Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Enzymatic Deacetylation of Chitin by Extracellular Chitin Deacetylase from a Newly Screened Mortierella sp. DY-52

  • Kim, Young-Ju (Glucosamine Saccharide Materials-National Research Laboratory (GSM-NRL), Division of Applied Bioscience and Biotechnology, Institute of Agricultural Science and Technology, Chonnam National University) ;
  • Zhao, Yong (Glucosamine Saccharide Materials-National Research Laboratory (GSM-NRL), Division of Applied Bioscience and Biotechnology, Institute of Agricultural Science and Technology, Chonnam National University) ;
  • Oh, Kyung-Taek (Glucosamine Saccharide Materials-National Research Laboratory (GSM-NRL), Division of Applied Bioscience and Biotechnology, Institute of Agricultural Science and Technology, Chonnam National University) ;
  • Nguyen, Van-Nam (Glucosamine Saccharide Materials-National Research Laboratory (GSM-NRL), Division of Applied Bioscience and Biotechnology, Institute of Agricultural Science and Technology, Chonnam National University) ;
  • Park, Ro-Dong (Glucosamine Saccharide Materials-National Research Laboratory (GSM-NRL), Division of Applied Bioscience and Biotechnology, Institute of Agricultural Science and Technology, Chonnam National University)
  • Published : 2008.04.30
Download PDF
⟨ Previous Next ⟩

Abstract

Among more than a hundred colonies of fungi isolated from soil samples, DY-52 has been screened as an extracellular chitin deacetylase (CDA) producer. The isolate was further identified as Mortierella sp., based on the morphological properties and the nucleotide sequence of its 18S rRNA gene. The fungus exhibited maximal growth in yeast peptone glucose (YPD) liquid medium containing 2% of glucose at pH 5.0 and $28^{\circ}C$ with 150 rpm. The CDA activity of DY-52 was maximal (20 U/mg) on the 3rd day of culture in the same medium. The CDA was inducible by addition of glucose and chitin. The enzyme contained two isoforms of molecular mass 50 kDa and 59 kDa. This enzyme showed a maximal activity at pH 5.5 and $60^{\circ}C$. In addition, it had a pH stability range of 4.5-8.0 and a temperature stability range of $4-40^{\circ}C$. The enzyme was enhanced in the presence of $Co^{2+}$ and $Ca^{2+}$. Among various substrates tested, WSCT-50 (water-soluble chitin, degree of deacetylation 50%), glycol chitin, and crab chitosan (DD 71-88%) were deacetylated. Moreover, the CDA can handle N-acetylglucosamine oligomers $(GlcNAc)_{2-7}$.

Keywords

References

  1. Alfonso, C., O. M. Nuero, F. Santamaria, and F. Reyes. 1995. Purification of a heat-stable chitin deacetylase from Aspergillus nidulans and its role in cell wall degradation. Curr. Microbiol. 30: 49-54 https://doi.org/10.1007/BF00294524
  2. Araki, Y. and E. Ito. 1975. A pathway of chitosan formation in Mucor rouxii. Eur. J. Biochem. 55: 71-78 https://doi.org/10.1111/j.1432-1033.1975.tb02139.x
  3. Aruchami, M., N. Gowri, and G. Sundara-Rajulu. 1986. Chitin deacetylases in invertebrates, pp. 263-265. In R. A. A. Muzzarelli, C. Jeuniaux, and G. W. Gooday (eds.), Chitin in Nature and Technology. Plenum Press, New York, U.S.A.
  4. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  5. Cai, J., J. H. Yang, Y. M. Du, L. H. Fan, Y. L. Qiu, J. Li, and J. F. Kennedy. 2006. Purification and characterization of chitin deacetylase from Scopulariopsis brevicaulis. Carbohydr. Polym. 1-7
  6. Chang, K. L., G. Tsai, J. Lee, and W. R. Fu. 1997. Heterogeneous N-deacetylation of chitin in alkaline solution. Carbohydr. Res. 303: 327-332 https://doi.org/10.1016/S0008-6215(97)00179-1
  7. Gao, X., T. Katsumoto, and K. Onoder. 1995. Purification and characterization of chitin deacetylase from Absidia coerulea. J. Biochem. 117: 257-263 https://doi.org/10.1093/jb/117.2.257
  8. Hekmat, O., K. Tokuyasu, and S. G. Withers. 2003. Subsite structure of the endo-type chitin deacetylase from Deuteromycete, Colletotrichum lindemuthianum: An investigation using studystate kinetic analysis and MS. Biochem. J. 374: 369-380 https://doi.org/10.1042/BJ20030204
  9. Imoto, T. and K. Yagishita. 1971. A simple activity measurement of lysozyme. Agric. Biol. Chem. 35: 1154-1156 https://doi.org/10.1271/bbb1961.35.1154
  10. Jeuniaux, C. 1982. La chitine dans le regne animal. Bull. Soc. Zool. Fr. 107: 363-386
  11. Kadokura, K., A. Rokutani, M. Yamamoto, T. Ikegami, H. Sugita, S. Itoi, W. Hakamata, T. Oku, and T. Nishio. 2006. Purification and characterization of Vibrio parahaemolyticus extracellular chitinase and chitin oligosaccharide deacetylase involved in the production of heterodisaccharide from chitin. Appl. Microbiol. Biotechnol. 75: 357-365 https://doi.org/10.1007/s00253-006-0831-6
  12. Kafetzopoulos, D., A. Martinou, and V. Bouriotis. 1993. Bioconversion of chitin to chitosan: Purification and characterization of chitin deacetylase from Mucor rouxii. Proc. Natl. Acad. Sci. USA 90: 2564-2568
  13. Kauss, H., W. Jeblick, and D. H. Young. 1983. Chitin deacetylase from the plant pathogen Colletotrichum lindemuthianum. Plant Sci. Lett. 28: 231-236 https://doi.org/10.1016/S0304-4211(83)80014-5
  14. Kuk, J. H., W. J. Jung, K. Y. Kim, and R. D. Park. 2005. Enzymatic characteristics and applications of microbial chitin deacetylase. Kor. J. Microbiol. Biotechnol. 33: 9-15
  15. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 https://doi.org/10.1038/227680a0
  16. Martinou, A., D. Koutsioulis, and V. Bouritiotis. 2002. Expression, purification, and characterization of a cobalt-activated chitin deacetylase (Cda2p) from Saccharomyces cerevisiae. Prot. Expr. Purif. 24: 111-116 https://doi.org/10.1006/prep.2001.1547
  17. Martinou, A., I. Tsigos, and V. Bouriotis. 1997. Preparation of chitosan by enzymatic deacetylation, pp. 501-505. In R. A. A. Muzzarelli and M. G. Peter (eds.), Chitin Handbook. Eur. Chitin Soc., Atec, Grottammare, Italy
  18. Nahar, P., V. Ghormade, and V. Deshpande. 2004. The extracellular constitutive production of chitin deacetylase in Metarhizium anisopliae: Possible edge to entomopathogenic fungi in the biological control of insect pests. J. Invertebr. Pathol. 85: 80-88 https://doi.org/10.1016/j.jip.2003.11.006
  19. Ruiz-Herrera, J. 1978. The distribution and quantitative importance of chitin in fungi, pp. 11-21. In R. A. A. Muzzarelli and E. R. Pariser (eds.), Proceedings of the 1st International Conference on Chitin and Chitosan, Massachusetts Institute of Technology, Cambridge
  20. Sannan, T., K. Kurita, and Y. Iwakura. 1975. Studies on chitin. 1. Solubility change by alkaline treatment and film casting. Makromol. Chem. 176: 1191-1195 https://doi.org/10.1002/macp.1975.021760426
  21. Shin, W. S., J. C. Kil, and G. M. Park. 2006. Antiprotozoal activity of deacetylated chitosan oligosaccharide (dp 2-8) on Trichomonas vaginalis. J. Microbiol. Biotechnol. 16: 1984-1989
  22. Srinivasan, V. R. 1998. Biotransformation of chitin to chitosan. US Patent 5739015
  23. Stevens, W. F., N. N. Win, C. H. Ng, C. Pichyangkura, and S. Chandrkrachang. 1997. Towards technical biocatalytic deacetylation of chitin, pp. 40-47. In A. Domard, G. A. F. Roberts, and K. M. Varum (eds.), Advances in Chitin Science, Vol. 2. Jacques Andre, Lyon
  24. Toei, K. and T. Kokara. 1976. A conductometric method for colloid titrations. Anal. Chem. Acta 83: 59-65 https://doi.org/10.1016/S0003-2670(01)84631-6
  25. Tokuyasu, K., M. O. Kameyama, and K. Hayashi. 1996. Purification and characterization of extracellular chitin deacetylase from Colletotrichum lindemuthianum. Biosci. Biotech. Biochem. 60: 1598-1603 https://doi.org/10.1271/bbb.60.1598
  26. Trudel, J. and A. Asselain. 1990. Detection of chitin deacetylase activity after polyacrylamide gel electrophoresis. Anal. Biochem. 189: 249-253 https://doi.org/10.1016/0003-2697(90)90116-Q
  27. Tsigos, I. and V. Bouriotis. 1995. Purification and characterization of chitin deacetylase from Colletotrichum lindemuthianum. J. Biol. Chem. 270: 26286-26291 https://doi.org/10.1074/jbc.270.44.26286
  28. Win, N. N. and W. F. Stevens. 2001. Shrimp chitin as substrate for fungal chitin deacetylase. Appl. Microbiol. Biotechnol. 57: 334-341 https://doi.org/10.1007/s002530100741
  29. White, T. J., T. Bruns, S. Lee, and J. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, pp. 315-322. In M. Innis, D. Gelfand, J. Sninsky, and T. White (eds.), A Guide to Methods and Applications. Academic Press, San Diego, U.S.A.