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

Applied Microbiology and Biotechnology volume 97, pages 5801–5813 (2013)Cite this article

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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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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  1. Mélanie Fortin

    Present address: Centre d’expertise en spectrométrie de masse clinique Waters—CHUS, Centre Hospitalier Universitaire de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, J1H 5N4, Québec, Canada

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  1. Centre d’Étude et de Valorisation de la Diversité Microbienne, Département de Biologie, Faculté des Sciences, Université de Sherbrooke, 2500 boulevard de l’Université, Sherbrooke, J1K 2R1, Québec, Canada

    Mina Zitouni, Mélanie Fortin, Dominick Matteau, Sébastien Rodrigue & Ryszard Brzezinski

  2. Analytical Research Group, Wissenschaftszentrum Weihenstephan, Technische Universität München, Weihenstephaner Steig 23, 85354, Freising-Weihenstephan, Germany

    Romy K. Scheerle & Thomas Letzel

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  1. Mina Zitouni
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  2. Mélanie Fortin
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  7. Ryszard Brzezinski
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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