Applied Biochemistry and Biotechnology

, Volume 175, Issue 6, pp 2872–2882 | Cite as

Impact of Enzyme Loading on the Efficacy and Recovery of Cellulolytic Enzymes Immobilized on Enzymogel Nanoparticles

  • Ashani Samaratunga
  • Olena Kudina
  • Nurun Nahar
  • Andrey Zakharchenko
  • Sergiy Minko
  • Andriy Voronov
  • Scott W. Pryor
Article

Abstract

Cellulase and β-glucosidase were adsorbed on a polyacrylic acid polymer brush grafted on silica nanoparticles to produce enzymogels as a form of enzyme immobilization. Enzyme loading on the enzymogels was increased to a saturation level of approximately 110 μg (protein) mg−1 (particle) for each enzyme. Enzymogels with varied enzyme loadings were then used to determine the impact on hydrolysis rate and enzyme recovery. Soluble sugar concentrations during the hydrolysis of filter paper and Solka-Floc with the enzymogels were 45 and 53 %, respectively, of concentrations when using free cellulase. β-Glucosidase enzymogels showed lower performance; hydrolyzate glucose concentrations were just 38 % of those using free enzymes. Increasing enzyme loading on the enzymogels did not reduce net efficacy for cellulase and improved efficacy for β-glucosidase. The use of free cellulases and cellulase enzymogels resulted in hydrolyzates with different proportions of cellobiose and glucose, suggesting differential attachment or efficacy of endoglucanases, exoglucanases, and β-glucosidases present in cellulase mixtures. When loading β-glucosidase individually, higher enzyme loadings on the enzymogels produced higher hydrolyzate glucose concentrations. Approximately 96 % of cellulase and 66 % of β-glucosidase were recovered on the enzymogels, while enzyme loading level did not impact recovery for either enzyme.

Keywords

Cellulase Enzyme immobilization Enzyme recovery Enzymatic hydrolysis Enzymogels 

References

  1. 1.
    Huber, G. W., Iborra, S., & Corma, A. (2006). Synthesis of transportation fuels from biomass: chemistry, catalysts, and engineering. Chemical Reviews, 106, 4044–4098.CrossRefGoogle Scholar
  2. 2.
    García, V., Päkkilä, J., Ojamo, H., Muurinen, E., & Keiski, R. L. (2011). Challenges in biobutanol production: how to improve the efficiency? Renewable and Sustainable Energy Reviews, 15, 964–980.CrossRefGoogle Scholar
  3. 3.
    Wyman, C. E. (2007). What is (and is not) vital to advancing cellulosic ethanol. Trends in Biotechnology, 25, 153–157.CrossRefGoogle Scholar
  4. 4.
    Wyman, C. E. (1994). Ethanol from lignocellulosic biomass: technology, economics, and opportunities. Bioresource Technology, 50, 3–15.CrossRefGoogle Scholar
  5. 5.
    Brittain, W. J., & Minko, S. (2007). A structural definition of polymer brushes. Journal of Polymer Science Part A: Polymer Chemistry, 45, 3505–3512.CrossRefGoogle Scholar
  6. 6.
    Czeslik, C., Jackler, G., Steitz, R., & von Grünberg, H.-H. (2004). Protein binding to like-charged polyelectrolyte brushes by counterion evaporation. The Journal of Physical Chemistry B, 108, 13395–13402.CrossRefGoogle Scholar
  7. 7.
    Dai, J., Bao, Z., Sun, L., Hong, S. U., Baker, G. L., & Bruening, M. L. (2006). High-capacity binding of proteins by poly(acrylic acid) brushes and their derivatives. Langmuir, 22, 4274–4281.CrossRefGoogle Scholar
  8. 8.
    de Vos, W. M., Biesheuvel, P. M., de Keizer, A., Kleijn, J. M., & Cohen Stuart, M. A. (2008). Adsorption of the protein bovine serum albumin in a planar poly(acrylic acid) brush layer as measured by optical reflectometry. Langmuir, 24, 6575–6584.CrossRefGoogle Scholar
  9. 9.
    Chen, H., & Hsieh, Y.-L. (2005). Enzyme immobilization on ultrafine cellulose fibers via poly(acrylic acid) electrolyte grafts. Biotechnology and Bioengineering, 90, 405–413.CrossRefGoogle Scholar
  10. 10.
    Hollmann, O., & Czeslik, C. (2006). Characterization of a planar poly(acrylic acid) brush as a materials coating for controlled protein immobilization. Langmuir, 22, 3300–3305.CrossRefGoogle Scholar
  11. 11.
    Wang, X., Xu, J., Li, L., Wu, S., Chen, Q., Lu, Y., Ballauff, M., & Guo, X. (2010). Synthesis of spherical polyelectrolyte brushes by thermo-controlled emulsion polymerization. Macromolecular Rapid Communications, 31, 1272–1275.CrossRefGoogle Scholar
  12. 12.
    Kudina, O., Zakharchenko, A., Trotsenko, O., Tokarev, A., Ionov, L., Stoychev, G., Puretskiy, N., Pryor, S., Voronov, A., & Minko, S. (2014). Highly efficient phase boundary biocatalysis with enzymogel nanoparticles. Angewandte Chemie International Edition, 53, 483–487.CrossRefGoogle Scholar
  13. 13.
    Simionescu, C. I., Dumitriu, S., Popa, M., Dumitriu, M., & Moldovan, F. (1984). Bioactive polymers. 28. Immobilization of invertase on carboxymethyl cellulose acid chloride. Polymer Bulletin, 12, 369–374.CrossRefGoogle Scholar
  14. 14.
    Gianfreda, L., & Bollag, J. M. (1994). Effect of soils on the behavior of immobilized enzymes. Soil Science Society of America Journal, 58, 1672–1681.CrossRefGoogle Scholar
  15. 15.
    Ghose, T. K. (1987). Measurement of cellulase activities. Pure and Applied Chemistry, 59, 257–268.Google Scholar
  16. 16.
    Nieves, R. A., Ehrman, C. I., Adney, W. S., Elander, R. T., & Himmel, M. E. (1997). Survey and analysis of commercial cellulase preparations suitable for biomass conversion to ethanol. World Journal of Microbiology and Biotechnology, 14, 301–304.CrossRefGoogle Scholar
  17. 17.
    DiCosimo, R., McAuliffe, J., Poulose, A. J., & Bohlmann, G. (2013). Industrial use of immobilized enzymes. Chemical Society Reviews, 42, 6437–6474.CrossRefGoogle Scholar
  18. 18.
    Bayramoglu, G., & Arica, M. Y. (2010). Reversible immobilization of catalase on fibrous polymer grafted and metal chelated chitosan membrane. Journal of Molecular Catalysis B: Enzymatic, 62, 297–304.CrossRefGoogle Scholar
  19. 19.
    Srere, P. A., & Ovadi, J. (1990). Enzyme-enzyme interactions and their metabolic role. FEBS Letters, 268, 360–364.CrossRefGoogle Scholar
  20. 20.
    Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.CrossRefGoogle Scholar
  21. 21.
    Ionov, L., & Minko, S. (2012). Mixed polymer brushes with locking switching. ACS Applied Materials & Interfaces, 4, 483–489.CrossRefGoogle Scholar
  22. 22.
    Stöber, W., Fink, A., & Bohn, E. J. (1968). Controlled growth of monodisperse silica spheres in the micron size range. Journal of Colloid and Interface Science, 26, 62–69.CrossRefGoogle Scholar
  23. 23.
    Zor, T., & Seliger, Z. (1996). Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Analytical Biochemistry, 236, 302–308.CrossRefGoogle Scholar
  24. 24.
    Sun, L., Dai, J., Baker, G. L., & Bruening, M. L. (2006). High-capacity, protein-binding membranes based on polymer brushes grown in porous substrates. Chemistry of Materials, 18, 4033–4039.CrossRefGoogle Scholar
  25. 25.
    Jain, P., Sun, L., Dai, J., Baker, G. L., & Bruening, M. L. (2007). High-capacity purification of his-tagged proteins by affinity membranes containing functionalized polymer brushes. Biomacromolecules, 8, 3102–3107.CrossRefGoogle Scholar
  26. 26.
    Gautrot, J. E., Huck, W. T. S., Welch, M., & Ramstedt, M. (2009). Protein-resistant NTA-functionalized polymer brushes for selective and stable immobilization of histidine-tagged proteins. ACS Applied Materials & Interfaces, 2, 193–202.CrossRefGoogle Scholar
  27. 27.
    Minko, S. (2006). Responsive polymer brushes. Journal Macromolecular Science, 46, 397–420.CrossRefGoogle Scholar
  28. 28.
    Cantarel, B. L., Coutinho, P. M., Rancurel, C., Bernard, T., Lombard, V., & Henrissat, B. (2009). The carbohydrate-active enzymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Research, 37, D233–D238.CrossRefGoogle Scholar
  29. 29.
    Huang, X. M., Yang, Q., Liu, Z. H., Fan, J. X., Chen, X. L., Song, J. Z., & Wang, Y. (2010). Cloning and heterologous expression of a novel endoglucanase gene egVIII from Trichoderma viride in Saccharomyces cerevisiae. Applied Biochemistry and Biotechnology, 162, 103–115.CrossRefGoogle Scholar
  30. 30.
    Thrane, C., Tronsmo, A., & Jensen, D. F. (1997). Endo-1,3-beta-glucanase and cellulase from Trichoderma harzianum: purification and partial characterization, induction of and biological activity against plant pathogenic Pythium spp. European Journal of Plant Pathology, 103, 331–344.CrossRefGoogle Scholar
  31. 31.
    Gregg, D. J., & Saddler, J. N. (1996). Factors affecting cellulose hydrolysis and the potential of enzyme recycle to enhance the efficiency of an integrated wood to ethanol process. Biotechnology and Bioengineering, 51, 375–383.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Ashani Samaratunga
    • 1
  • Olena Kudina
    • 2
  • Nurun Nahar
    • 1
  • Andrey Zakharchenko
    • 3
  • Sergiy Minko
    • 3
  • Andriy Voronov
    • 2
  • Scott W. Pryor
    • 1
  1. 1.Department of Agricultural and Biosystems EngineeringNorth Dakota State UniversityFargoUSA
  2. 2.Department of Coatings and Polymeric MaterialsNorth Dakota State UniversityFargoUSA
  3. 3.Nanostructured Materials LaboratoryUniversity of GeorgiaAthensUSA

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