Part:BBa_K2380005
Chitinase A1
Chitinase A1 (ChiA1) is a hydrolase isolated from the organism Bacillus circulans WL-12. The chiA gene is 1983 base pairs long and translates into an enzyme with a molecular weight of approximately 69 kDa. ChiA1's main function is to break down glycosidic bonds between N-Acetylglucosamine (GlcNAc) units in a chitin oligomer.
Usage and Biology
Chitin is a molecule found in fungal cell walls [1]. Many plants possess enzymes, so-called chitinases, which are able to break down chitin, thus helping with its digestion. These enzymes play a role in defense mechanisms of plants, in case of fungal infections [2]. Even in human tissues chitinases appear, where they defend the human body against parasites [3]. In bacteria chitinases mostly appear as exoenzymes carried in cellulosomes, thus making them virulence factors [4]. Chitinases break down the glycosidic bonds between chitin monomer units, and are classified as hydrolases.
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 [5].
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[5].
For the expression of that basic part, two composite parts were designed.
For constitutive expression ChiA was fused to a Anderson promoter. https://parts.igem.org/Part:BBa_K2380006
For inducible expression a araC/Pbad promoter system was added. https://parts.igem.org/Part:BBa_K2380007
For more informations about this Part, visit the Wiki: http://2017.igem.org/Team:TU_Darmstadt/project/chitinase
Results
Fig. 5: SDS-Page
Lane 1: 24 hours after induction
Lane 2: 3 hours after induction
Lane 3: Before induction
Lane 4: Empty BL21 cells
To verify if ChiA1 is produced in E.coli 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.
References
[1] Stefanie Nicole Hamer, Stefan Cord-Landwehr, Xevi Biarnés, Antoni Planas, Hendrik Waegeman, Bruno Maria Moerschbacher, and Staphan Kolkenbrock (2015) Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases
DOI: 10.1038/srep08716
[2] John G. Verburg and Q. Khai Huynh (1990) Purification and Characterization of an Antifungal Chitinase from Arabidopsis thaliana. Plant Physiol. 95, 450-455
DOI: https://doi.org/10.1104/pp.95.2.450
[3] Paoletti MG, Norberto L., Damini R., and Musumeci S. (2007) Human gastric juice contains chitinase that can degrade chitin
DOI: 10.1159/000104144
[4] Frederiksen RF, Paspaliari DK, Larsen T., Storgaard BG, Larsen MH, Ingmer H., Palcic MM, Leisner JJ (2013) Bacterial chitinases and chitin-binding proteins as virulence factors
[5] Sylvain Cottaz, Eric Samain (2005) Genetic engineering of Escherichia coli for the production of NI,NII-diacetylchitobiose (chitinbiose) and its utilization as a primer for the synthesis of complex carbohydrates. Metabolic Engineering 7, 311–317
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 150
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 256
Illegal AgeI site found at 952
Illegal AgeI site found at 1240
Illegal AgeI site found at 1303
Illegal AgeI site found at 1366
Illegal AgeI site found at 1696 - 1000COMPATIBLE WITH RFC[1000]
| None |