Sheila Romo-Sánchez, Conrado Camacho, Héctor L. Ramirez, María Arévalo-Villena^*

Show more
Food Science and Technology, University of Castilla-La Mancha (UCLM), Albacete, Spain.
Biotechnology Center, University of Matanzas, Matanzas, Cuba.
Show less

Abstract: Industrial applications require enzymes highly stable and economically viable in terms of reusability. Enzyme immobilization is an exciting alternative to improve the stability of enzymatic processes. The immobilization of two commercial enzymes is reported here (cellulase and xylanase) using three chemical methods (adsorption, reticulation, and crosslinking-adsorption) and two polymeric supports (alginate-chitin and chitosan-chitin). The optimal pH for binding was 4.5 for cellulase and 5.0 for xylanase, and the optimal enzyme concentrations were 170 μg/mL and 127.5 μg/mL respectively, being the chitosan and the ideal support. In some cases, a low concentration of crosslinking agent (glutaraldehyde) improved stability of the immobilization process. Biotechnological characterization showed that the reusability of enzymes was the most striking finding, particularly of immobilized cellulase using glutaraldehyde, which after 19 cycles retained 64% activity. These results confirm the economic and biotechnical advantages of enzyme immobilization for a range of industrial applications.

Keywords: Cellulase, Crosslinking-Adsorption, Immobilization, Reticulation, Xylanase

Cite this paper: Romo-Sánchez, S. , Camacho, C. , Ramirez, H. and Arévalo-Villena, M. (2014) Immobilization of Commercial Cellulase and Xylanase by Different Methods Using Two Polymeric Supports. Advances in Bioscience and Biotechnology, 5, 517-526. doi: 10.4236/abb.2014.56062.

References

[1]   Díaz, A., Caro, I., Ory, I. and Blandino, A. (2005) Recovery of Hydrolytic Enzymes Obtained by Solid State Fermentation of Grape Pomace with Aspergillus awamori. Chemical Engineering Transactions, 7, 75.

[2]   Datta, S., Christena, L.R. and Rajaram, Y.R.S. (2013) Enzyme Immobilization: An Overview on Techniques and Support Materials. Biotech, 3, 1-9.

[3]   Cao, L. (2006) Carrier-Bound Immobilized Enzymes: Principles, Application and Design. Willey-VCH, New Jersey.

[4]   Arya, S.K. and Srivastava, S.K. (2006) Kinetics of Immobilized Cyclodextrin Gluconotransferase Produced by Bacillus macerans ATCC 8244. Enzyme and Microbial Technology, 39, 507-510.
http://dx.doi.org/10.1016/j.enzmictec.2005.12.019

[5]   Katchalski-Katzir, E. (1993) Immobilized Enzymes: Learning from Past Successes and Failures. Trends in Biotechnology, 11, 471-478.
http://dx.doi.org/10.1016/0167-7799(93)90080-S

[6]   Roy, I. and Gupta, M.N. (2006) Bioaffinity Immobilization. In: Jose, M.G., Ed., Immobilization of Enzymes and Cells, 2nd Edition, Humana Press Inc., New York.
http://dx.doi.org/10.1007/978-1-59745-053-9_10

[7]   D’Souza, S.F. (1998) Immobilized Enzymes in Bioprocess. Current Science, 77, 69-79

[8]   Butt, M.S., Tahir-Nadeem, M., Ahmad, Z. and Sultan, M.T. (2008) Xylanases and Their Applications in Baking Industry. Food Technology and Biotechnology, 46, 22-31.

[9]   Simon, O., Politz, O. and Borriss R. (1993) Improving the Characteristics of Bacterial β-Glucanases by Construction of Hybrid Enzymes. Proceedings of the 1st Symposium of Enzymes in Animal Nutrition, Kartause Ittingen, 13-16 October 1993, 22-28.

[10]   Liu, J., Yuan, X., Zeng, G., Shi, J. and Chen, S. (2006) Effect of Biosurfactant on Cellulase and Xylanase Production by Tricoderma viride in Solid Substrate Fermentation. Process Biochemistry, 41, 2347-2351.
http://dx.doi.org/10.1016/j.procbio.2006.05.014

[11]   Qu, Y., Zhu, M., Liu, K., Bao, X. and Lin, J. (2006) Studies on Cellulosic Ethanol Production for Sustainable Supply of Liquid Fuel in China. Biotechnology Journal, 1, 1235-1240.
http://dx.doi.org/10.1002/biot.200600067

[12]   Shen, X. and Xia, L. (2006) Lactic Acid Production from Cellulosic Material by Synergistic Hydrolysis and Fermentation. Applied Biochemistry and Biotechnology, 133, 252-262.
http://dx.doi.org/10.1385/ABAB:133:3:251

[13]   Cao, N.J., Xia, Y.K., Gong, C.S. and Tsao, G.T. (1997) Production of 2,3-Butanediol from Pretreated Corn Cob by Klebsiella oxytoca in the Presence of Fungal Cellulase. Applied Biochemistry and Biotechnology, 63-65, 129-139.
http://dx.doi.org/10.1007/BF02920419

[14]   Baxter, A., Dillon, M., Taylor, K.D.A. and Roberts, G.A.F. (1992) Improved Method for i.r. Determination of the Degree of N-Acetylation of Chitosan. International Journal of Biological Macromolecules, 14, 166-169.
http://dx.doi.org/10.1016/S0141-8130(05)80007-8

[15]   Kurita, K., Sannan, T. and Iwakura, Y. (1977) Studies on Chitin, 4: Evidence for Formation of Block and Random Copolymers of N-Acetyl-D-Glucosamine and D-Glucosamine by Hetero- and Homogeneous Hydrolyses. Die Makromolekulare Chemie, 178, 3197-3202.
http://dx.doi.org/10.1002/macp.1977.021781203

[16]   Beppu, M.M., Vieira, R.S., Aimoli, C.G. and Santana, C.C. (2007) Crosslinking of Chitosan Membranes Using Glutaraldehyde: Effect on Ion Permeability and Water Absorption. Journal of Membrane Science, 301, 126-130.
http://dx.doi.org/10.1016/j.memsci.2007.06.015

[17]   Gómez, L., Ramirez, H.L., Cabrera, G., Simpson, B.K. and Villalonga, R. (2008) Immobilization of Invertase-Chitosan Conjugate on Hyaluronic-Acid-Modified Chitin. Journal of Food Biochemistry, 32, 264-277.
http://dx.doi.org/10.1111/j.1745-4514.2008.00170.x

[18]   Bernath, F.R. and Venkatasubramanian, K. (1986) Methods of Enzyme Immobilization. In: Demain, A.L. and Solomon, N.A., Eds., Manual of Industrial Microbiology and Biotechnology, Vol. 19, 230-247.

[19]   Miller, G.L. (1959) Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Analytical Chemistry, 31, 426-428.
http://dx.doi.org/10.1021/ac60147a030

[20]   Bradford, M.M. (1976) A Rapid and Sensitive for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry, 72, 248-254.
http://dx.doi.org/10.1016/0003-2697(76)90527-3

[21]   Arévalo-Villena, M., úbeda-Iranzo, J.F., Gundllapalli, S.B., Cordero Otero, R.R. and Briones-Pérez, A.I. (2006) Characterization of an Exocellular β-Glucosidase from Debaryomyces pseudopolymorphus. Enzyme and Microbial Technology, 39, 229-234.
http://dx.doi.org/10.1016/j.enzmictec.2005.10.018

[22]   Chang, M.Y. and Juang, R.S. (2007) Use of Chitosan-Clay Composite as Immobilization Support for Improved Activity and Stability of β-Glucosidase. Biochemical Engineering Journal, 35, 93-98.
http://dx.doi.org/10.1016/j.bej.2007.01.003

[23]   Romo-Sánchez, S., Arévalo-Villena, M., García-Romero, E., Ramirez, H.L. and Briones-Pérez, A. (2013) Immobilization of β-Glucosidase and Its Application for Enhancement of Aroma Precursors in Muscat Wine. Food and Bioprocess Technology, 7, 1381-1392.
http://dx.doi.org/10.1007/s11947-013-1161-1

[24]   Busto, M.D., Ortega, N. and Perez-Mateos, M. (1997) Effect of Immobilization on the Stability of Bacterial and Fungal β-D-Glucosidase. Process Biochemistry, 32, 441-449.
http://dx.doi.org/10.1016/S0032-9592(96)00104-5

[25]   Pal, A. and Khanum, F. (2011) Covalent Immobilization of Xylanase on Glutaraldehyde Activated Alginate Beads Using Response Surface Methodology: Characterization of Immobilized Enzyme. Process Biochemistry, 46, 1315-1322.
http://dx.doi.org/10.1016/j.procbio.2011.02.024

[26]   Zhou, J. (2010) Immobilization of Cellulase on a Reversibly Soluble-Insoluble Support: Properties and Application. Journal of Agricultural and Food Chemistry, 58, 6741-6746.
http://dx.doi.org/10.1021/jf100759c

[27]   Chen, S.H., Yen, Y.H., Wang, C.L. and Wang, S.L. (2003) Reversible Immobilization of Lysozyme via Coupling Reversibly Soluble Polymer. Enzyme and Microbial Technology, 33, 643-649.
http://dx.doi.org/10.1016/S0141-0229(03)00186-8

[28]   Dincer, A. and Telefoncu, A. (2007) Improving the Stability of Cellulase by Immobilization on Modified Polyvinyl Alcohol Coated Chitosan Beads. Journal of Molecular Catalysis B: Enzymatic, 45, 10-14.
http://dx.doi.org/10.1016/j.molcatb.2006.10.005

[29]   Nagar, S., Mittal, A., Kumar, D., Kumar, L. and Gupta, V.K. (2012) Immobilization of Xylanase on Glutaraldehyde Activated Aluminum Oxide Pellets for Increasing Digestibility of Poultry Feed. Process Biochemistry, 47, 1402-1410.
http://dx.doi.org/10.1016/j.procbio.2012.05.013

[30]   Akkaya, B., Sahin, F., Demirel, G. and Tümtürk, H. (2009) Functional Polymeric Supports for Immobilization of Cholesterol Oxidase. Biochemical Engineering Journal, 43, 333-337.
http://dx.doi.org/10.1016/j.bej.2008.11.003

[31]   Monier, M., El-Sokkaty, A.M.A. and Sarhan, A.A. (2010) Immobilization of Candida rugosa Lipase on Modified Natural Wool Fibers. Reactive and Functional Polymers, 70, 122-128.
http://dx.doi.org/10.1016/j.reactfunctpolym.2009.11.004

[32]   Buchholz, K. (1992) Immobilized Enzymes. Kinetics, Efficiency, and Applications. International Journal of Chemical Engineering, 32, 1-13.

[33]   Kapoor, M. and Kuhad, R.C. (2007) Immobilization of Xylanase from Bacillus pumilus Strain MK001 and Its Application in Production of Xylo-Oligosaccharides. Applied Biochemistry and Biotechnology, 142, 125-138.
http://dx.doi.org/10.1007/s12010-007-0013-8

[34]   Wu, L., Yuan, X. and Sheng, J. (2005) Immobilization of Cellulase in Nanofibrous PVA Membranes by Electrospinning. Journal of Membrane Science, 250, 167-173.
http://dx.doi.org/10.1016/j.memsci.2004.10.024