Browse > Article
http://dx.doi.org/10.9713/kcer.2011.49.6.840

Optimization of Anion-exchange Chromatography for the Separation of Agarase from Culture Broth of Pseudoalteromonas sp.  

Kim, Yu-Na (Department of Chemical & Biological Engineering and ERI, Gyeongsang National University)
Lee, Jae-Ran (Department of Chemical & Biological Engineering and ERI, Gyeongsang National University)
Kim, Mu-Chan (Department of Marine Environmental Engineering, Gyeongsang National University)
Kim, Sung-Bae (Department of Chemical & Biological Engineering and ERI, Gyeongsang National University)
Chang, Yong-Keun (Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology)
Hong, Soon-Kwang (Division of Bioscience and Bioinformatics, Myong Ji University)
Kim, Chang-Joon (Department of Chemical & Biological Engineering and ERI, Gyeongsang National University)
Publication Information
Korean Chemical Engineering Research / v.49, no.6, 2011 , pp. 840-845 More about this Journal
Abstract
Degradation products of agarose are biologically active and thus used as an ingredient in pharmaceuticals or functional cosmetics. Furthermore, it has been strongly considered as a substrate for bio-ethanol fermentation. Recently, we isolated new agarase-producing microorganism, Pseudoalteromonas sp. from south sea of Korea. In this study, we aimed to separate and purify the agarase from culture broth of this strain. Separation of agarase was performed by ion- exchange chromatography on DEAE-Sepharose resin. Equilibrium pH and volume ratio of resin to the amount of protein were optimized for the efficient adsorption of protein. 410 ${\mu}g$ of protein was completely adsorbed to 3 mL of resin at pH 7.5. The total amount of eluted protein increased as NaCl concentration increased to 400 mM at isocratic elution. Agarase was separated by linear gradient elution of NaCl (0~1,000 mM). Three major protein peaks were observed and the presence or absence of agarase in these eluted proteins was measured by Lugol's staining technique. Only six eluted protein fractions showed strong agarase activity.
Keywords
Pseudoalteromonas sp.; Agarase; Separation; Anion-Exchange Chromatography; Optimization;
Citations & Related Records
Times Cited By KSCI : 1  (Citation Analysis)
연도 인용수 순위
1 Lahaye, M. and Rochas, C., "Chemical Structure and Physicochemical Properties of Agar," Hydrobiol., 221, 137-148(1991).   DOI   ScienceOn
2 Michel, G., Nyval-Collen, P., Barbeyron, T., Czjzek, M. and Helbert, W., "Bioconversion of Red Seaweed Galactans: A Focus on Bacterial Agarases and Carrageenases," Appl. Microbiol. Biotechnol., 71, 23-33(2006).   DOI   ScienceOn
3 Ting, X. and Kim, S. M., "Agarase: Review of Major Sources, Categories, Purification Method, Enzyme Chracteristics and Applications," Mar. Drugs, 8, 200-218(2010).   DOI   ScienceOn
4 Flament, D., Barbeyron, T., Jam, M., Potin, P., Czjzek, M., Kloareg, B. and Michel, G., "Alpha-agarase Define a New Family of Glycoside Hydrolases, Distinct from Beta-agarase Families," Appl. Environ. Microbiol., 73(14), 4691-4694(2007).   DOI   ScienceOn
5 Ohta, Y., Hatada, Y., Miyazaki, M., Nogi, Y., Ito, S. and Horikoshi, K., "Purification and Characterization of a Novel a-agarase from a Thalassomonas sp.," Curr. Microbiol., 50, 212-216 (2005).   DOI   ScienceOn
6 Potin, P., Richard, C., Rochas, C. and Kloareg, B., "Purification and Characterization of the $\alpha$-agarase from Alteromonas agarlyticus (Cataldi) comb. nov. Strain GJ1B," Eur. J. biochem., 214, 599- 607 (1993).   DOI   ScienceOn
7 Lakshmikanth, M., Manohar, S., Souche, Y. and Lalitha, J., "Extracellular $\beta$-agarase LSL-1 Producing Neoagarobiose from a Newly Isolated Agar-liquefying Soil Bacterium, Acinetobacter sp., AG LSL-1", World J. Microbiol. Biotechnol., 22, 1087-1094 (2006).   DOI   ScienceOn
8 Wang, J., Mou, H., Jiang, X. and Guan, H., "Characterization of a Novel $\beta$-agarase from Marine Alteromonas sp. SY37-12 and Its Degrading Products", Appl. Microbiol. Biotechnol., 71, 833-839 (2006).   DOI   ScienceOn
9 Jam, M., Flament, D., Allouch, J., Potin, P., Thion, L., Kloareg, B., Czjzek, M., Helbert, W., Michel, G. and Barbeyron, T., "The Endo-$\beta$-agarases AgaA and AgaB from the Marine Bacterium Zobellia galactanivorans: Two Paralogue Enzymes with Different Molecular Organizations and Catalytic Behaviours", Biochem. J., 385, 703-713(2005).   DOI   ScienceOn
10 Schroeder, D. C., Jaffer, M. A. and Coyne, V. E., "Investigation of the Role of a $\beta$(1-4) Agarase Produced by Pseudoalteromonas gracillis B9 in Eliciting Disease Symptoms in the Red Alga Gracilaria Gracillis", Microbiol., 149, 2919-2929(2003).   DOI   ScienceOn
11 Hatada, Y., Ohta, Y. and Horikoshi, K., "Hyperproduction and Application of $\alpha$-agarase to Enzymatic Enhancement of Antioxidant Activity of Porphyran," J. Agric. Food Chem., 54, 9895- 9900(2006).   DOI   ScienceOn
12 Chen, H.-M., Zheng, L. and Yan, X.-J., "The Preparation and Bioactivity Research of Agaro-Oligosaccharides," Food Technol. Biotechnol. 43(1), 29-36(2005).
13 Shin, M. H., Lee, D. Y., Wohlgemuth, G., Choi, I.-G., Fiehn, O. and Kim, K. H., "Global Metabolite Profiling of Agarose Degradation by Saccharophagus degradans 2-40," New Biotechnol. 27(2), 156-168(2010).   DOI   ScienceOn
14 Lee, S.-M., Yu, B. J., Kim, Y. M., Choi, S.-J., Ha, J.-M., Lee, J.- H., "Production of Bio-ethanol from Agar using Saccharomyces cerevisiae," J. Kor. Ind. Eng. Chem. 20(3), 290-295(2009).
15 Lim, D.-J., Kim, B.-J., Bae, S.-K., Kim, J.-D. and Kong, J.-Y., "Immobilization of Agarase for the Agarooligosaccharide Production," Kor. J. Appl. Microbiol. Biotechnol. 27(3), 208-214 (1999).
16 Lakshmikanth, M., Manohar, S. and Lalitha, J., "Purification and Characterization of $\beta$-agarase from Agar-liquefying Soil Bacterium, Acinetobacter sp., AG LSL-1," Proc. Biochem. 44, 999-1003 (2009).   DOI   ScienceOn
17 Sugano, Y., Terada, I., Arita, M., Noma, M. and Matsumoto, T., "Purification and Characterization of a New Agarase from a Marine Bacterium, Vibrio sp. Strain JT0107," Appl. Environ. Microbiol., 59(5), 1549-1554(1993).
18 Hassairi, I., Amar, R. B., Nonus, M. and Gupta, B. B., "Production and Separation of $\alpha$-agarase from Altermonas agarlyticus Strain GJ1B," Biores. Technol., 79, 47-51(2001).   DOI   ScienceOn
19 Ghadi, S. C., Muraleedharan, U. D. and Jawaid, S., "Screening for Agarolytic Bacteria and Development of a Novel Method for in Situ Detection of Agarase," J. Mar. Biotechnol. 5, 194-200 (1997).
20 Toussaint, G., Ding, L. H., Jaffrina, M. Y., Hassairib, I. and Nonus, M., "Recovery of $\alpha$-agarase Enzyme from Fermentation Broths by Membrane Crossflow Filtration, " Sep. Sci. Technol, 35(6), 759- 809(2000).
21 Fu, W., Han, B., Duan, D., Liu, W. and Wang, C., "Purification and Characterization of Agarase from a Marine Bacterium Vibrio sp. F-6," J. Ind. Microbiol. Biotechnol. 35, 915-922(2008).   DOI   ScienceOn
22 Fu, X. T., Lin, H. and Kim, S. M., "Purification and Characterization of a Novel $\beta$-agarase, AgaA34, from Agarivorans albus YKW-34," Appl. Microbiol. Biotechnol. 78, 265-273(2008).   DOI   ScienceOn
23 Levison, P. R., "Large-scale Ion-exchange Column Chromatography of Proteins Comparison of Different Formats, " J. Chromatogr. B, 790, 17-33(2003).   DOI   ScienceOn
24 Yamamoto, S. and Ishihara, T., "Ion-exchange Chromatography of Proteins Near the Isoelectric Points," Chromatogr. A, 852, 31- 36(1999).   DOI   ScienceOn
25 Johansson, H. J., Jagersten, C. and Shiloach, J., "Large Scale Recovery and Purification of Periplasmic Recombinant Protein from E. coli Using Expanded Bed Adsorption Chromatography Followed by New Ion Exchange Media," J. Biotechnol., 48, 9-14 (1996).   DOI   ScienceOn
26 Kupke, T., Stevanovic, S., Sahl, H.-G. and Gotz, F., "Purification and Characterization of EpiD, a Flavoprotein Involved in the Biosynthesis of the Lantibiotic Epidermin," J. Bacteriol., 174, 5354- 5361(1992).   DOI