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Biochemical and molecular characterization of a thermostable chitosanase produced by the strain Paenibacillus sp. 1794 newly isolated from compost

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Abstract

Chitosan raises a great interest among biotechnologists due to its potential for applications in biomedical or environmental fields. Enzymatic hydrolysis of chitosan is a recognized method allowing control of its molecular size, making possible its optimization for a given application. During the industrial hydrolysis process of chitosan, viscosity is a major problem; which can be circumvented by raising the temperature of the chitosan solution. A thermostable chitosanase is compatible with enzymatic hydrolysis at higher temperatures thus allowing chitosan to be dissolved at higher concentrations. Following an extensive micro-plate screening of microbial isolates from various batches of shrimp shells compost, the strain 1794 was characterized and shown to produce a thermostable chitosanase. The isolate was identified as a novel member of the genus Paenibacillus, based on partial 16S rDNA and rpoB gene sequences. Using the chitosanase (Csn1794) produced by this strain, a linear time course of chitosan hydrolysis has been observed for at least 6 h at 70 °C. Csn1794 was purified and its molecular weight was estimated at 40 kDa by SDS-PAGE. Optimum pH was about 4.8, the apparent K m and the catalytic constant kcat were 0.042 mg/ml and 7,588 min−1, respectively. The half-life of Csn1794 at 70 °C in the presence of chitosan substrate was >20 h. The activity of chitosanase 1794 varied little with the degree of N-acetylation of chitosan. The enzyme also hydrolyzed carboxymethylcellulose but not chitin. Chitosan or cellulose-derived hexasaccharides were cleaved preferentially in a symmetrical way (“3 + 3”) but hydrolysis rate was much faster for (GlcN)6 than (Glc)6. Gene cloning and sequencing revealed that Csn1794 belongs to family 8 of glycoside hydrolases. The enzyme should be useful in biotechnological applications of chitosan hydrolysis, dealing with concentrated chitosan solutions at high temperatures.

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References

  1. Aam BB, Heggset EB, Norberg AL, Sørlie M, Vårum KM, Eijsink VGH (2010) Production of chitooligosaccharides and their potential applications in medicine. Mar Drugs 8:1482–1517. doi:10.3390/md8051482

  2. Adachi W, Sakihama Y, Shimizu S, Sunami T, Fukazawa T, Suzuki M, Yatsunami R, Nakamura S, Takénaka A (2004) Crystal structure of family GH-8 chitosanase with subclass II specificity from Bacillus sp. K17. J Mol Biol 343:785–795. doi:10.1016/j.jmb.2004.08.028

  3. Bensing BA, Meyer BJ, Dunny GM (1996) Sensitive detection of bacterial transcription initiation sites and differentiation from RNA processing sites in the pheromone-induced plasmid transfer system of Enterococcus faecalis. Proc Natl Acad Sci U S A 93:7794–7799

  4. Blanchard J, Fukamizo T, Boucher I, Brzezinski R (2001) High level expression of the chitosanase from Streptomyces sp. N174 and study of its intramolecular interactions. Recent Res Dev Microbiol 5:111–121

  5. Boucher I, Dupuy A, Vidal P, Neugebauer WA, Brzezinski R (1992) Purification and characterization of a chitosanase from Streptomyces N174. Appl Microbiol Biotechnol 38:188–193

  6. Bradford MM (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. doi:10.1016/0003-2697(76)90527-3

  7. Bueno A, Vazquez de Aldana CR, Correa J, del Rey F (1990) Nucleotide sequence of a 1,3-1,4-β-glucanase-encoding gene in Bacillus circulans WL-12. Nucl Acids Res 18:4248

  8. Chen X, Zhai C, Kang L, Li C, Yan H, Zhou Y, Yu X, Ma L (2012) High-level expression and characterization of a highly thermostable chitosanase from Aspergillus fumigatus in Pichia pastoris. Biotechnol Lett 34:689–694. doi:10.1007/s10529-011-0816-0

  9. Crini G, Guibal É, Morcellet M, Torri G, Badot P-M. (2009) Chitine et chitosane. Préparation, propriétés et principales applications. In : Crini G, Badot P-M, Guibal É (ed) Chitine et chitosane. Du biopolymère à l’application, Presses universitaires de Franche-Comté, pp 19–54

  10. Dahllöf I, Baillie H, Kjelleberg S (2000) rpoB-based microbial community analysis avoids limitations inherent in 16S rRNA gene intraspecies heterogeneity. Appl Environ Microbiol 66:3376–3380. doi:10.1128/AEM.66.8.3376-3380.2000

  11. Dai T, Tanaka M, Huang Y-Y, Hamblin MR (2011) Chitosan preparations for wounds and burns: antimicrobial and wound-healing effects. Expert Rev Anti Infect Ther 9:857–879. doi:10.1586/eri.11.59

  12. Da Mota FF, Gomes EA, Paiva E, Rosado AS, Seldin L (2004) Use of rpoB gene analysis for identification of nitrogen-fixing Paenibacillus species as an alternative to the 16S rRNA gene. Lett Appl Microbiol 39:34–40. doi:10.1111/j.1472-765X.2004.01536.x

  13. Dennhart N, Fukamizo T, Brzezinski R, Lacombe-Harvey M-È, Letzel T (2008) Oligosaccharide hydrolysis by chitosanase enzymes monitored by real-time electrospray ionization-mass spectrometry. J Biotechnol 134:253–260. doi:10.1016/j.jbiotec.2008.02.004

  14. Desbrieres J (2002) Viscosity of semiflexible chitosan solutions: influence of concentration, temperature and role of intermolecular interactions. Biomacromolecules 3:342–349. doi:10.1021/bm010151+

  15. Domard A (2011) A perspective on 30 years research on chitin and chitosan. Carbohydr Pol 84:696–703. doi:10.1016/j.carbpol.2010.04.083

  16. Ekowati E, 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. doi:10.1385/MB:33:2:93

  17. Evans PD, Cook SN, Riggs PD, Noren CJ (1995) LITMUS: multipurpose cloning vector with a novel system for bidirectional in vitro transcription. Biotechniques 19:130–135

  18. Fullbrook PD (1996) Practical limits and prospects (kinetics). In: Godfrey T, West S (eds) Industrial enzymology. Macmillan Press, London, pp 503–540

  19. Ghinet MG, Gagnon J, Lacombe-Harvey M-È, Brzezinski R (2009) Molecular weight modulates the antimicrobial effect of chitosan on Escherichia coli. In: Rustichelli F, Caramella C, Şenel S, Vårum KM (eds) Advances in chitin sciences, vol. XI, Venice, Italy

  20. Ghinet MG, Roy S, Poulin-Laprade D, Lacombe-Harvey M-E, Morosoli R, Brzezinski R (2010) Chitosanase from Streptomyces coelicolor A3(2): biochemical properties and role in protection against antibacterial effect of chitosan. Biochem Cell Biol 88:907–916. doi:10.1139/o10-109

  21. Hamdine M, Heuzey M-C, Bégin A (2005) Effect of organic and inorganic acids on concentrated chitosan solutions and gels. Int J Biol Macromol 37:134–142

  22. Hirano S, Ohe Y (1975) A facile N-acylation of chitosan with carboxylic anhydrides in acidic solutions. Carbohydr Res 41:C1–C2

  23. Hoell IA, Vaaje-Kolstad G, Eijsink VGH (2010) Structure and function of enzymes acting on chitin and chitosan. Biotechnol Genet Eng Rev 27:331–366

  24. Il’ina AV, Varlamov VP (2003) Effect of the degree of acetylation of chitosan on its enzymatic hydrolysis with the preparation celloviridin G20kh. Appl Biochem Microbiol 39:239–242. doi:10.1023/A:1023515308584

  25. Isogawa D, Fukuda T, Kuroda K, Kusaoke H, Kimoto H, Suye S, Ueda M (2009) Demonstration of catalytic proton acceptor of chitosanase from Paenibacillus fukuinensis by comprehensive analysis of mutant library. Appl Microbiol Biotechnol 85:95–104. doi:10.1007/s00253-009-2041-5

  26. Kimoto H, Kusaoke H, Yamamoto I, Fujii Y, Onodera T, Taketo A (2002) Biochemical and genetic properties of Paenibacillus glycosyl hydrolase having chitosanase activity and discoidin domain. J Biol Chem 277:14695–14702. doi:10.1074/jbc.M108660200

  27. Kong M, Chen XG, Xing K, Park HJ (2010) Antimicrobial properties of chitosan and mode of action: a state of the art review. Int J Food Microbiol 144:51–63. doi:10.1016/j.ijfoodmicro.2010.09.012

  28. Lacombe-Harvey M-È, Fukamizo T, Gagnon J, Ghinet MG, Dennhart N, Letzel T, Brzezinski R (2009) Accessory active site residues of Streptomyces sp. N174 chitosanase: variations on a common theme in the lysozyme superfamily. FEBS J 276:857–869

  29. Lavertu M, Xia Z, Serreqi AN, Berrada M, Rodrigues A, Wang D, Buschmann MD, Gupta A (2003) A validated 1H NMR method for the determination of the degree of deacetylation of chitosan. J Pharm Biomed Anal 32:1149–1158. doi:10.1016/S0731-7085(03)00155-9

  30. Lee YS, Yoo JS, Chung SY, Lee YC, Cho YS, Choi YL (2006) Cloning, purification, and characterization of chitosanase from Bacillus sp. DAU101. Appl Microbiol Biotechnol 73:113–121. doi:10.1007/s00253-006-0444-0

  31. Lever M (1972) A new reaction for colorimetric determination of carbohydrates. Anal Biochem 47:273–279. doi:10.1016/0003-2697(72)90301-6

  32. Muzzarelli RAA, Lough C, Emanuelli M (1987) The molecular weight of chitosans studied by laser light-scattering. Carbohydr Res 164:433–442. doi:10.1016/0008-6215(87)80146-5

  33. Nonomura H, Hayakawa M (1988) New methods for selective isolation of soil actinomycetes. In: Okami Y, Beppu T, Ogawara H (eds) Biology of actinomycetes ’88. Japan Scientific Societies Press, Tokyo, pp 288–293

  34. Notredame C, Higgins DG, Heringa J (2000) T-Coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol 302:205–217. doi:10.1006/jmbi.2000.4042

  35. Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nature Methods 8:785–786. doi:10.1038/nmeth.1701

  36. Přenosil JE, Dunn IJ, Heinzle E (1987) Biocatalyst reaction engineering. In: Kennedy JF (ed) Biotechnology, vol 7a, Enzyme technology. VCH, Weinheim, pp 489–545

  37. Pscheidt B, Glieder A (2008) Yeast cell factories for fine chemical and API production. Microb Cell Fact 7:25. doi:10.1186/1475-2859-7-25

  38. Roberts GAF (1992) Chitin chemistry. MacMillan, London

  39. Roy S, Gagnon J, Fortin M, Ghinet MG, LeHoux J-G, Dupuis G, Brzezinski R (2007) Quantitative fluorometric analysis of the protective effect of chitosan on thermal unfolding of catalytically active native and genetically-engineered chitosanases. Biochim Biophys Acta—Proteins Proteom 1774:975–984. doi:10.1016/j.bbapap. 2007.05.016

  40. Rwei SP, Chen TY, Cheng YY (2005) Sol/gel transition of chitosan solutions. J Biomater Sci Polymer Edn 16:1433–1445. doi:10.1163/156856205774472290

  41. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor

  42. Scheerle RK, Grassmann J, Letzel T (2011) Enzymatic conversion continuously monitored with a robotic nanoESI-MS tool: experimental status. Anal Methods 3:822–830. doi:10.2116/analsci.28.607

  43. Schep GP, Shepherd MG, Sullivan PA (1984) Purification and properties of a β-1,6-glucanase from Penicillium brefeldianum. Biochem J 223:707–714

  44. Shinoda S, Kanamasa S, Arai M (2012) Cloning of an endoglycanase gene from Paenibacillus cookii and characterization of the recombinant enzyme. Biotechnol Lett 34:281–286. doi:10.1007/s10529-011-0759-5

  45. Sorlier P, Denuzière A, Viton C, Domard A (2001) Relation between the degree of acetylation and the electrostatic properties of chitin and chitosan. Biomacromolecules 2:765–772. doi:10.1021/bm015531+

  46. Yoon H-G, Ha S-C, Lim Y-H, Cho H-Y (1998) New thermostable chitosanase from Bacillus sp.: purification and characterization. J Microbiol Biotechnol 8:449–454

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

  48. Zitouni M, Fortin M, Thibeault J-S, Brzezinski R (2010) A dye-labelled soluble substrate for the assay of endo-chitosanase activity. Carbohydr Polym 80:522–525. doi:10.1016/j.carbpol.2009.12.012

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Acknowledgments

Work at Université de Sherbrooke was supported by a Strategic Grant to RB and Discovery Grants to RB and SR from Natural Science and Engineering Council of Canada. Work at TU München was supported by the Arbeitsgemeinschaft industrieller Forschungsvereinigungen, Köln, Germany (project no. 16203N). The authors thank Dr. Brian Talbot for critical reading of the manuscript. A provisional patent on behalf the Université de Sherbrooke, involving MZ, MF, and RB has been filed regarding the application of this enzyme for chitosan hydrolysis.

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Correspondence to Ryszard Brzezinski.

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Zitouni, M., Fortin, M., Scheerle, R.K. et al. Biochemical and molecular characterization of a thermostable chitosanase produced by the strain Paenibacillus sp. 1794 newly isolated from compost. Appl Microbiol Biotechnol 97, 5801–5813 (2013). https://doi.org/10.1007/s00253-012-4483-4

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Keywords

  • Chitosan
  • Chitosanase
  • Oligosaccharide
  • Thermostability
  • Cleavage pattern
  • Paenibacillus