Difference between revisions of "Part:BBa K2380041"

 
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<h2>Biology and Usage</h2>
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===Usage and Biology===
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<h3>Enzyme properties</h3>
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<p>A chitin deacetylase isolated from the gram-negative proteobacteria [1] Sinorhizobium meliloti (strain 1021) [3]. The gene itself is 653 base pairs long and translates into a hydrolase with a molecular weight of approximately 24,4 kDa [2]. It solely deacetylates the first position of the non-reducing end in a chitin oligomer, changing the acetyl-group to an amino-group via hydrolysis. Expressed in E. coli the enzyme accumulates within the cell in insoluble inclusion bodies and must be purified to gain functionality. The enzymes optimal working conditions are in surroundings of pH 9 and temperatures ranging around 37 degrees Celsius. </p>
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<h3>Application</h3>
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<p>Chemically or biologically modified chitosan oligomers allow for a multitude of applications in modern day projects ranging from agriculture to applied medicine. Due to its stability, easy handling and inexpensive raw material chitosan can be produced in large scales and used as a substrate for further modifications. Thanks to its antibacterial features usage in clinical environments is also possible. </p>
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<h3>Purification</h3>
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<p>During enzyme expression it is important to consider that the protein must be either be purified and refolded before usage or be used with a weak promotor to allow for a significant amount of soluble NodB within the cell. Otherwise it will as already described accumulate in inclusion bodies and therefore be unusable [4]. </p>
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<h2>References</h3>
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<p>[1] Muriel Gargaud (Editor-in-Chief), Ricardo Amils, Jose ́ Cernicharo Quintanilla, Henderson James (Jim) Cleaves II, William M. Irvine, Daniele L. Pinti and Michel Viso (Eds.) (2011); Encyclopedia of Astrobiology, Springer-Verlag Berlin Heidelberg, DOI: 10.1007/978-3-642-11274-4, Pages<br>
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[2] Hamer, S.N. et al. Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases (2015). Sci. Rep. 5, 8716; DOI:10.1038/srep08716 <br>
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[3] Alex Bateman (PI), Cathy Wu, and Ioannis Xenarios; UniProtKB - P02963 (NODB_RHIME); http://www.uniprot.org/uniprot/P02963; last visited: 10/19/2017<br>
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[4] Rémi Chambon, Stéphanie Pradeau, Sébastien Fort, Sylvain Cottaz, Sylvie Armand (2011); High yield production of Rhizobium NodB chitin deacetylase and its use for in vitro synthesis of lipo-chitinoligosaccharide precursors; Carbohydrate Research 442, 25-30; DOI: 10.1016/j.carres.2017.02.007<p/>
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Revision as of 18:34, 20 October 2017


Chitin deacetylase NodB

Chitin deacetylase (CDA) NodB from Sinorhizobium meliloti. The enzyme is a hydrolase and deacetylates chitin to chitosan. It solely deacetylates the first position of the non-reducing end in a chitin oligomer. We used it for the production of chitosan with a defined deacetylation pattern. The CDA NodB forms inclusion bodies in E.coli.


Biology and Usage

Enzyme properties

A chitin deacetylase isolated from the gram-negative proteobacteria [1] Sinorhizobium meliloti (strain 1021) [3]. The gene itself is 653 base pairs long and translates into a hydrolase with a molecular weight of approximately 24,4 kDa [2]. It solely deacetylates the first position of the non-reducing end in a chitin oligomer, changing the acetyl-group to an amino-group via hydrolysis. Expressed in E. coli the enzyme accumulates within the cell in insoluble inclusion bodies and must be purified to gain functionality. The enzymes optimal working conditions are in surroundings of pH 9 and temperatures ranging around 37 degrees Celsius.

Application

Chemically or biologically modified chitosan oligomers allow for a multitude of applications in modern day projects ranging from agriculture to applied medicine. Due to its stability, easy handling and inexpensive raw material chitosan can be produced in large scales and used as a substrate for further modifications. Thanks to its antibacterial features usage in clinical environments is also possible.

Purification

During enzyme expression it is important to consider that the protein must be either be purified and refolded before usage or be used with a weak promotor to allow for a significant amount of soluble NodB within the cell. Otherwise it will as already described accumulate in inclusion bodies and therefore be unusable [4].

References</h3>

[1] Muriel Gargaud (Editor-in-Chief), Ricardo Amils, Jose ́ Cernicharo Quintanilla, Henderson James (Jim) Cleaves II, William M. Irvine, Daniele L. Pinti and Michel Viso (Eds.) (2011); Encyclopedia of Astrobiology, Springer-Verlag Berlin Heidelberg, DOI: 10.1007/978-3-642-11274-4, Pages
[2] Hamer, S.N. et al. Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases (2015). Sci. Rep. 5, 8716; DOI:10.1038/srep08716
[3] Alex Bateman (PI), Cathy Wu, and Ioannis Xenarios; UniProtKB - P02963 (NODB_RHIME); http://www.uniprot.org/uniprot/P02963; last visited: 10/19/2017
[4] Rémi Chambon, Stéphanie Pradeau, Sébastien Fort, Sylvain Cottaz, Sylvie Armand (2011); High yield production of Rhizobium NodB chitin deacetylase and its use for in vitro synthesis of lipo-chitinoligosaccharide precursors; Carbohydrate Research 442, 25-30; DOI: 10.1016/j.carres.2017.02.007<p/> Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]