Effect of cross-linked enzyme aggregates in hierarchically mesocellular mesoporous magnetic silica preparation conditions towards enzyme activity retention


  • Nurul Jannah Sulaiman Universiti Teknologi Malaysia
  • Roshanida Abdul Rahman Universiti Teknologi Malaysia
  • Rosli Md Illias Universiti Teknologi Malaysia




Fractional factorial design, Optimization, Magnetic silica, Cellulase, Xylanase


This work aimed to optimize ten preparation factors that might influence the cellulase and xylanase activity retention of cross-linked enzyme aggregates in hierarchically mesocellular mesoporous magnetic silica. The factors were optimized using the fractional factorial design (210-5). The optimized output was occurred at 2 mL of enzyme amount, magnet-to-enzyme ratio of 1:0.15, enzyme adsorption at 26°C and 162 rpm in 40 min, enzyme-to-precipitant ratio of 1:11, 0.05% (v/v) of glutaraldehyde concentration, and cross-linking process at 37°C and 300 rpm in 2 h. The factors were examined to observe the effect of every factor towards cellulase and xylanase acrivity retention.

Author Biographies

Nurul Jannah Sulaiman, Universiti Teknologi Malaysia

Department of Bioprocess and Polymer Engineering, Faculty of Chemical and Energy Engineering

Roshanida Abdul Rahman, Universiti Teknologi Malaysia

Department of Bioprocess and Polymer Engineering, Faculty of Chemical and Energy Engineering

Rosli Md Illias, Universiti Teknologi Malaysia

Department of Bioprocess and Polymer Engineering, Faculty of Chemical and Energy Engineering


Agyei, D., He, L. (2015). Evaluation of cross-linked enzyme aggregates of Lactobacillus cell-envelope proteinases, for protein degradation. Food and Bioproducts Processing, 94, 59–69.

Arakawa, T., Kita, Y., Timasheff, S. N. (2007). Protein precipitation and denaturation by dimethyl sulfoxide. Biophysical Chemistry, 131, 62–70.

Azevedo, H., Bishop, D., Cavaco-Paulo, A. (2000). Effects of agitation level on the adsorption, desorption, and activities on cotton fabrics of full length and core domains of EGV (Humicola insolens) and CenA (Cellulomonas fimi ). Enzyme and Microbial Technology, 27, 325–329.

Baig, K., Turcotte, G., Doan, H. (2016). Adsorption of cellulose enzymes on lignocellulosic materials and influencing factors : A Review. International Journal of Waste Resources, 6(3), 27–31.

Cetinus, S. A., Öztop, H. N. (2003). Immobilization of catalase into chemically crosslinked chitosan beads. Enzyme and Microbial Technology, 32, 889–894.

Chen, H., Zhang, Q., Dang, Y., Shu, G. (2013). The effect of glutaraldehyde cross-linking on the enzyme activity of immobilized β-galactosidase on chitosan bead. Advanced Journal of Food Science and Technology, 5(7), 932–935.

Cui, J. D., Li, L. L., Bian, H. J. (2013). Immobilization of cross-linked phenylalanine ammonia lyase aggregates in microporous silica gel. PloS One, 8(11), e80581.

Deveci, I., Dogac, Y. I., Teke, M., Mercimek, B. (2015). Synthesis and characterization of chitosan / TiO2 Composite beads for improving stability of porcine pancreatic lipase. Applied Biochemistry and Biotechnology, 175(October), 1052–1068.

Ghose, T. K. (1987). Measurement of cellulase activities. Pure and Applied Chemistry, 59(2), 257—268.

Ghose, T. K., Bisaria, V. S. (1987). Measurement of hemicellulase activities part 1 : Xylanases. Pure and Applied Chemistry, 59(12), 1739-1752.

Gupta, K., Jana, A. K., Kumar, S., Maiti, M. (2013). Immobilization of amyloglucosidase from SSF of Aspergillus niger by crosslinked enzyme aggregate onto magnetic nanoparticles using minimum amount of carrier and characterizations. Journal of Molecular Catalysis B: Enzymatic, 98, 30–36.

Illanes, A. (2008). Enzyme Biocatalysis: Principles and Applications. Valparaiso: Springer Science + Business Media B.V.

Jiang, Y., Shi, L., Huang, Y., Gao, J., Zhang, X., Zhou, L. (2014). Preparation of robust biocatalyst based on cross-linked enzyme aggregates entrapped in three-dimensionally ordered macroporous silica. ACS Applied Materials and Interfaces, 6(1), 2622–2628.

Jun, S.-H., Lee, J., Kim, B. C., Lee, J. E., Joo, J., Park, H., Lee, J. H., Lee, S.-M., Lee, D., Kim, S., Koo, Y.-M., Shin, C. H., Kim, S. W., Hyeon, T., Kim, J. (2012). Highly efficient enzyme immobilization and stabilization within meso-structured onion-like silica for biodiesel production. Chemistry of Materials, 24(5), 924–929.

Kazenwadel, F., Wagner, H., Rapp, B. E., Franzreb, M. (2015). Analytical methods. Analytical Methods, 7, 10291–10298.

Kim, D. W., Kim, T. S., Jeong, Y. K., Lee, J. K. (1992). Adsorption kinetics and behaviors of cellulase components on microcrystalline cellulose. Journal of Fermentation and Bioengineering, 73(6), 461–455.

Kim, Y. H., Lee, I., Choi, S. H., Lee, O. K., Shim, J., Lee, J., Kim, J., Lee, E. Y. (2013). Enhanced stability and reusability of marine epoxide hydrolase using ship-in-a-bottle approach with magnetically-separable mesoporous silica. Journal of Molecular Catalysis B: Enzymatic, 89, 48–51.

Kim, M. I., Kim, J., Lee, J., Jia, H., Na, H. B., Youn, J. K., Kwak, J. H., Dohnalkova, A., Grate, J. W., Wang, P., Hyeon, T., Park, H. G., Chang, H. N. (2007). Crosslinked enzyme aggregates in hierarchically-ordered mesoporous silica : A simple and effective method for enzyme stabilization. Biotechnology and Bioengineering, 96(2), 210–218.

Kramer, R. M., Shende, V. R., Motl, N., Pace, C. N., Scholtz, J. M. (2012). Toward a molecular understanding of protein solubility : Increased negative surface charge correlates with increased solubility. Biophysical Journal, 102(8), 1907–1915.

Kumar, L., Reddy, M. S., Managuli, R. S., K., P. G. (2015). Full factorial design for optimization, development and validation of HPLC method to determine valsartan in nanoparticles. Saudi Pharmaceutical Journal, 23(5), 549–555.

Lee, J., Na, H. B., Kim, B. C., Lee, J. H., Lee, B., Kwak, J. H., Hwang, Y., Park, J.-G., Gu, M. N., Kim, J., Joo, J., Shin, C.-H., Grate, J. W., Hyeon, T., Kim, J. (2009). Magnetically-separable and highly-stable enzyme system based on crosslinked enzyme aggregates shipped in magnetite-coated mesoporous silica. Journal of Materials Chemistry, 19(42), 7864.

Lee, S. (1999). Molecular Adsorption at Solid / Liquid Interfaces Using Self-Assembled Monolayer Films. Massachusetts Institute of Technology.

Li, B., Dong, S. L., Xie, X. L., Xu, Z. B., Li, L. (2012). Preparation and Properties of cross-linked enzyme aggregates of cellulase. Advanced Materials Research, 581–582, 257–260.

McCue, J. T. (2009). Theory and Use of Hydrophobic Interaction Chromatography in Protein Purification Applications. (R. R. Burgess & M. P. Deutscher, Eds.), Methods in Enzymology (2nd ed., Vol. 463). Massachusetts: Elsevier Inc.

Melo, R. R. D., Alnoch, R. C., Vilela, A. F. L., Souza, E. M. D., Krieger, N., Ruller, R., Sato, H. H., Mateo, C. (2017). New Heterofunctional supports based on glutaraldehyde-activation : A Tool for enzyme immobilization at neutral pH. Molecules, 22(1088), 1–18.

O’Neill, A., Araujo, R., Casal, M., Guebitz, G., Cavaco-paulo, A. (2007). Effect of the agitation on the adsorption and hydrolytic efficiency of cutinases on polyethylene terephthalate fibres. Enzyme and Microbial Technology, 40, 1801–1805.

Radva, D., Spanyol, J., Kosáry, J. (2011). Testing of the effect of reaction parameters on the enzyme immobilization by adsorption and cross-linking processes with kinetic desorption method. Food Technology and Biotechnology, 49(2), 257–262.

Salwanee, S., Aida, W. M. W., Mamot, S., Maskat, M. Y., Ibrahim, S. (2013). Effects of Enzyme concentration, temperature, ph and time on the degree of hydrolysis of protein extract from viscera of tuna (euthynnus affinis) by using alcalase. Sains Malaysiana, 42(3), 279–287.

Sukri, S. S. M., Munaim, M. S. A. (2017). Combination of entrapment and covalent binding techniques for xylanase immobilisation on alginate beads : Screening process parameters. Chemical Engineering Transactions, 56, 169–174.

Sulaiman, N. J., Rahman, R. A. (2016). New advancement on cross-linked enzyme aggregates within magnetically-separable mesoporous silica. Applied Mechanics and Materials, 818, 276–280.

Taskila, S., Ahokas, M., Järvinen, J., Toivanen, J., Tanskanen, J. P. (2017). Concentration and separation of active proteins from potato industry waste based on low-temperature evaporation and ethanol precipitation. Scientifica, 2017(5120947), 1-6.

Wang, S., Meng, X., Zhou, H., Liu, Y., Secundo, F., Liu, Y. (2016). Enzyme stability and activity in non-aqueous reaction systems : A mini review. Catalysts, 6(32), 1–16.

Zheng, Y., Zhang, S., Miao, S., Su, Z., Wang, P. (2013). Temperature sensitivity of cellulase adsorption on lignin and its impact on enzymatic hydrolysis of lignocellulosic biomass. Journal of Biotechnology, 166, 135–143.