholar 

  28. Paljevac M, Primožič M, Habulin M, Novak Z, Knez Z (2007) Hydrolysis of carboxymethyl cellulose catalyzed by cellulase immobilized on silica gels at low and high pressures. J Supercrit Fluids 43:74–80

    Article  CAS  Google Scholar 

  29. Andriani D, Sunwoo C, Ryu H-W, Prasetya B, Park D-H (2012) Immobilization of cellulase from newly isolated strain Bacillus subtilis TD6 using calcium alginate as a support material. Bioprocess Biosyst Eng 35:29–33

    Article  CAS  Google Scholar 

  30. Zarcula C, Corici L, Croitoru R, Ursoiu A, Peter F (2010) Preparation and properties of xerogels obtained by ionic liquid incorporation during the immobilization of lipase by the sol–gel method. J Mol Cat B: Enzymatic 65:79–86

    Article  CAS  Google Scholar 

  31. Kawakami K, Yoshida S (1996) Thermal stabilization of lipase by sol–gel entrapment in organically modified silicates formed on kieselguhr. J Ferment Bioeng 82:239–245

    Article  CAS  Google Scholar 

  32. Lowry OH, Rosebrough NJ, Farr AL, Randall RL (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    CAS  Google Scholar 

  33. Ghose TK (1987) Measurements of cellulase activities. Pure Appl Chem 2:257–268

    Article  Google Scholar 

  34. Miller GL (1972) Use of dinitrosalicylic acid reagent determination of reducing sugar. Anal Chem 31:426–428

    Article  Google Scholar 

  35. Berghem LER, Petterson LG, Axiö-Fredriksson U-B (1975) Characterization and enzymatic properties of a β-1,4-glucan cellobiohydrolase from Trichoderma viride. Eur J Biochem 53:55–62

    Article  CAS  Google Scholar 

  36. Corici LN, Frissen AE, van Zoelen D-J, Eggen IF, Peter F, Davidescu CM, Boeriu CG (2011) Sol-gel immobilization of Alcalase from Bacillus licheniformis for application in the synthesis of C-terminal peptide amides. J Mol Cat B: Enzymatic 73:90–97

    Article  CAS  Google Scholar 

  37. Turner MB, Spear SK, Huddleston JG, Holbrey JD, Rogers RD (2003) Ionic liquid salt-induced inactivation and unfolding of cellulase from Trichoderma reesei. Green Chem 5:443–447

    Article  CAS  Google Scholar 

  38. Zandoná Filho A, Siika-Aho M, Ramos LP (2006) Method for characterization of the enzyme profile and the determination of CBH I (Cel 7a) core protein in Trichoderma reesei cellulase preparations. World J Microbiol Biotechnol 22:821–825

    Article  Google Scholar 

  39. Bansal P, Hall M, Reallf MJ, Lee JH, Bommarius AS (2009) Modeling cellulase kinetics on lignocellulosic substrates. Biotechnol Adv 27:833–848

    Article  CAS  Google Scholar 

  40. Andrić P, Meyer SA, Jensen PA, Dam-Johansen K (2010) Effect of modeling of glucose inhibition and in situ glucose removal during enzymatic hydrolysis of pretreated wheat straw. Appl Biochem Biotechnol 160:280–297

    Article  Google Scholar 

  41. Bélafi-Bakó K, Koutinas A, Nemestóthy N, Gubicza L, Webb C (2006) Continuous enzymatic cellulose hydrolysis in a tubular membrane bioreactor. Enzyme Microb Technol 38:155–161

    Article  Google Scholar 

  42. Chang K, Thitikorn-amorn J, Chen S, Hsieh J, Ratanakhanokchai K, Huang P, Lin T, Chen S (2011) Improving the remaining activity of lignocellulolytic enzymes by membrane entrapment. Bioresour Technol 102:519–523

    Article  CAS  Google Scholar 

  43. Balsan G, Astolfi V, Benazzi T, Meireles MAA, Maugeri F, Di Luccio M, Dal Prá V, Mossi AJ, Treichel H, Mazutti MA (2012) Characterization of a commercial cellulase for hydrolysis of agroindustrial substrates. Bioprocess Biosyst Eng 35:1229–1237

    Article  CAS  Google Scholar 

  44. Bezerra RMF, Dias AA (2004) Discrimination among eight modified Michaelis-Menten kinetic models of cellulose hydrolysis with a large range of substrate/enzyme ratios. Appl Biochem Biotechnol 112:173–184

    Article  CAS  Google Scholar 

  45. Yu Y, Yuan J, Wang Q, Fan X, Wang P (2012) Covalent immobilization of cellulases onto a water-soluble-insoluble reversible polymer. Appl Biochem Biotechnol 166:1433–1441

    Article  CAS  Google Scholar 

  46. Li C, Yoshimoto M, Fukunaga K, Nakao K (2007) Characterization and immobilization of liposome-bound cellulase for hydrolysis of insoluble cellulose. Bioresour Technol 98:1366–1372

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was partially supported by the strategic grants POSDRU 2009, project ID 50783, of the Ministry of Labour, Family and Social Protection, and by the grant POSDRU/21/1.5/G/13798, inside POSDRU Romania 2007–2013, co-financed by the European Social Fund—Investing in People. Financial support of this work was provided by UEFISCDI through PNII-Parteneriate grant No. 21077, 2007–2010.

Author information

Authors and Affiliations

  1. Faculty of Industrial Chemistry and Environmental Engineering, University “Politehnica” of Timişoara, C. Telbisz 6, 300001, Timisoara, Romania

    Mihaela Ungurean, Cristina Paul & Francisc Peter

Authors
  1. Mihaela Ungurean
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cristina Paul
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Francisc Peter
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Francisc Peter.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ungurean, M., Paul, C. & Peter, F. Cellulase immobilized by sol–gel entrapment for efficient hydrolysis of cellulose. Bioprocess Biosyst Eng 36, 1327–1338 (2013). https://doi.org/10.1007/s00449-012-0835-9

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00449-012-0835-9

Keywords

Search

Navigation