Team:TU Darmstadt/project/chitin deacetylase
ChiTUcare
Chitin Deacetylases NodB and COD
Abstract
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 several 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 regulating this pattern using an orthogonal expression system, designer chitosan can be adjusted to the respective task. Our method allows the expression of each enzyme separately creating a defined deacetylation pattern.
What are chitin deacetylases?
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NodB
Introduction
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Mechanism
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Material/Methods
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Results and Discussion
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COD
Introduction
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Mechanism
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.
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 a 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.
