Difference between revisions of "Part:BBa K2380044"

Revision as of 15:43, 30 October 2017

Chitin deacetylase COD

Chitin deacetylase (CDA) from Vibrio cholerae. The enzyme is a hydrolase and deacetylates chitin to chitosan. It solely deacetylates the second position of the non-reducing end in a chitin oligomer. We used it for the production of chitosan with a defined deacetylation pattern. It is not part of the pathogenic activity of Vibrio cholerae.

Usage and Biology

In order to bring more variability in produced chitosans, we decided to implement a second chitin deacetylase. Because of its bacterial origin, we picked COD isolated from the gram-negative organism Vibrio cholerae, but it is not responsible for the pathogenicity of Vibrio cholerae in any way. The cod gene is 1296 base pairs long and translates into a hydrolase with a molecular weight of approximately 45,5 kDa [2]. Deacetylases target different units in a chitin molecule. Which unit is deacetylated depends on the chosen enzyme. In the case of COD, the second position from the non-reducing end is deacetylated [2][3]. The enzyme works optimally in surroundings with a pH of 8 and temperatures around 45 degrees Celsius [2][3]. It is already proven that more than one CDA can be expressed in the same organism [2]. In contrast to NodB CDA, COD does not deacetylate chitosan twice, even at high enzyme concentrations and after long incubation periods , making it a very specific enzyme and therefore a valuable tool to create designer chitosan[2]. CODs catalytic part is its N-terminal domain, while the other two domains make up carbohydrate-binding molecules. The catalytic domain correlates to a carbohydrate esterase domain (CDA) [3].

Results

Expression of the enzyme was successfully detected via SDS-PAGE (Fig. 3). For expression cod was fused to a T7 promoter (Part:BBa_K2380043) Production becomes visible three hours after IPTG induction, which further increases with longer incubation periods of the induced cultures (Fig. 3, pUPD-nodB, 6h and 24h). A comparison to cultures without induction (Fig. 3, pSB1C3-nodB) functions as a negative control.
This proves that COD can be produced in E. coli BL21. For further analysis of the enzymes activity, assays for acetic acid detection could be used as it´s a byproduct of the deacetylation process.

Figure 3: SDS-Page of chitin deacetylase COD protein. The arrow marks the region of expression. From left to right: Non transformed E. coli BL21 24h after and simultaneous (0h) to induced cultures, E. coli BL21 transformed with pUPD-nodB after being induced with IPTG for 24h, 6h, 3h and before induction (0h). Usage of PageRuler Prestained Protein Ladder 10 to 180 kDa from ThermoFischer Scientific.

References

[1] National Center for Biotechnology Information, U.S. National Library of Medicine (NCBI); Vibrio cholerae O1 biovar eltor str. N16961 chromosome I, complete sequence, GenBank: AE003852.1; https://www.ncbi.nlm.nih.gov/nuccore/AE003852.1?from=1355388&to=1356683&sat=4&sat_key=105780702; last visited: 09/01/2017
[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] Andrés, E. et.al., Albesa-Jové, D., Biarnés, X., Moerschbacher, B.M., Guerin, M., Planas, A. (2014) Structural Basis of Chitin Oligosaccharide Deacetylation; Angewandte Chemie International Edition, 53, 6882-6887; DOI: 10.1002/anie.201400220
[4] Li, X., Wang, L., Wang, X., Roseman, S. (2007) The Chitin Catabolic Cascade in the Marine Bacterium Vibrio Cholerae: Characterization of a Unique Chitin Oligosaccharide Deacetylase, Glycobiology, vol. 17, Issue 12, 1377–1387; DOI: 10.1093/glycob/cwm096

Sequence and Features