Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Enzymatic Production of High Molecular Weight Chitooligosaccharides Using Recombinant Chitosanase from Bacillus thuringiensis BMB171

  • Kang, Lixin (Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University) ;
  • Jiang, Sijing (Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University) ;
  • Ma, Lixin (Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University)
  • Received : 2017.12.14
  • Accepted : 2018.02.13
  • Published : 2018.03.28
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Abstract

The chitosanase gene (btbchito) of Bacillus thuringiensis BMB171 was cloned and heterologously expressed in the yeast Pichia pastoris. After purification, about 300 mg of recombinant chitosanase was obtained from the 1-1 culture medium with a specific activity of 240 units/mg. Results determined by the combined use of thin layer chromatography (TLC) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) showed that the chitooligosaccharides (COSs) obtained by chitosan (N-deacetylated by 70%, 80%, and 90%) hydrolysis by rBTBCHITO were comprised of oligomers, with degrees of polymerization (DP) mainly ranging from trimers to heptamers; high molecular weight chitopentaose, chitohexaose, and chitoheptaose were also produced. Hydrolysis products was also deduced using MS since the COSs (n) are complex oligosaccharides with various acetyl groups from one to two, so the non-acetyl COSs (GlcN)n and COSs with more acetyls (> 2) were not detected. The employment of this method in the production of high molecular weight COSs may be useful for various industrial and biological applications, and the activity of chitosanase has great significance in research and other applications.

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References

  1. Park JK, Chung MJ, Choi HN, Park YI. 2011. Effects of the molecular weight and the degree of deacetylation of chitosan oligosaccharides on antitumor activity. Int. J. Mol. Sci. 12: 266-277.
  2. Jeon YJ, Kim SK. 2002. Antitumor activity of chitosan oligosaccharides produced in ultrafiltration membrane reactor system. J. Microbiol. Biotechnol. 12: 503-507.
  3. Lee BC, Kim MS, Choi SH, Kim KY, Kim TS. 2009. In vitro and in vivo antimicrobial activity of water-soluble chitosan oligosaccharides against Vibrio vulnificus. Int. J. Mol. Med. 24: 327-333.
  4. Takano S. 2012. Progress in antimicrobial activities of chitin, chitosan and its oligosaccharides: A systematic study needs for food applications. Food Sci. Technol. Int. 18: 3-34. https://doi.org/10.1177/1082013211399195
  5. Liu HT, Li WM, Xu G, Li XY, Bai XF, Wei P, et al. 2009. Chitosan oligosaccharides attenuate hydrogen peroxide-induced stress injury in human umbilical vein endothelial cells. Pharmacol. Res. 59: 167-175. https://doi.org/10.1016/j.phrs.2008.12.001
  6. Jung MJ, Park JK, Park YI. 2012. Anti-inflammatory effects of lowmolecular weight chitosan oligosaccharides in IgE-antigen complex- stimulated RBL-2H3 cells and asthma model mice. Int. Immunopharmacol. 12: 453-459. https://doi.org/10.1016/j.intimp.2011.12.027
  7. Liang TW, Liu CP, Wu CH, Wang SL. 2013. Applied development of crude enzyme from Bacillus cereus in prebiotics and microbial community changes in soil. Carbohydr. Polym. 92: 2141-2148. https://doi.org/10.1016/j.carbpol.2012.11.097
  8. Wei XL, Wang YF, Zhu Q, Xiao JB, Xia WS. 2009. Effects of chitosan pentamer and chitosan hexamer in vivo and in vitro on gene expression and secretion of cytokines. Food Agr. Immunol. 20: 269-280. https://doi.org/10.1080/09540100903168157
  9. Wei XL, Wang YF, Xiao JB, Xia WS. 2009. Separation of chitooligosaccharides and the potent effects on gene expression of cell surface receptor CR3. Int. J. Biol. Macromol. 45: 432-436. https://doi.org/10.1016/j.ijbiomac.2009.07.003
  10. Feng J, Zhao LH, Yu QQ. 2004. Receptor-mediated stimulatory effect of oligochitosan in macrophages. Biochem. Bioph. Res. Co. 317: 414-420. https://doi.org/10.1016/j.bbrc.2004.03.048
  11. Panda SK, Kumar S, Tupperwar NC, Vaidya T, George A, Rath S, et al. 2012. Chitohexaose activates macrophages by alternate pathway through TLR4 and blocks endotoxemia. PLoS Pathog. 8: e1002717. https://doi.org/10.1371/journal.ppat.1002717
  12. Xiong C, Wu H, Wei P, Pan M, Tuo Y, Kusakabe I, et al. 2009. Potent angiogenic inhibition effects of deacetylated chitohexaose separated from chitooligosaccharides and its mechanism of action in vitro. Carbohydr. Res. 344: 1975-1983.
  13. Kadowaki O, Ogasawara A, Watanabe T, Mikami T, Matsumoto T, Misawa Y, et al. 2007. Chitohexose induce the yeast proliferation of Candida albicans. Biol. Pharm. Bull. 30: 583-584. https://doi.org/10.1248/bpb.30.583
  14. Zhang X, Li K, Liu S, Xing R, Yu H, Chen X, et al. 2016. Size effects of chitooligomers on the growth and photosynthetic characteristics of wheat seedlings. Carbohydr. Polym. 138: 27-33. https://doi.org/10.1016/j.carbpol.2015.11.050
  15. Oh C, De Zoysa M, Kang DH, Lee Y, Whang I, Nikapitiya C, et al. 2011. Isolation, purification, and enzymatic characterization of extracellular chitosanase from marine bacterium Bacillus subtilis CH2. J. Microbiol. Biotechnol. 21: 1021-1025.
  16. Pechsrichuang P, Yoohat K, Yamabhai M. 2013. Production of recombinant Bacillus subtilis chitosanase, suitable for biosynthesis of chitosan-oligosaccharides. Bioresour. Technol. 127: 407-414. https://doi.org/10.1016/j.biortech.2012.09.130
  17. Gao XA, Ju WT, Jung WJ, Park RD. 2008. Purification and characterization of chitosanase from Bacillus cereus D-11. Carbohydr. Polym. 72: 513-520.
  18. Goo BG, Park JK. 2014. Characterization of an alkalophilic extracellular chitosanase from Bacillus cereus GU-02. J. Biosci. Bioeng. 117: 684-689. https://doi.org/10.1016/j.jbiosc.2013.11.005
  19. Wang SL, Chao CH, Liang TW, Chen CC. 2009. Purification and characterization of protease and chitinase from Bacillus cereus TKU006 and conversion of marine wastes by these enzymes. Mar. Biotechnol. 11: 334-344. https://doi.org/10.1007/s10126-008-9149-y
  20. Ekowati C, Hariyadi P, Witarto AB, Hwang JK, Suhartono MT. 2006. Biochemical Characteristics of Chitosanase From the Indonesian Bacillus licheniformis MB-2. Mol. Biotechnol. 33: 93-102. https://doi.org/10.1385/MB:33:2:93
  21. Saito J, Kita A, Higuchi Y, Nagata Y, Ando A, Miki K. 1999. Crystal structure of chitosanase from Bacillus circulans MH-K1 at 1.6-AA resolution and its substrate recognition mechanism. J. Biol. Chem. 274: 30818-30825. https://doi.org/10.1074/jbc.274.43.30818
  22. Tohru K, Osamu K, Shigeru D, Koki H. 2011. Characterization of chitosanase of a deep biosphere Bacillus strain. Biosci. Biotechnol. Biochem. 75: 669-673.
  23. Lee HS, Jang JS, Choi SK, Lee DW, Kim EJ, Jung HC, et al. 2007. Identication and expression of GH-8 family chitosanases from several Bacillus thuringiensis subspecies. FEMS Microbiol. Lett. 277: 133-141. https://doi.org/10.1111/j.1574-6968.2007.00944.x
  24. Zhang GM, Hu Y, Zhuang YH. 2006. Molecular cloning and heterologous expression of an alkaline xylanase from Bacillus pumilus HBP8 in Pichia pastoris. Biocatal. Biotransform. 24: 371-379. https://doi.org/10.1080/10242420600768771
  25. Wu DC, Qu LZ, Fu Y, Li J, Jiang LY, Chen XJ, et al. 2016. Expression and purification of the kinase domain of PINK1 in Pichia pastoris. Protein Expr. Purif. 128: 67-72. https://doi.org/10.1016/j.pep.2016.08.010
  26. Bradford MM. 1976. Arapid and sensitive method for the quantification of microgram quanlities of proteins utlizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  27. Kang LX, Chen XM, Fu L, Ma LX. 2012. Recombinant expression of chitosanase from Bacillus subtilis HD145 in Pichia pastoris. Carbohydr. Res. 352: 37-43. https://doi.org/10.1016/j.carres.2012.01.025
  28. Camarillo RC, Perez OS, Avelizapa NGR, Ramirez MG, Avelizapa LIR. 2004. Chitosanase activity in Bacillus thuringiensis. Folia Microbiol. 49: 94-96. https://doi.org/10.1007/BF02931653
  29. Chang WT, Chen YC, Jao CL. 2007. Antifungal activity and enhancement of plant growth by Bacillus cereus grown on shellfish chitin wastes. Bioresour. Technol. 98: 1224-1230. https://doi.org/10.1016/j.biortech.2006.05.005
  30. Liang TW, Liu CP, Wu CH, Wang SL. 2013. Applied development of crude enzyme from Bacillus cereus in prebiotics and microbial Community changes in soil. Carbohydr. Polym. 92: 2141-2148.
  31. Liang TW, Hsieh JL, Wang SL. 2012. Production and purification of a protease, a chitosanase, and chitin oligosaccharides by Bacillus cereus TKU022 fermentation. Carbohydr. Res. 362: 38-46. https://doi.org/10.1016/j.carres.2012.08.004
  32. Kurakake M, You S, Nakagawa K, Sugihara M, Komaki T. 2000. Properties of chitosanase from Bacillus cereus S1. Curr. Microbiol. 40: 6-9. https://doi.org/10.1007/s002849910002
  33. Liang TW, Chen YY, Pan PS, Wang SL. 2014. Purification of chitinase/ chitosanase from Bacillus cereus and discovery of an enzyme inhibitor. Int. J. Biol. Macromol. 63: 8-14. https://doi.org/10.1016/j.ijbiomac.2013.10.027

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