The chitinase is an enzyme whose ability to break down glycosidic bonds in chitin brings more variability into the molecules. Since not just the grade and pattern of deacetylation, but also the amount of connected chitin monomers influences the entire molecule´s behavior , there is a great limitation of the properties and the bioactivity of the products. Its possible implementation in the project shows the future prospects of how chitins and chitosans with all kind of properties can be produced in E. coli.
Chitin is a molecule found in fungal cell walls . Many plants possess enzymes, so-called chitinases, which are able to break down chitin and thus help along with its digestion. These enzymes play a role in defense mechanisms of plants in case of fungal infections . Even in human tissues chitinases appear, where they defend us against parasites . Chitinases break down the glycosidic bonds between chitin monomer units and are classified as hydrolases.
In our project, we decided to focus mainly on chitosan pentamers, synthesized and deacetylated by NodC and NodB respectively. However, chitosan's bioactivity and properties are also defined by its polymer length. For this reason, we want to give a first look at how our project could be extended.
The enzyme we use is the ChiA1 from Bacillus circulans . In the strain WL-12, the gene chiA encodes, together with chiB, chiC and chiD, an entire chitinase-system which primarily degrades chitin. This system is made up of at least six different chitinases , in which ChiA1 is believed to be the key enzyme .
The chiA gene is 1983 base pairs long and translates into an enzyme with a molecular weight of approximately 69.5 kDa .
Since ChiA1 has been successfully expressed together with our chitin synthase NodC , we originally decided on this specific enzyme. Its enzymatic activity has also previously been tested on chitin pentamers. ChiA1 breaks down these chitin pentamers in two dimers and one N-acetylglucosamine (GlcNAc) unit . Its implementation would then give us a bigger variety of chitosan molecules.
The N-terminal domain in the ChiA1 is responsible for its catalytic activity. The C-terminal domain plays an important role in the hydrolysis of chitin, and is the reason ChiA1 has such a high affinity to the substrate   .
A Chitinase's function is to break down chitin-oligomers into smaller molecules. ChiA1 turns chitinpentaose into two molecules of chitinbiose and one molecule of GlcNAc, by attacking the glycosidic bonds between the monomers .
We ordered the chiA gene via IDT sequencing. First, we inserted this gene into the pUPD vector using a GoldenBraid assembly as this is a simple and fast cloning method . For cloning the chiA gene into the pSB1C3 vector, we used the BioBrick system .
After cutting the pUPD vector, which contains the chiA gene, and the pSB1C3 vector, with the restriction enzymes XbaI and PstI, dephosphorylating the backbone and subsequent ligation, we inserted the chiA gene succesfully into the pSB1C3 vector with an Anderson promoter with defined cleavage sites (BBa_K2380025).
We used the same protocols inserting the chiA gene successfully into the pSB1C3 vector with an inducible promoter system. We altered the restriction enzymes to NheI and PstI for the pUPD vector with the chiA gene in order to exchange the Anderson promoter which is located on the pUPD vector. We have cut the pSB1C3 vector containing an AraC promoter system (BBa_K808000) with the restriction enzymes SpeI and PstI, dephosphorylated it and succesfully inserted the chiA gene via subsequent ligation. As NheI and SpeI are complementary the finalized construct does contain the BioBrick retriction sites.
Both the pSB1C3 vectors, containing the chiA gene, have the RBS BBa_K2380024. After that, we transformed both plasmids into E. coli Top10 cells and BL21 cells. We verified the validity via eurofins tube sequencing, using a Mini-Prep-Kit first for DNA preparation.
We induced the E. coli BL21 cells, containing the pSB1C3 vector with the AraC promoter system, with arabinose to start expression. In order to validate the successful expression, we performed a SDS-Page.
Results and Discussion
To verify if ChiA1 is produced in BL21 cells containing the pSB1C3-AraC-chiA, a SDS-Page was performed. As we have described before, the page shows the expected results and proves that ChiA1 was successfully produced in E. coli BL21 cells.
Chitosan is a derivative produced from chitin. As previously mentioned, chitosan's properties and bioactivity are heavily dependent on three factors: degree and pattern of deacetylation as well as its degree of polymerisation. The first two characteristics can be influenced and controlled via chitin deacetylases. Bringing variability into the length, however, is easiest done by implementing chitinases, whose ability to break down chitin would mean shorter chitosan molecules in turn.
Since chitosan is such a versatile molecule, the ability to select the influencing factors beforehand, means a great advantage over normally used methods.
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|||Image of 1ITX ( Three-dimensional structure of the catalytic domain of chitinase A1 from Bacillus circulans WL-12 at a very high resolution Matsumoto, T., Nonaka, T., Hashimoto, M., Watanabe, T., Mitsui, Y. CRDT - 2002/02/13 12:00 AID - 10.2210/pdb1itx/pdb [doi] SO - http://www.rcsb.org/pdb/explore/explore.do?structureId=1ITX)created with The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.|
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