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Biochemical characteristics of chitosanase from the Indonesian Bacillus licheniformis MB-2

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Abstract

Bacillus licheniformis MB-2, isolated from a hot spring water in Manado, Indonesia, secreted a unique chitosanase. Media consisted of 0.24% chitosan, 0.25% casiton, 1% MgSO4, 1.4% K2HPO4, 0.02% CaCl2·2H2O, 0.002% FeSO4·7H2O (w/v) was used for enzyme production. Purification of the enzyme through the hydrophobic interaction chromatography system (butyl Sepharose 4 FF) resulted in two major active fractions; the F2 fraction was shown as a single band at both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram analysis with apparent molecular mass of 75 kDa. The enzyme worked best at 70°C and pH between 6.0 and 7.0. When incubated at 70, 80, and 90°C, the t1/2 values were 26.56, 18.44, and 16.74 min, respectively with the k constant being at 0.026, 0.037, and 0.04/min. When heated at 90°C, the enzyme retained its activity up to 8 h in the presence of 1mM MnCl2. The enzyme's activity was unaffected by the presence of 1 M NaCl and 6 M urea but was decreased by 2 M of guanidine hydrochloride. Albeit the enzyme did not degrade colloidal and glycol chitin, it hydrolyzed glycol chitosan up to 0.8% and colloidal chitosan up to 11%. The 85% deacetylated (DDA) soluble chitosan was the most susceptible to this enzyme, followed by 90% and 100% DDA chitosan. The K m app values of the 85, 90, and 100% DDA soluble chitosans were found as 0.23, 0.24, and 0.58 mg/mL, whereas the Vmax values were 843, 668, and 261 U/mg, respectively. The hydrolysis products of F2 chitosanase at 24 h incubation (70°C) were pentasaccharide (GlcN)5 and hexasaccharide (GlcN)6. The prelimiaary test showed inhibitory effect of chitooligosaccharides resulted from enzymatic degradation toward Pseudomonas aeruginosa, Salmonella typhimurium. Listeria monocytogenes, Bacillus cereus, Escherichia coli, and Staphylococcus aureus.

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References

  1. Sanford, P. T. (2003) World market of chitin and its derivatives. In: Advances in Chitin Science, vol. VI (Varum, K. M., Domard, A., and Smidsrod, O, eds.). Trondheim, Norway.

  2. Fukamizo, T. and Brzezinski, R. (1997) Chitosanase from Streptomyces sp. strain N174; a comparative review of its structure and function. Biochem. Cell Biol. 75, 687–696.

    Article  PubMed  CAS  Google Scholar 

  3. Izume, M., Nagae, S., Kawagishi, H., Mitsutomi, M., and Ohtakara, A. (1992) Action pattern of Bacillus sp. No. 7-M chitosanase on partially N-acetylated chitosan. Biosci. Biotechnol. Biochem. 56, 448–453.

    PubMed  CAS  Google Scholar 

  4. Tanabe, T., Morinaga, K., Fukamizo, T., and Mitsutomi, M. (2003) Novel chitosanase from Streptomyces griseus HUT 6037 with transglycosylation activity. Biosci. Biotechnol. Biochem. 67, 354–364.

    Article  PubMed  CAS  Google Scholar 

  5. Choi, Y. J., Kim, E. J., Piao, Z., Yun, Y. C., and Shin, Y. C. (2004) Purification and characterization of chitosanase from Bacillus sp. strain KCTC 0377BP and its application for the production of chitosan oligosaccharides. Appl. Env. Microbiol. 70(8), 4522–4531.

    Article  CAS  Google Scholar 

  6. Henrissat, B. (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 293, 781–788.

    Google Scholar 

  7. Henrissat, B. and Bairoch, A. (1993) New families in the classification of glycosyl hydrolases based on amino acids sequences similarities. Biochem. J. 3, 781–788.

    Google Scholar 

  8. Henrissat, B. and Bairoch, A. (1996) Updating the sequence-based classification of glycosyl hydrolases. Biochem. J. 316, 695–696.

    PubMed  Google Scholar 

  9. Jayanti, J. F. L. (2002) Thermostable chitinase and chitin deacetylase from Manado isolates. Report Thesis, Department of Food Technology and Human Nutrition, Bogor Agricultural University.

  10. Trudel, J. and Asselin, A. (1989) Detection of chitinase activity after polyacrylamide gel electrophoresis. Anal. Biochem. 178, 302–366.

    Article  Google Scholar 

  11. Park, J. K., Shimono, K., Ochiai, N., et al. (1999) Purification, characterization and gene analysis of a chitosanase (Cho A) from Matsuebacter chitosanotabidus 3001. J. Bateriol. 181, 6642–6649.

    CAS  Google Scholar 

  12. Cullimore, D. R. (2000) Practical Atlas for Bacterial Identification, 2nd ed. CRC Press LLC., Boca Raton, FL.

    Google Scholar 

  13. Sambrook, J., Fritsch, E. F., Maniatis, T. (2001) Molecular Cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Google Scholar 

  14. Van de Peer, Y., and de Watcher, R. (1993) Treecon: a software package for the construction and drawing of evolution trees. Comput. Appl. Biosci. 9, 177–182.

    PubMed  Google Scholar 

  15. Yoon, H. G., Kim, H. Y., Kim, H. K., Hong, B. S., Shin, D. H., and Cho, H. Y. (2001) Thermostable chitosanase from Bacillus sp. strain CK4: its purification, characterization and reaction patterns. Biosci. Biotechnol. Biochem. 65, 802–809.

    Article  PubMed  CAS  Google Scholar 

  16. Uchida, Y. and Ohtakara, A. (1998) Chitosanase from Bacillus species. Methods Enzymol. 161, 501–506.

    Article  Google Scholar 

  17. Guttenberger, M. (1994) Protein Determination. Cell Biology: A Laboratory Manual, vol. 3. (Celis, J. E., ed.) Academic Press.

  18. Smith, B. J. (1984) SDS Polyacrylamide Gel Electrophoresis of Proteins. Methods in Molecular Biology, vol. 1 (Walker, J. M., ed.), Humana Press, Totowa, NJ.

    Google Scholar 

  19. Grenier, J., Trudel, J., and Asselin, A. (1997) Gel Electrophoretic Analysis of Chitinase, Chitosanase and Chitin Deacetylase, Chitin Handbook (Muzzarelli, R. A. A. and Peter, M. G., eds.) Atec, Grottammare, Italy.

    Google Scholar 

  20. Spindler, K.D. and Rapp, C. (1997) Detection of Chitin Degrading Enzymes on Gels. Chitin Handbook (Muzzarelli, R. A. A. and Peter, M. G., eds.) Atec, Grottammare, Italy.

    Google Scholar 

  21. Carson, C. F. and Riley, T. V. (1995) Antimicrobial activity of the major component of essential oil of melaleuca alternifolia. J. Appl. Bacteriol. 78, 264–269.

    PubMed  CAS  Google Scholar 

  22. Somashekar, D. and Joseph, R. (1996) Chitosanases: properties and applications: a review. Bioresource Technol. 55, 35–45.

    Article  CAS  Google Scholar 

  23. Cheng, C. Y. and Li, Y. K. (2000) An Aspergillus chitosanase with potential for large-scale preparation of chitosan oligosaccharides. Biotechnol. Appl. Biochem. 32, 197–203.

    Article  PubMed  CAS  Google Scholar 

  24. Boucher, I. A., Dupuy, P., Vidal, W. A., Neugebauer, and Brzezinski, R. (1992) Purification and characterisation of a chitosanase from Streptomyces N 174. Appl. Microbiol. Biotechnol. 38, 188–193.

    Article  CAS  Google Scholar 

  25. Pedraza-Reyes, M. and Gutierrez-Corona F. (1997) The bifunctional enzyme chitosanase-cellulase produced by the gram negative microorganism Mycobacter sp. AL-1 is highly similar to Bacillus subtilis endoglucanases. Arch. Microbiol. 168, 321–327.

    Article  PubMed  CAS  Google Scholar 

  26. Yoshihara, K., Hosokawa, J., Kubo, T., Nishiyama, M., and Koba, Y. (1992) Purification and properties of a chitosanase from Pseudomonas sp. H-14. Biosci. Biotech. Biochem. 56(6), 972–973.

    CAS  Google Scholar 

  27. Kurakake, M., Yo-U, S., Nakagawa, K., Sugihara, M., and Komaki, T. (2000) Properties of chitosanase from Bacillus cereus S1. Curr. Microbiol. 40, 6–9.

    Article  PubMed  CAS  Google Scholar 

  28. Seino, H., Tsukuda, K., and Shimasue, Y. (1991) Properties and action pattern of a chitosanase from Bacillus sp. PI-7S. Agric. Biol. Chem. 55. 2421–2423.

    CAS  Google Scholar 

  29. Yabuki, M. (1988) Characterization of chitosanase produced by Bacillus circulans MH-K1. Chitin and Chitosan (Gudmund Skjak-Braek, T. Anthousen, P. Sanford, eds.) Proceeding from the 4th Int. Conf. on Chitin and Chitosan. Norway, August 22–24. 1988.

  30. Yamasaki, Y., Hayashi, i., Ohta, Y., Nakagawa, T., Kawamukai, M., and Matsuda, H. (1992) Purification and mode of action of chitosanolytic enzyme from Enterobacter sp. G-1. Biosci. Biotechnol. Biochem. 57, 444–449.

    Google Scholar 

  31. Kimoto, H., Kusaoke, H., Yamamoto, I., Fujii, Y., Onodera, T., and Taketo, A. (2002) Biochemical and genetic properties of Paenibacillus glycosyl hydrolase having chitosanase activity and discoidin domain. J. Biol. Chem. 277, 14695–14702.

    Article  PubMed  CAS  Google Scholar 

  32. Okajima, S., Ando, A., Shinoyama, H., and Fujii, T. (1994) Purification and characterization of an extracellular chitosanase produced by Amycolatopsis sp. CsO-2. J. Ferment. Bioeng. 77, 617–620.

    Article  CAS  Google Scholar 

  33. Shimosaka, M., Nogawa, M., Ohno, Y., and Okazaki, M. (1993) Chitosanase from the plant pathogenic fungus, Fusarium solani f.sp. phaseoli: purification and some properties. Biosci. Biotech. Biochem. 57, 231–235.

    Article  CAS  Google Scholar 

  34. Vieille, C., Epting, K. L., Kelly, R. M., and Zeikus, J. G. (2001) Bivalent cations and amino acid composition contribute to the thermostability of B. licheniformis xylose isomerase. Eur. J. Biochem. 6268, 268–6301.

    Google Scholar 

  35. Vielle, C. and Zeikus, G. J. (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65(1), 1–43.

    Article  Google Scholar 

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Authors and Affiliations

  1. Center for Marine and Fisheries Product Processing and Biotechnology, Jl. KS Tubun, Petamburan VI, Jakarta, Indonesia

    Chasanah Ekowati

  2. Department of Food Science and Technology, Bogor Agricultural University, Darmaga Campus, PO Box 220, 16002, Bogor, Indonesia

    Purwiyatno Hariyadi & Maggy Thenawidjaja Suhartono

  3. Research Center for Biology and Biotechnology Bogor Agricultural University, Darmaga Campus, PO Box 220, 16002, Bogor, Indonesia

    Purwiyatno Hariyadi & Maggy Thenawidjaja Suhartono

  4. Research Center for Biotechnology, Indonesian Institute of Science, Indonesia

    Arief B. Witarto

  5. Bioproducts Resources Center, Yonsei University, Korea

    Jae Kwan Hwang

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  1. Chasanah Ekowati
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  2. Purwiyatno Hariyadi
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Correspondence to Maggy Thenawidjaja Suhartono.

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Ekowati, C., Hariyadi, P., Witarto, A.B. et al. Biochemical characteristics of chitosanase from the Indonesian Bacillus licheniformis MB-2. Mol Biotechnol 33, 93–102 (2006). https://doi.org/10.1385/MB:33:2:93

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