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

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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. 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

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Material/Methods

Faucibus sed lobortis aliquam lorem blandit. Lorem eu nunc metus col. Commodo id in arcu ante lorem ipsum sed accumsan erat praesent faucibus commodo ac mi lacus. Adipiscing mi ac commodo. Vis aliquet tortor ultricies non ante erat nunc integer eu ante ornare amet commetus vestibulum blandit integer in curae ac faucibus integer non. Adipiscing cubilia elementum.

Results and Discussion

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Regulation of both enzymes

In order to achieve various degrees and patterns of deacetylation for our designer chitosan, different chitin deacetylases are regulated via an orthogonal T7-Split-Polymerase system. This allows induction with rapamycin or blue light. Mutants of the T7-promotor (N4) were used to increase regulation of CDAs and to minimize the expression of nodB, thus preventing aggregation of NodB inclusion bodies within the cell.
By individually controlling the expression rate of each enzyme, hybrid oligomers can be produced, as both enzymes can work simultaneously. Chitin oligomers are deacetylated at specific positions by different chitin deacetylases. We aim to achieve predefined chitosan oligomers based on expression level regulation.

For further details on how our T7-Split-Polymerase regulation system works and how it is implemented, visit the following link:
Regulation System

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 deactylases 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.

References