Team:TU Darmstadt/project/chitin deacetylase
ChiTUcare
Chitin Deacetylases NodB and COD
Chitosan is a polymeric product of deacetylated chitin, which exists in a wide variety of patterns differing in their degree of deacetylation. Our goal is to design chitosan oligomers with a specific pattern of deacetylation. It can then be used for the production of our hydrogels.
We implemented chitin deacetylases originating from the organisms Sinorhizobium meliloti (NodB) and Vibrio cholerae (COD) into our E. coli cells. These enzymes deacetylate chitin individually. NodB targets the first position of the non-reducing end, while COD works similarly on the second unit. By implementing an orthogonal expression system to regulate the patterns, designer chitosan could be adjusted to the respective task. This would allow the expression of each enzyme separately, creating a defined deacetylation pattern.
What are Chitin Deacetylases?
Chitin deacetylases (CDA) mostly occur in marine bacteria, few in insects, and several in fungi [1].
In fungi, for example, CDAs are involved in cell wall formation, sporulation, and catabolism of chitin oligosaccharides. Many plant fungal pathogens secrete CDAs during plant infection. Plants only detect fungal infections by registering chitin. Fungi “turn invisible” by deacetylating chitin into chitosan and thus, outwit the plant defence system [2].
The CDAs generate chitosan oligomers from chitin by deacetylating the N-acetylglucosamine units of the substrate [3]. During deacetylation, acetic acid is cleaved off from a glucosamine unit. Some CDAs may even deacetylate chitosan, creating a double deacetylated oligomer [2].
Chitin deacetylases belong to the carbohydrate esterase family 4. All family members, including NodB protein and chitin deacetylases, share the same primary structure called “NodB homology domain” or “polysaccharide deacetylase domain” [4].
In medical applications and plant protection, CDAs are used for designing antifungal and antibacterial biofilms [2].
NodB - Sinorhizobium meliloti
Introduction
The CDA NodB is isolated from the organism Sinorhizobium meliloti (strain 1021) [6], which belongs to the family of gram-negative proteobacteria [5]. Rhizobium sp. often form a root endosymbiosis with legumes in nature. Through this, nitrogen assimilation in legumes is provided. For cell signalling the microbial partners and plants exchange diffusible molecules, the so-called nodulation factors (Nod factors) [7]. Belonging to these Nod factors are NodC, NodB and NodA.
For further information to NodC visit the following link Chitin Synthase
The nodB gene is 653 base pairs long and translates into a hydrolase with a molecular weight of approximately 24.4 kDa [2].
The enzyme works optimally in surroundings with a pH of 9 and temperatures reaching 37 degrees Celsius [2]. It solely deacetylates the first position of the non-reducing end in a chitin oligomer [2]. Due to regioselectivity [7], monomers are not deacetylated by NodB, therefore chains of dimers up to hexamers are converted to mono-deacetylated chitosan oligomers [7].
If NodB is incubated for a long time with the substrate and high enzyme concentrations, the possibility of double-deacetylated oligomers arises. However, the emerging amount is insignificant [2].
Mechanism
As explained before, CDAs occur in many different organisms and produce chitosan out of chitin to outwit plant defence systems.
NodB deacetylates the first N-acetyl-D-glucosamine unit (GlcNAc) of the non-reducing end [2]. Deacetylation describes hydrolysis of the acetamido group in the GlcNAc units, thus generating acetic acid und D-glucosamine (GlcN) [1].
Material/Methods
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Results and Discussion
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COD -Vibrio Cholerae
Introduction
In order to bring more variability in our 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 O1 biovar El Tor str. N16961 [10]. It is not part of its pathogenic activity. During our conversation with Professor Mörschbacher, he informed us that the expression of COD in E.coli has been successfully performed by himself and his group. In addition, it was already proven that both NodB and COD could be expressed in the same organism [2].
The cod gene is 1296 base pairs long and translates into a hydrolase with a molecular weight of approximately 45.5 kDa [2].
The enzyme works optimally in surroundings with a pH of 8 and temperatures reaching 45 degrees Celsius [2][4].
As mentioned before, 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][4][9]. If both enzymes – COD and NodB – are active at once we are able to create a deacetylation pattern involving the first two units.
In contrast to NodB, COD does not deacetylate chitosan twice, after long incubation periods [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) [4].
Mechanism
COD deacetylates the second N-acetyl-D-glucosamine unit (GlcNAc) of the non-reducing end [2], thus generating acetic acid and D-glucosamine (GlcN).
Material/Methods
We ordered the cod gene via IDT sequencing and inserted this gene into the pSB1C3 vector through a BioBrick system and verified this via sequencing. The cod was fused to a T7-promoter (BBa_I719005). The vector includes the RBS BBa_K2380024.
Results and Discussion
The cod gene was successfully expressed in E. coli Bl21 after induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The expression temperature was reduced to 30 degrees Celsius.
Analysis utilizing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed single bands for the COD enzyme (approximately 45.5 kDa).
Further approach would include adding His-tags and purification via affinity chromatography. To test the enzyme activity the amount of released acetic acid during enzymatic reaction can be measured, using an acetic acid assay kit or via thin-layer chromatography.
Considering the importance of using two CDAs an orthogonal expression system could be implemented to create multiple patterns of deacetylation.
Designer Future
The implementation of these two deacetylases shows the potential our approach can hold. Since each enzyme possesses an unique pattern of deacetylation, the chitosan pentamers will vary as well. Through our regulation system, involving the T7-split polymerases, the activity of each enzyme can be controlled. Thus, we are able to directly influence which deacetlyase is active at what time. Accordingly, we have the possibility to know which deacetylation pattern the pentamers will have. This is a stark contrast to chemical productions, where the properties of chitosan are random.
While our project only involves two deacetylases at this time, others could be introduced as well. In the future, new additions would open up new alternatives for deacetylation patterns. Since bioactivity of chitosan is hugely dependent on its patterns and degree of deacetylation, our approach allows to predetermine these factors of the product. Production would be possible at a much lower cost and with higher specificity than with current standard methods.
Group picture
![Members of the group chitin deacetylases]()
References
| [1] | Zhao, Y. et.al. , Park, R.-D., Muzzarelli, A.A. (2010) Chitin Deacetylases: Properties and Applications; Marine Drugs, 8(1), 24-46; DOI: 10.3390/md8010024 |
| [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] | Hamer, S.N. et.al., Moerschbacher, B. M., Kolkenbrock, S. (2014) Enzymatic sequencing of partially acetylated chitosan oligomers; Carbohydrate Research, 392, 16–20; DOI: 10.1016/j.carres.2014.04.006 |
[4] | 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 |
[5] | Gargaud M. et.al., Amils, R., Cernicharo Quintanilla, J. , Cleaves II, H.J., Irvine, W.M., Pinti, D., Viso, M. (Eds.) (2011) Encyclopedia of Astrobiology, Springer-Verlag Berlin Heidelberg; DOI: 10.1007/978-3-642-11274-4 |
[6] | Bateman, A., Wu, C., Xenarios, I.; UniProtKB - P02963 (NODB_RHIME); http://www.uniprot.org/uniprot/P02963; last visited: 10/19/2017 |
[7] | Chambon, R., Pradeau, S., Fort, S., Cottaz, S., Armand, S. (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 |
[8] | ...quelle von kristalstruktur von nodB... | [9] | 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 |
[10] | 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 |
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